Note: Descriptions are shown in the official language in which they were submitted.
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
COATED PARTICLES, METHODS OF MAKING AND USING
DESCRIPTION
BACKGROUND OF THE INVENTION
Field of the Ihventio~z
l 0 The present invention relates to coated particles and to methods of
malting and using
them. These coated particles have application in the targeting and release of
one or more
materials into selected environments, the absorption of one or more materials
from selected
environments and the adsorption of one or more materials from selected
environments.
RELATED ART
Two particle technologies-polymer-coated particles and liposomes-are of
general
interest.
Polymer-coated particles have been very impbrtant in the development of useful
microparticles and of controlled-release vehicles generally. In certain
circumstances
polymers have coating and spreading properties that provide for good
encapsulation of
2 0 various matrices, and they are available in a range of chemistries and
molecular weights.
Certain polymeric coatings are of such utility and low toxicity that approval
has been
obtained for their use even in injectable products within the pharmaceutical
industry, most
notably polylactic-glycolic acid copolymers, and the usefulness of polymeric
coatings in oral
products is well-established, as in the cases of Eudragits, gelatin, and a
number of natural
2 5 gums. In many settings in fact, microparticle coatings are tacitly assumed
to be polymers.
However, polymer-coated particles exhibit several limitations, as the
flattened and
diffuse response of their polymer coatings to chemical and physical triggers
indicates. This is
due to two factors. First, the high molecular weight of polymers reduces their
diffusion
-1-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
coefficients and their kinetics of solubilization. Second, the neighboring
group effect
broadens the curves representing the chemical responses to triggers such as,
i~te~ alia, pH,
salinity, oxidation and reduction, ionization, etc. (The neighboring group
effect indicates that
chemical changes in one monomeric unit of a polymer significantly alter the
parameters
governing chemical transitions in each of the neighboring monomeric units.)
Further, most
polymers are collections of chemical species of broadened molecular weight
distribution. In
addition, for a given application of the polymer coated particle only a
limited number of
suitable polymers are frequently available. This is due to a number of
factors: regulatory
issues: the coating processes often entail harsh chemical and/or physical
conditions, such as
solvents, free radicals, elevated temperatures, dessication or drying, and/or
macroscopic
shearing forces needed to form the particles; the limited mechanical and
thermal stabilities of
the polymeric coatings in industrial applications; and adverse environmental
impacts in large
scale applications of polymer-coated particles, such as in agricultural use.
Liposomes also exhibit a number of limitations. Among these axe their physical
and
chemical instabilities. The release of a material disposed within the liposome
is usually
dependent on the destabilization of the structure of the liposome. In
particular, the absence of
porosity precludes the pore-controlled release of such materials. The dual
requirements of 1)
physical stability of the liposome until release is desired on the one hand
and 2) release of
materials by bilayer destabilization when release is desired on the other, are
problematic. (The
2 0 term liposomes is frequently interchanged with the term vesicles and is
usually reserved for
vesicles of glycerophospholipids or other natural lipids. Vesicles are self
supported closed
bilayer assemblies of several thousand lipid molecules (amphiphiles) that
enclose an aqueous
interior volume. The lipid bilayer is a two-dimensional fluid composed of
lipids with their
hydrophilic head groups exposed to the aqueous solution and their hydrophobic
tails
2 5 aggregated to exclude water. The bilayer structure is highly ordered yet
dynamic because of
the rapid lateral motion of the lipids within the plane of each half of the
bilayer.) See O'Brien.
D.F. and Rarnaswami, V. (1989) in Mark-Bikales-Overberger-Menges Encyclopedia
of
Polymer Science and Engineering. Vol. 17, Ed. John Wiley & Inc., p. 108.
-2-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
SUMMARY OF THE INVENTION
It is an object of the invention to provide coated particles that are suitable
for
solubilizing or containing a wide variety of materials, including materials
sensitive to
physical, chemical or biological deterioration.
It is an object of the invention to provide coated particles that release one
or more
material disposed within a matrix in their internal cores without requiring
the destabilization
of that matrix.
It is an object of the invention to provide coated particles covering a wide
range of
physical and chemical properties, particularly in the selection of the
coating, such that a user
can substantially preselect the coating and release characteristics.
It is an object of the invention to provide coated particles that sharply
initiate the
release or absorption of one or more materials to or from a selected
environment in response
to one or more physical or chemical triggers.
It is an object of the invention to provide a wide variety of coated particle
systems that
can be tailored to the particular physical, chemical and biological
requirements of their
contemplated use, such as mechanical and thermal stability in industrial
applications of the
coated particles or freedom from adverse environmental impact in large scale
application of
the coated particles in agricultural use.
It is an object of the invention to provide coated particles that provide, if
desired, a
2 0 porous coating that permits pore-controlled release of material disposed
within them or pore-
controlled absorption of materials disposed without them.
It is a further object of the invention to provide coated particles that can
incorporate
targeting moieties such as antibodies, lectins, receptors, and complementary
nucleic acids, for
targeting the particles to specific sites, either before or after the coating
releases, as well as
2 5 other bioactive materials such as absorption enhancers, adjuvants,
adsorption inhibitors, or
pharmaceutical actives themselves.
It is a further object of the invention to provide coated particles that can
be produced
by a process that is flexible and can be adapted to a wide range of actives,
coatings, and
matrices. .
5 0 It is a further obj ect of the invention to provide coated particles that
have a
-3-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
polymerized interior matrix which is more permanent chemically,
thermodynamically, and
structurally than their unpolymerized counterparts.
It is a still further object of the invention to provide coated particles that
can be made
by a simple process, including, preferably, without entailing harsh physical
and/or chemical
conditions.
The foregoing and other objects are provided by a coated particle that
comprises a.n
internal core comprising a matrix and an exterior coating. The matrix consists
essentially of at
least one nanostructured liquid phase, or at least one nanostructured liquid
crystalline phase
or a combination of the two and the exterior coating comprises a nonlamellar
material that is
a nonlamellar crystalline material, a nonlamellar amorphous material, or a
nonlamellar semi-
crystalline material.
In a preferred embodiment, the coated particle may be made by
1. providing a volume of the matrix that includes at least one chemical
species having
a moiety capable of forming a nonlamellar material upon reaction with a second
moiety and
2. contacting the volume with a fluid containing at least one chemical species
having
the second moiety under nonlamellar solid material-forming conditions so as to
react the first
moiety with the second moiety, and subdividing the volume into particles by
the application
of energy to the volume, or performing this subdivision into particles before,
and/or after, the
chemical reaction.
2 0 Alternatively, the coated particle can be made by one of the following
processes:
providing a volume of the matrix that includes a material in solution in it
that
is capable of forming a nonlamellar material that is insoluble in the matrix
and causing the
aforesaid material to become insoluble in the matrix and subdividing the
volume into
particles by the application of energy to the volume;
2 5 dispersing particles of said matrix into a fluid that includes at least
one
chemical species having a moiety capable of forming a nonlamellar material
upon reaction or
association with a second moiety and adding to said dispersion at least one
chemical species
having said second moiety to react said first moiety with said second moiety;
dispersing particles of said matrix into a fluid that includes at least one
3 0 chemical species having a moiety capable of forming a nonlamellar material
upon reaction or
association with a second moiety, adding to said dispersion at least one
chemical species
-4-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
having said second moiety to react said first moiety with said second moiety,
and subdividing
the resulting material into particles by the application of energy to said
material;
dispersing a volume of said matrix in a form of said nonlamellar material
selected from the group consisting of liquefied form, solution, or fluid
precursor, and
solidifying said nonlamellar material by a techniques selected from the group
consisting of
cooling, evaporating a volatile solvent, or implementing a chemical reaction;
or
dispersing or dissolving a volume of said matrix in a liquid comprising said
nonlamellar material in solution or dispersed form and comprising also a
volatile solvent, and
spray-drying said solution or dispersion.
Or, a combination of these methods can be applied.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graphic representation, in vertical section, illustrating a coated
particle of the
present invention comprising an internal core comprising a 2 by 2 by 2 unit
cell matrix and an
exterior coating;
Fig. 2 is a graphic representation, in section, illustrating a coated particle
of the present
invention;
Fig. 3 is a scanning electron microscope micrograph of coated particles of the
present
invention;
Fig. 4 is a scanning electron microscope micrograph of other coated particles
of the present
2 0 invention;
Fig. 5 is a graph of the measured volume-weighted cumulative particle size
distribution for
coated particles of the present invention on a volume-weighted particle
diameter versus
cumulative particle size basis;
Fig. 6 is a graph of measured small-angle X-ray scattering intensity versus
wave vector q of
2 5 coated particles of the present invention;
Fig. 7 is a graph of detector counts versus elution time in minutes for a
control using high
pressure liquid chromatography; and
Fig. 8 is a graph of detector counts versus elution time in minutes for coated
particles of the
present invention using high pressure liquid chromatography.
-5-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Figure 9. A phase-contrast optical micrograph of PLGA-coated microparticles
dispersed in
water, showing the core-shell structure.
Figure 10. On the left is a PLGA-coated cubic phase, made according to the
instant
invention, soaking in linalool, which is a non-solvent for PLGA but a solvent
for the cubic
phase. On the right, the same cubic phase was soaked in linalool under
identical conditions,
demonstrating that the cubic phase dissolves in the linalool when not coated.
Figure 11. A large (5 mm) particle of coated cubic phase in which the coating
consists of
amorphous trehalose, obtained by freeze-drying a dispersion of Arlatone G-
based cubic phase
in a trehalose solution.
DETAILED DESCRIPTION OF THE PREFERRED
EMBODIMENTS
As illustrated in Figs. 1 and 2, a coated particle 1 used in the present
invention
comprises an internal pore 10 and a coating 20 exterior to it (hereinafter
"exterior coating
20"). The internal core 10 comprises a matrix consisting essentially of a
nanostructured
material selected from the group consisting of
a. at least one nanostructured liquid phase,
b. at least one nanostructured liquid crystalline phase and
c. a combination of
i. at least one nanostructured liquid phase and
ii. at least one nanostructured liquid crystalline phase.
Alternatively, the interior could be a composition that yields one of these
phases upon contact
with water or other aqueous fluid.
The liquid phase material and the liquid crystalline phase material may either
contain
2 5 solvent (lyotropic) or not contain solvent (thermotropic). The exterior
coating 20 comprises a
nonlamellar material. The term "exterior coating" as used herein is intended
to indicate that
the coating 20 is exterior to the internal core 10 and is not intended to be
limited to meaning
that the exterior coating 20 is the most exterior coating of the coated
particle 1. For instance,
in many of the Examples given herein, a surfactant-rich layer is present at
the outer surface of
3 0 the non-lamellax exterior coating. And in other embodiments presented, an
antibody ox other
-6-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
bioactive material will be adsorbed to, or extending out from, this non-
lamellar exterior
coating.
Nanostructured liquid phase and nanostructured liquid crystalline phase
possess
unique properties that are not only important in making possible the easy
production of
particles according to the present invention, but also yield highly desirable
solubilization,
stability, and presentation properties and other capabilities in the final
coated particles of the
present invention.
As for the exterior coating 20, non-lamellax structures which exhibit bonding
and/or
packing rigidity that extends in all three dimensions are strongly preferred
in the present
invention over lamellar materials, due to the well-known physical and chemical
limitations
and instabilities of lamellar, and more generally layered, structures, as
exemplified by, for
example, (a) the instability (even when acquiescent) of emulsions which have
droplets coated
with lamellar liquid crystalline layers, (b) the chemical instability upon
removal of guest
molecules in certain Wemer complexes, and (c) the dramatically inferior
haxdness and shear
modulus for graphite as compared with diamond.
Coated particles 1 used in the present invention may be from 0.1 micron to 30
microns
or above in mean caliper diameter, and preferably from about 0.2 micron to
about 5 microns
in mean caliper diameter. Macroscopic particles can be made as well, i.e.,
particles with sizes
measured in millimeters or even larger, as exemplified in Examples 39 and 40;
the ability to
2 0 make particles of this larger size could open up applications of the
present invention in, for
example, depot delivery systems for sustained release upon implantation. The
coated particle
1 used may also be provided with a stabilizing layer on its exterior, i.e.
outside the exterior
coating 20 as desired, such as a polyelectrolyte or surfactant monolayer to
prevent
agglomeration of coated particles 1.
2 5 The coated particles 1 used in the present invention have application in a
variety of
modalities of use. The coated particle 1 may, upon release of the exterior
coating 20, absorb
one or more materials from a selected environment, adsorb one or more
materials from a
selected environment or release one or more materials, such as active agents
disposed in the
matrix, into a selected environment, and/or target specific sites for the
intended release or
3 0 ad/absorption. Alternatively, certain exterior coatings possessing
porosity, such as inclusion
compounds and zeolites, do not require release in order to effect the
absorption or release of a
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
material of interest into or out of the matrix, and in some such cases very
high selectivity can
be obtained by the use of properly tuned pore characteristics. In cases where
the particles are
used to adsorb a compound or compounds of interest, neither porosity nor
release of the
exterior coating 20 are required, but porosity can provide for a very large
increase in
adsorption capacity by allowing the adsorbed material to diffuse into the
matrix, making the
adsorption sites in the exterior coating 20 available to adsorb new material.
In a preferred
embodiment, an additional material, such as an active agent, may be disposed
within the
matrix for release into a selected environment.
Coating: In the present context of particles, a "coating" is composed of a
material which
behaves as a solid in the common sense, and in the engineering viewpoint, of
the term
"solid", namely that it exhibits a rigidity and permanence that contrasts
sharply with low-
viscosity liquids, and thus represents a significant diffusional barrier to
the passage of
compounds across that material, in a way that is intuitively different from
any protection that
a low-viscosity liquid layer could provide. This common sense understanding of
the terms
"liquid" and "solid" differs fundamentally from the strict scientific
definitions, which refer
only to the existence or non-existence of long-range atomic order. Thus, while
an amorphous
material such as PMMA (Plexiglass) or ordinary glass-particles of which make
up an
everyday coating known as cexamie glaze-may technically be a liquid, for the
purposes of
simplifying nomenclature in the context of this invention these materials will
be referred to as
2 0 solids, as they would in ordinary life outside of the physics laboratory.
The matrix is
a. thermodynamically stable
b. nanostructured and
c, a liquid phase or liquid crystalline phase or a combination thereof.
2 5 Nanostructured: The terms "nanostructure" or"nanostructured" as used
herein in the context
of the structure of a material refer to materials the building blocks of which
have a size that is
on the order of nanometers (10-9 meter) or tens of nanometers (10 x 10-9
meter). Generally
speaking, any material that contains domains or particles 1 to 100 nm
(nanometers) across, or
layers or filaments of that thickness, can be considered a nanostructured
material. (See also
_g_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Dagani, R., "Nanostructured Materials Promise to Advance Range of
Technologies."
November 23, 1992 C&E News 1 ~ (1992).) The term is meant to exclude so-called
"ceramic
glasses" which are crystalline materials in which the crystallite size is so
small that one may
not observe peaks in wide-angle x-ray diffraction and which some physicists
may refer to as
nanostructured materials: the nanostructured liquid and liquid crystalline
phases that are
defined herein are characterized by nanoscale domains which are clearly
distiniguished from
neighboring domains by large differences in local chemical composition, and do
not include
materials in which neighboring domains have essentially the same local
chemical
composition amd differ only in lattice orientation. Thus, by the term 'domain'
as used herein it
is meant a spatial region which is characterized by a particular chemical
makeup which is
clearly distinguishable from that of neighboring domains: often such a domain
is hydrophilic
(hydrophobic) which contrasts with the hydrophobicity (hydrophilicity) of
neighboring
domains: in the context of this invention the characteristic size of these
domains is in the
nanometer range. (The term 'microdomain' is often used to indicate domains
whose size range
is micron or na.nometer scale.)
Nanostructured liquids and liquid crystals: Nanostructured liquid phases and
liquid
crystalline phases, which provide the matrix of the internal cores 10 of the
coated particles 1
in the present invention, possess unique collections of properties that are
not only crucial in
making possible the production of particles of the present invention, but also
yield highly
2 0 desirable solubilization, stability, and presentation properties and
capabilities in the final
coated particles. As discussed in more detail below in the discussion of
particle production
processes, in order that a material provide for ready dispersibility with one
of the processes
described herein, it is desirable for the material to be of very low
solubility in water
(otherwise it will tend to dissolve during the dispersing process, limiting
dispersibility), yet,
2 5 at the same time it should contain water - both for the purpose of
solubilizing water-soluble
reactants used in dispersing and for malting possible the solubilization of a
large range of
active compounds.
In particular, for solubilization of hydrophilic (especially charged) and
amphiphilic
compounds, and for the maintenance of not only solubilization but also proper
conformation
3 0 and activity, of sensitive compounds of biological origin such as
proteins, the interior matrix
should contain substantial concentrations of water or other polar solvent. In
teens of
-9-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
establishing versatility in coating selection, a great many (perhaps a
majority) of the
compounds listed as useful coatings in the present invention require reactants
that are soluble
only in polar solvents: Furthermore, the use of organic solvents for
solubilization is in most
cases inconsistent with the present matrices and/or with active biological
compounds such as
proteins (used in the present invention as actives or as targeting agents),
and in any case is
highly disfavored from regulatory, environmental, and health considerations.
These two
requirements of water-insolubility and solubilization of water-soluble
compounds are, of
course, working in opposite directions and are difficult to resolve in a
single, inexpensive,
and safe material.
Very effective systems for satisfying such solubilization requirements are
provided by
lipid - water systems, in which microdomains are present which are very high
in water
content, and simultaneously hydrophobic domains are in very close contact with
the aqueous
domains.The presence of aqueous domains circumvents precipitation tendencies
encountered
in systems where water structure is interrupted by the presence of high
loadings of co-
solvents or co-solutes, as, for example, in concentrated aqueous polymer
solutions. At the
same time the proximity of hydrophobic domains provides for effective
solubilization of
amphiphilic compounds (and hydrophobic as well).
Nanostructured liquid and liquid crystalline phases are synthetic or
semisynthetic
materials which adopt these solubilization characteristics, and provide pure,
well-
2 0 characterized, easily produced, and typically inexpensive matrices that
also have the
following desirable properties:
a) versatility in chemical systems forming nanostructured liquid phases and
nanostructured liquid crystalline phases, ranging from biological lipids that
are ideal for
biomolecules, to hardy fluorosurfactants, to glycolipids that bind bacteria,
to surfactants with
2 5 ionic or reactive groups, etc. This provides for applicability over a wide
range of conditions
and uses;
b) the unsurpassed ability of nanostructured liquid phases and nanostructured
liquid
crystalline phases to: i) solubilize a wide range of active compounds
including many
traditionally difficult compounds such as Paclitaxel and biopharmaceuticals,
circumventing
3 0 the need for toxic and increasingly regulated organic solvents; ii)
achieve high concentrations
of actives with uncompromised stability, and iii) provide the biochemical
environment that
-10-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
preserves their structure and function;
c) true thermodynamic stability, which greatly reduces instabilities common
with
other vehicles, such as precipitation of active agents, breaking of emulsions,
vesicle fusion,
etc., and,
d) the presence of a porespace with preselectable pore size in the nanometer
range,
facilitating further control of the release kinetics even after triggered
release of the coating,
particularly in the release of proteins and other biomacromolecules.
The desired properties of the nanostructured material of the internal core 10
derive
from several related concepts regarding materials that can be described with
respect to
surfactants by use of the terms "polar," "apolar," "amphiphile," "surfactant"
and the "polar-
apolar interface, and analogously with respect to block copolymer systems, as
described
below.
Polar: polar compounds (such as water) and polar moieties (such as the charged
head groups
on ionic surfactants or on lipids) are water-loving or hydrophilic: "polar"
and "hydrophilic" in
the context of the present invention are essentially synonymous. In terms of
solvents, water is
not the only polar solvent. Others of importance in the context of the present
Invention are:
glycerol, ethylene glycol, formamide, N-methyl formamide, dimethylformamide,
ethylammonium nitrate, acetamide, N-methylacetamide, dimethylacetamide, N-
methyl
sydnone, and polyethylene glycol. Note that one of these (polyethylene glycol)
is actually a
2 0 polymer, thereby illustrating the range of possibilities. At sufficiently
low molecular weights,
polyethylene glycol (PEG) is a liquid, and although PEG has not been
extensively studied as a
polar solvent in combination with surfactants, it has been found that PEG does
form
nanostructured liquid phases and liquid crystalline phases in combination
with, for example,
surfactants such as BRIJ-type surfactants, which are nonionic surfactants with
PEG head
2 5 groups ether-linked to alkane chains. More generally, in terms of polar
groups in hydrophilic
and amphiphilic molecules (including but not limited to polar solvents and
surfactants), a
number of polar groups are tabulated below, in the discussion of which polar
groups are
operative as surfactant head groups and which are not.
Apolar. An apolar compound is a compound that has no dominant polar group.
Apolar (or
3 0 hydrophobic, or alternatively, "lipophilic") compounds include not only
the
paraffinic/hydrocarbon l alkane chains of surfactants, but also modifications
of them, such as
-11-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
perfluorinated alkanes, as well as other hydrophobic groups such as the fused-
ring structure in
cholic acid as found in bile salt surfactants, or phenyl groups as form a
portion of the apolar
group in Triton-type surfactants, and oligomer and polymer chains that run the
gamut from
polyethylene (which represents a long alkane chain) to hydrophobic polymers
such as
hydrophobic polypeptide chains in novel peptide-based surfactants that have
been
investigated. A listing of some apolar groups and compounds is given below, in
the
discussion of useful components of the nanostructured phase interior. An
apolar compound
will be lacking in polar groups, a tabulation of which is included herein, and
will generally
have an octanol-water partition coefficient greater than about 100, and
usually greater than
about 1,000.
Amphiphile: an amphiphile can be defined as a compound that contains both a
hydrophilic
and a lipophilic group. See D. H. Everett. Pure and Applied Chemistry, vol.
31, no. 6, p.
611,1972. It is important to note that not every amphiphile is a surfactant.
For example,
butanol is an amphiphile, since the butyl group is lipophilic and the hydroxyl
group
hydrophilic, but it is not a surfactant since it does not satisfy the
definition, given below.
There exist a great many amphiphilic molecules possessing functional groups
which are
highly polar and hydrated to a measurable degree, yet which fail to display
surfactant
behavior. See R. Laughlin, Advances in liquid crystals, vol. 3. p. 41, 1978.
Surfactant: A surfactant is an amphiphile that possesses two additional
properties. First, it
2 0 significantly modifies the interfacial physics of the aqueous phase (at
not only the air-water
but also the oil-water and solid-water interfaces) at unusually low
concentrations compared to
nonsurfactants. Second, surfactant molecules associate reversibly with each
other (and with
nmnerous other molecules) to a highly exaggerated degree to form
thermodynamically stable,
macroscopically one-phase, solutions of aggregates or micelles. Micelles are
typically
2 5 composed of many surfactant molecules (10's to 1000's) and possess
colloidal dimensions.
See R. Laughlin, Advances in liquid crystals, vol. 3, p. 41, 1978. Lipids and
polar lipids in
particular, often are considered as surfactants for the purposes of discussion
herein, although
the term 'lipid' is normally used to indicate that they belong to a subclass
of surfactants which
have slightly different characteristics than compounds which are normally
called surfactants
3 0 in everyday discussion. Two characteristics which frequently, though not
always, are
possessed by lipids are first, they are often of biological origin, and
second, they tend to be
-12-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
more soluble in oils and fats than in water. Indeed, many compounds referred
to as lipids
have extremely low solubilities in water, and thus the presence of a
hydrophobic solvent may
be necessary in order for the interfacial tension-reducing properties and
reversible self
association to be most clearly evidenced, for lipids which are indeed
surfactants. Thus, for
example, such a compound will strongly reduce the interfacial tension between
oil and water
at low concentrations, even though extremely low solubility in water might
make observation
of surface tension reduction in the aqueous system difficult. Similarly, the
addition of a
hydrophobic solvent to a lipid-water system might make the determination of
self association
into nanostructured liquid phases and nanostructured liquid crystalline phases
a much simpler
matter, whereas difficulties associated with high temperatures might make this
difficult in the
lipid-water system.
Indeed, it has been in the study of nanostructured liquid crystalline
structures that the
commonality between what had previously been considered intrinsically
different --'lipids'
and 'surfactants' -- came to the forefront, and the two schools of study
(lipids, coming from
the biological side, and surfactants, coming from the more industrial side)
came together as
the same nanostructures were observed in lipids as for all surfactants. In
addition, it also came
to the forefront that certain synthetic surfactants, such as
dihexadecyldimethylammonium
bromide, which were entirely of synthetic, non-biological origin, showed
'lipid-like' behavior
in that hydrophobic solvents were needed for convenient demonstration of their
surfactancy.
2 0 On the other end, certain lipids such as lysolipids, which are clearly of
biological origin,
display phase behavior more or less typical of water-soluble surfactants.
Eventually, it
became clear that for purposes of discussing and comparing self association
and interfacial
tension-reducing properties, a more meaningful distinction was between single-
tailed and
double-tailed compounds, where single-tailed generally implies water-soluble
and double-
2 5 tailed generally oil soluble.
Thus, in the present context, any amphiphile which at very low concentrations
lowers
interfacial tensions between water and hydrophobe, whether the hydrophobe be
air or oil, and
which exhibits reversible self association into nanostructured micellar,
inverted micellar, or
bicontinuous morphologies in water or oil or both, is a surfactant. The class
of lipids simply
3 0 includes a subclass consisting of surfactants which are of biological
origin.
Polar-apolar interface: In a surfactant molecule, one can find a dividing
point (or in
-13-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
some cases two points, if there are polar groups at each end, or even more
than two, as in
Lipid A, which has seven acyl chains and thus seven dividing points per
molecule), in the
molecule that divide the polar part of the molecule from the apolar part. In
any nanostructured
liquid phase or nanostructured liquid crystalline phase, the surfactant forms
monolayer or
bilayer films: in such a film, the locus of the dividing points of the
molecules describes a
surface that divides polar domains from apolar domains: this is called the
"polar-apolar
interface" or "polar-apolar dividing surface." For example, in the case of a
spherical micelle,
this surface would be approximated by a sphere lying inside the outer surface
of the micelle,
with the polar groups of the surfactant molecules outside the surface and
apolar chains inside
it. Care should be taken not to confuse this microscopic interface with
macroscopic interfaces
separating two bulk phases that are seen by the naked eye.
Bicontinuous: In a bicontinuous structure, the geometry is described by two
distinct, multiply
-connected, intertwined subspaces each of which is continuous in all three
dimensions;
thus, it is possible to traverse the entire span of this space in any
direction even if the path is
restricted to one or other of the two subspaces. In a bicontinuous structure,
each of the
subspaces is rich in one type of material or moiety, and the two subspaces are
occupied by
two such materials or moieties each of which extends throughout the space in
all three
dimensions. Sponge, sandstone, apple, and many sinters are examples of
relatively permanent
though chaotic bicontinuous structures in the material realm. In these
particular examples,
2 0 one of the subspaces is occupied by a solid that is more or less
deformable and the other
subspace, though it may be referred to as void, is occupied by a fluid.
Certain lyotropic liquid
crystalline states axe also examples, one subspace being occupied by
amphiphile molecules
oriented and aggregated into sheet-lilce arrays that are ordered
geometrically, the other
subspace being occupied by solvent molecules. Related liquid crystalline
states that contain
2 5 two incompatible kinds of solvent molecules, e.g. hydrocarbon and water,
present a further
possibility in which one subspace is rich in the first solvent, the other in
the second, and the
surface between lies within a multiply connected stratum rich in oriented
surfactant
molecules. Certain equilibrium microemulsion phases that contain comparable
amounts of
hydrocarbon and water as well as amphiphilic surfactant may be chaotic
bicontinuous
3 0 structures, maintained in a permanent state of fluctuating disorder by
thermal motions, for
they give no evidence of geometric order but there is compelling evidence for
multiple
-14-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
continuity. Bicontinuous morphologies occur also in certain phase-segregated
block
copolymers. See Anderson. D.M., Davis. H.T., Nitsche. J.C.C. and Scriven. L.E.
(1900)
Advances in Chemical Physics, 77:337.
Chemical criteria: A number of criteria have been tabulated and discussed in
detail by
Robert Laughlin for determining whether a given polar group is functional as a
surfactant
head group, where the definition of surfactant includes the formation in water
of
nanostructured phases even at rather low concentrations. R. Laughlin, Advances
in Liquid
Crystals, pp. 3-41, 1978.
T'he following listing given by Laughlin gives some polar groups which are not
operative as surfactant head groups - and thus, for example, an alkane chain
linked to one of
these polar groups would not be expected to form nanostructured liquid or
liquid crystalline
phases - are: aldehyde, ketone, carboxylic ester, carboxylic acid, isocyanate,
amide, acyl
cyanoguanidine, acvl guanyl urea, acyl biuret, N.N-dimethylamide,
nitrosoalkane,
nitroalkane, nitrate ester, nitrite ester, nitrone, nitrosamine, pyridine N-
oxide, nitrile,
isonitrile, amine borane, amine haloborane, sulfone, phosphine sulfide, arsine
sulfide,
sulfonamide, sulfonamide methylimine, alcohol (monofunctional), ester
(monofunctional),
secondary amine, tertiary amine, mercaptan, thioether, primary phosphine,
secondary
phosphine, and tertiary phosphine.
Some polar groups which are operative as surfactant head groups, and thus, for
2 0 example, an alkane chain linked to one of these polar groups would be
expected to form
nanostructured liquid and liquid crystalline phases, are:
a. Anionics: carboxylate (soap), sulfate, sulfamate, sulfonate, thiosulfate,
sulfinate,
phosphate, phosphonate, phosphinate, nitroamide, tris(alkylsulfonyl)methide,
xanthate;
~ 5 b. Cationics: ammonium, pyridinium, phosphonium, sulfonium, sulfoxonium;
c. Zwiterionics: ammonio acetate, phosphoniopropane sulfonate, pyridinioethyl
sulfate;
d. Semipolars: amine oxide, phosphonyl, phosphine oxide, arsine oxide,
sulfoxide,
sulfoximine, sulfone diimine, ammonio amidate.
3 0 Laughlin also demonstrates that as a general rule, if the enthalpy of
formation of a 1:1
association complex of a given polar group with phenol (a hydrogen bonding
donor) is less
-l5-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
than 5 kcal, then the polar group will not be operative as a surfactant head
group.
In addition to the polar head group, a surfactant requires an apolar group,
and again
there are guidelines for an effective apolar group. For alkane chains, which
are of course the
most common, if n is the number of carbons, then n must be at least 6 for
surfactant
association behavior to occur, although at least 8 or 10 is the usual case.
Interestingly
octylamine, with n = 8 and the amine head group which is just polar enough to
be effective as
a head group, exhibits a lamellar phase with water at ambient temperature, as
well as a
nanostructured L2 phase. Warnhelm. T., Bergenstahl. B., Henriksson. U.,
Malmvilc. A.-C.
and Nilsson. P. (1987) J. of Colloid and Interface Sci. 118:233. Branched
hydrocarbons yield
basically the same requirement on the low n end: for example, sodium 2-
ethylhexylsulfate
exhibits a full range of liquid crystalline phases. Winsor, P.A. (1968) Chem.
Rev. 68:1.
However, the two cases of linear and branched hydrocarbons are vastly
different on the high n
side. With linear, saturated alkane chains, the tendency to crystallize is
such that for n greater
than about 18, the Krafft temperature becomes high and the temperature range
of
nanostructured liquid and liquid crystalline phases increases to high
temperatures, near or
exceeding 100°C. In the context of the present invention, for most
applications this renders
these surfactants considerably less useful than those with n between 8 and 18.
With the
introduction of unsaturation or branching in the chains, the range of n can
increase
dramatically. The case of unsaturation can be illustrated with the case of
lipids derived from
2 0 fish oils, where chains with 22 caxbons can have extremely low melting
points due to the
presence of as many as 6 double bonds, as in docosahexadienoic acid and its
derivatives,
which include monoglycerides, soaps, etc. Furthermore, polybutadiene of very
high MW is an
elastomeric polymer at ambient temperature, and block copolymers with
polybutadiene
blocks are well known to yield nanostructured liquid crystals. Similarly, with
the introduction
2 5 of branching one can produce hydrocarbon polymers such as
polypropyleneoxide (PPO)
which serves as the hydrophobic block in a number of amphiphilic block
copolymer
surfactants of great importance, such as the Pluronic series of surfactants.
Substitution of
fluorine for hydrogen, in particular the use of perfluorinated chains, in
surfactants generally
lowers the requirement on the minimal value of n, as exemplified by lithium
3 0 perfluourooctanoate (n=8), which displays a full range of liquid
crystalline phases, including
an intermediate phase which is fairly rare in surfactant systems. As discussed
elsewhere, other
-16-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
hydrophobic groups, such as the fused-ring structure in the cholate soaps
(bile salts), also
serve as effective apolar groups, although such cases must generally be
treated on a case by
case basis in terms of determining whether a particular hydrophobic group will
yield
surfactant behavior.
For single-component block copolymers, relatively simple mean-field
statistical
theories are sufficient to predict when nanostructure liquid phase and liquid
crystalline phase
materials will occur and these are quite general over a wide range of block
copolymers. If x is
the FloryHuzuins interaction parameter between polymer blocks A and B, and N
is the total
index of polymerization defined as the number of statistical units or monomer
units in the
polymer chain, consistently with the definition of the interaction parameter
of the block
copolymer, then nanostructure liquid and liquid crystalline phases are
expected when the
product x N is greater than 10.5. Leibler, L. (1980) Macromolecules 13:1602.
For values
comparable to but larger than this critical value of 10.5, ordered
nanostructured (liquid
crystalline) phases can occur, including ever, bicontinuous cubic phases.
Hajduk,. D.A.,
Harper, P.E., Gruner, S.M., Honeker, C.C., I~im, G., Thomas, E.L. and Fetters,
L. J. (1994)
Macromolecules 27:4063.
The nanostructured liquid phases of utility.
The nanostructured liquid phase material suitable for the nanostructured
material of
the matrix may be
a. a nanostructured L1 phase material,
b. a nanostructured L2 phase material.
c. a nanostructured microemulsion or
d. a nanostructured L3 phase material.
The nanostructured liquid phases are characterized by domain structures
composed of
2 5 domains of at least a first type and a second type (and in some cases
three or even more types)
having the following properties:
a) the chemical moieties in the first type domains are incompatible with those
in the
second type domains (and in general, each pair of different domain types axe
mutually
incompatible) such that they do not mix under the given conditions but rather
remain as
3 0 separate domains: for example, the first type domains could be composed
substantially of
-17-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
polar moieties such as water and lipid head groups, while the second type
domains could be
composed substantially of apolar moieties such as hydrocarbon chains: or,
first type domains
could be polystyrene-rich, while second type domains are polyisoprene-rich and
third type
domains are polyvinylpyrrolidone-rich;
b) the atomic ordering within each domain is liquid-like rather than solid-
like, i.e., it
lacks lattice-ordering of the atoms; this would be evidenced by an absence of
sharp Bragg
peak reflections in wide-angle x-ray diffraction;
c) the smallest dimension (e.g., thickness in the case of layers, diameter in
the case of
cylinder-like or sphere-like domains) of substantially all domains is in the
range of
nanometers (viz., from about 1 to about 100 nm); and
d) the organization of the domains does not exhibit long-range order nor
conform to any
periodic lattice. This is evidenced by the absence of sharp Bragg reflections
in small-angle x-
ray scattering examination of the phase. Furthermore, as seen below, if high
viscosity and
birefringence are both lacking, this is strong evidence of a liquid, as
opposed to liquid
crystalline, phase.
With respect to each of the liquid phases, systems based on surfactants, where
the two
types of domains in the nanostructured liquid are 'polar' and' apolar' are
initially discussed.
Generally, following that, systems based on block copolymers are discussed. In
these systems
the terms 'polar' and 'apolar' may or may not be applicable, but there exist
domain types 'A',
2 0 'B', etc., where as defined above (in the definition of a nanostructure
liquid) domain types 'A'
and 'B' are immiscible with respect to each other.
Ll phase: In an L1 phase that occurs in a system based on surfactants, the
curvature
of the polar-apolar interface is toward the apolar (non-polar) regions,
generally resulting in
particles -- normal micelles -- that exist in a water-continuous medium. (Here
"water" refers
2 5 to any polar solvent). When these micelles transform from spherical to
cylindrical as
conditions or compositions change, they can start to fuse together and
bicontinuity can result.
In addition to the water continuity, the hydrophobic domains can connect up to
form a
sample-spanning network: this can still be an L1 phase. In addition, there are
examples of L1
phases that show evidence of having no microstructure whatsoever. That is,
there are no
3 0 micelles, no well-defined domains, just surfactant molecules co-mingled in
a structureless,
one-phase liquid solution that is thus not a nanostructured material. These
"structureless
-18-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
solutions" can sometimes be changed to nanostructured phases by simple change
in
composition without any phase change in between. In other words,
thermodynamics does not
dictate a phase boundary between a structureless solution and a nanostructured
phase. This is,
of course, in contrast with the case of a transition between a phase having
long-range order (a
liquid crystal or a crystal) and a phase lacking long-range order (a liquid),
where a phase
boundary is required by thermodynamics.
For L1 phases that occur in systems based on block copolymers, the terms
'polar' and
apolar may not apply, but in any case there are two (or in some cases more)
domain types; we
make the convention that the curvature of the A/B interface is toward A
domains, so that a
typical nanostructure would consist of particles, often sphere-like, of domain
type A located
in a continuum of B domains. As an example, in polystyrene-polyisoprene
diblock
copolymers, if the volume fraction of polystyrene blocks is very low, say 10%,
then the usual
microstructure will be polystyrene-rich spheres in a continuous polyisoprene
matrix.
Contrariwise, polyisoprene-rich spheres in a polystyrene-continuous matrix
would be the
likely structure for a 10% polyisoprene PS-PI diblock.
Identification of the nanostructured Ll phase. Since the L1 phase is a liquid
phase,
techniques have been developed to distinguished the nanostructured Ll phase
from
unstructured solution liquid phases. In addition to the experimental probes
that are discussed
below, there is a well-known body of knowledge that provides criteria by which
one can
2 0 determine a priori whether a given system should be expected to form
nanostructured phases
instead of simple unstructured solutions.
Since the forniation of nanostructured liquid phases and nanostructured liquid
crystalline phases is one requirement in the definition of a surfactant, in
the discrimination of
a nanostructured liquid from an unstructured solution it is extremely valuable
to have criteria
2 5 for determining whether a given compound is in fact a surfactant, criteria
which provide for a
number of tests for surfactancy in addition to methods discussed below for
directly analyzing,
the liquid in question. A number of criteria have been discussed by Robert
Laughlin in
Advances in Liquid Crystals, 3:41, 1978. To begin with, Laughlin lists
chemical criteria for
determining a priori whether a given compound will be a surfactant, and this
was discussed in
3 0 detail above. If, based on these criteria, a compound is expected to be a
true surfactant, then
the compound is expected to form nanastructured phases in water. In addition,
with such a
-19-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
compound in the presence of water and hydrophobe, nanostructured phases are
also expected
to form normally, incorporating at least a portion of the hydrophobe present.
In the event that a non-surfactant amphiphile is added to such a system, and
in
particular an amphiphilic organic solvent such as a short-chained alcohol,
dioxane,
tetrahydrofuran, dimethylformamide, acetonitrile, dimethylsulfoxide, etc.,
then structureless
liquids could form as the action of the organic solvent will generally be to
disrupt colloidal
aggregates and cosolubilize all the components.
Laughlin also goes on to discuss a number of criteria based on physical
observations.
One well-known criteria is the critical micelle concentration (CMC) which is
observed in
surface tension measurements. If the surface tension of an aqueous solution of
the compound
in question is plotted as a function of the concentration, then at very low
concentrations, the
surface tension will be seen to drop off sharply if the added compound is
indeed a surfactant.
Then, at a particular concentration known as the CMC, a sharp break will occur
in this plot as
the slope of the line decreases drastically, to the right of the CMC, so that
the surface tension
decreases much less with added surfactant. The reason is that above the CMC,
added
surfactant goes almost entirely into the creation of micelles rather than to
the air-water
interface.
A second criterion tabulated by Laughlin is the liquid crystal criterion: if
the
compound forms liquid crystals at high concentrations, then it must be a
surfactant and will
2 0 form liquid crystalline phases at concentrations lower than those at which
the occur. In
particular, the L1 phase is usually found at concentrations of surfactant just
lower than those
that form normal hexagonal, or in some cases normal non-bicontinuous cubic
phase liquid
crystals.
Another criterion discussed by Laughlin is based on the temperature
differential
2 5 between the upper limit of the I~rafft boundary plateau and the melting
point of the anhydrous
compound. The Krafft boundary is a curve in the phase diagram of the binary
system with
compound and water: below he I~rafft line are crystals- and above the Krafft
line the crystals
melt, so that there is a dramatic increase in solubility over a very narrow
temperature range
along the I~rafft line. In the case of a true surfactant, this temperature
differential is
3 0 substantial: for example, in sodium palmitate, the melting point of the
anhydrous compound
is 288°C, while the Krafft line has its plateau at 69°C, so that
the differential is 219°C.
-20-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
i
Laughlin goes on to discuss the case of dodecylamine, which has a temperature
differential of
14°C, and has a small region in the phase diagram corresponding to
liquid crystals, thus
indicating a modest degree of association colloid behavior. In contrast,
neither
dodecylmethylamine nor dodecanol exhibit association behavior of the
surfactant type, and
both have zero temperature differential.
As in the case of liquid crystals, as discussed herein, given a material there
are a
number of experimental probes one can use to determine whether or not the
material, in this
case a liquid, is nanostrutured, and these will be discussed in the context of
the L1 phase,
although they apply to all nanostructured liquids, with the appropriate
modifications. In such
a determination, it is best to combine as many of these characterizations as
feasible.
As with all the liquid phases, the L1 phase is optically isotropic in the
absence of
flow. It does not give a splitting in the zH NMR bandshape with deuterated
surfactant.
Also, in examination with crossed polarizing filters, the L1 phase of
surfactant
systems does not generally give birefringence even under moderate flow
conditions. The
situation with respect to birefringence in the case of block copolymer-based
systems is
complicated by the possibility of strain birefringence, so this is not a
reliable method in that
case.
Returning to the surfactant-based L1 phase, viscosity is generally quite low,
considerably lower than in liquid crystals in the same system.
2 0 Using pulsed-gradient NMR to measure the effective self diffusion
coefficients of the
various components, one finds that the self diffusion of surfactant, and any
added hydrophobe
is very low, typically on the order of 10-'3 m'-/sec or less (unless the phase
is bicontinuous: see
below). This is because the primary means for diffusion of surfactant and
hydrophobe is by
diffusion of entire micelles, which is very slow. Also, the diffusion rates of
surfactant and of
2 5 hydrophobe should be nearly the same, for the same reason.
Small-angle x-ray scattering (SAXS) does not give sharp Bragg peaks in the
nanometer range (nor any range), of course. However, analysis of the entire
curve by several
methods from the literature can give the length scale of the nanostructure. By
analyzing the
falloff of intensity at low wave numbers (but not too low compared to the
inverse of the
3 0 surfactant molecule length), one can determine the apparent radius of
gyration: one plots
intensity, versus the square of the wave number, and takes the slope to deduce
R~ (the so-
-21-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
called Guinier plot). The radius of gyration is then related to the dimensions
of the micellar
units by standard well-known formulae. This will fall in the range of
nanometers. In addition,
by plotting the product of intensity times the square of the wave number
versus the wave
number - the so-called 'Hosemann plot'- one 'will find a peak that can also be
related to the
dimensions of the micelles; this has the advantage that it is less sensitive
to interactions
between micelles than is the radius of gyration.
For surfactant-based L1 phases which are bicontinuous, the above will change
as
follows: First, the viscosity can increase considerably when bicontinuity
occurs, do to the
rigidity of the surfactant film, which is continuous. Also, the self diffusion
rate of the
surfactant and even of added hydrophobe (which can be deliberately added to a
binary system
as a marker) can increase dramatically, approaching or even exceeding the
values in a
lamellas phase in the same system. And while SAXS analyses, both the radius of
gyration and
the Hosemann plot, will give resulting dimensions in the nanometer range,
these must be
interpreted as characteristic length scales of the bicontinuous domain
structure, rather than as
dimensions of discrete particles. In some models, such as the interconnected
cylinders model
of the author's thesis, or the Talmon-Prager model, a bicontinuous domain
structure is
represented as made up of units which although seemingly'particles' are in
reality only
building blocks for construction of a model bicontinuous geometry.
For Ll phases in block copolymer-based systems, this same SAXS analysis holds.
In
2 0 contrast, NMR bandshape and self diffusion measurements in general do not
carry over, nor
do surface tension measurements. However, vapor transport measurements have
been used in
the past in place of NMR self diffusion, in particular, if one can find a gas
which is
preferentially soluble in one of the domain types but not in the other(s),
then one can test for
continuity of those domains by measuring the transport of that gas through the
sample. If this
2 5 is possible, then transport through the continuous domains (type B) in the
micellar phase
should be only slightly slower than that in the pure B polymer, whereas gas
transport for a gas
confined to A domains should be very low.
The shear modulus of a block copolymer-based micellar phase is determined
largely
by that of the polymer block forming the continuous domains, polymer B in our
convention.
3 0 Thus, for example, in a PS-PI diblock which is 10% PS, so that PS micelles
form in a
continuous PI matrix, the shear modulus would be close to that of pure
polyisoprene with
-22-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
only a slight increase due to the presence of the PS micelles. Interestingly,
in the reverse case,
with 90% PS and thus PI micelles in a continuous PS matrix, the elastomeric PI
micelles can
provide a shock-absorbing component which can improve the fracture
characteristics over
those of pure, glassy polystyrene.
L2 phase: This phase is the same as the L1 phase except that the roles of the
polar
region and the apolar region are reversed: the curvature of the polar-apolar
interface is toward
the polar domains, the interior of the micelles (if they exist) is water
and/or other polar
moieties, and the apolar domains (typically alkane chains of a lipid) form a
continuous matrix
-- although it is possible for the polar domains also to connect up to form a
bicontinuous L2
phase. As above, this phase can be either nanostructured or structureless.
Identification of the nanostructured L2 hase. The guidelines for making a
phase
identification of the nanostructured L2 phase are the same as those given
above for the L1
phase, with the following modifications. We need only discuss the surfactant-
based L2 phase,
since in the block copolymer-based systems the two types of micellar phases (A
in B, and B
in A) are equivalent, and above we discussed the identification of the
micellar phase in block
copolymer systems.
First, L2 phases are generally more prominent when the HLB is low, for example
with
ethoxylated alcohol surfactants having a small number of ethylene oxide groups
(usually 5 or
less, with typical alkyl chain lengths), or with double-chained surfactants.
In terms of phase
2 0 behavior, they generally occur at higher surfactant concentrations than
even the reversed
liquid crystalline phases: a location that is very common is for the L2 phase
to border the
reversed hexagonal phase at higher surfactant concentrations. For L 1 phases
which are not
bicontinuous, it is the water self diffusion which is very low, and
measurement of the
diffusion coefficient (by pulsed-gradient NMR, for example) should give a
number on the
2 5 order of 10-" m2/sec or less. Also, a Hosemann plot will give the size of
the reversed
micelles, which will essentially be the water domain size.
Microemulsion: A microemulsion may be defined as a thermodynamically stable,
low viscosity, optically isotropic liquid phase containing oil (apolar
liquid), water (polar
liquid), and surfactant. See also Danielsson. I. and Lindman. B. (1981)
Colloids and
3 0 Surfaces, 3:391. Thermodynamically stable liquid mixtures of surfactant,
water and oil are
usually referred to as microemulsions. While being macroscopically
homogeneous, they are
-23-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
structured on a microscopic length scale (10-1,000 Angstrom) into aqueous and
oleic
microdomains separated by a surfactant-rich film. See Skurtveit, R. and
Olsson, U. (1991) J.
Phys. Chem. 95:5353. A key defining feature of a microemulsion is that it
contains an "oil"
(apolar solvent or liquid), in addition to water and surfactant; it is always
microstructured by
definition. In general, because of the strong tendency for oil and water to
phase segregate, in
the absence of an organic solvent capable of co-solubilizing oil and water
(such as ethanol,
THF, dioxane, DMF, acetonitrile, dimethylsulfoxide, and a few others), a
clear, single-phase
liquid containing water and surfactant must be a microemulsion, and one can
safely conclude
on that basis alone that the phase is nanostructured. Note that a
microemulsion can also be an
Ll or L2 phase especially, if it contains well-defined micelles; however, if
it is an L 1 phase
then the micelles are necessarily swollen with oil. The microemulsion is a
nanostructured
liquid phase. If a liquid with "oil," water and surfactant has a
characteristic domain size larger
than the nanometer range, that is, in the micron range, then it is no longer a
microemulsion
but rather a "miniemulsion" or plain emulsion; both of the latter are non-
equilibrium. The
term microemulsion was introduced, despite the fact that L1 and L2 phases can
contain oil,
and can even be bicontinuous, because it is fairly common for three-component
oil-water-
surfactant/lipid systems to evolve continuously from water-continuous to
bicontinuous to oil-
continuous with no phase boundaries in between. In this case, it does not make
sense to try to
set a dividing point between the "L1 " and "L2" regions of the phase diagram;
so instead, one
2 0 just refers to the whole region as "microemulsion" -- recognizing that at
the high-water-
content end of this region the structure is that of an oil-swollen L1 phase,
and at the high-oil-
content end of this region the structure is that of an L2 phase. (In terms of
Venn diagrams,
there are overlaps between microemulsions and L1 and L2 phases, though not
between L1
and L2 phases). As discussed below, the microstructure of microemulsions is
quite generally
2 5 describable in terms of a monolayer film of surfactant that divides oil-
rich domains from
water-rich domains. This surfactant/lipid-rich dividing film can enclose to
form micelles, or
connect up into a network structure to form a bicontinuous microemulsion.
It must be pointed out that an emulsion is not a nanostructured liquid, as the
term is
applied herein. To begin with, the characteristic length scale in an emulsion,
which essentially
3 0 is the average size of an emulsion droplet, is generally much larger than
the characteristic
length scale in a nanostructured liquid, and falls in the range of microns
instead of
-24-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
nanometers. While recent efforts to produce emulsions with submicron droplet
sizes have
given rise to smaller droplet emulsions and to the advent of the term
"miniemulsion", there
remain crucial differences which exclude emulsions and miniemulsions from the
realm of
nanostructured liquid phases as applied herein. The nanostructured liquid
phases described
herein, including microemulsions, exist at thermodynamic equilibrium, in
contrast to
emulsions which are not equilibrium phases but only metastable materials.
Furthermore, a
nanostructured liquid which is acquiescent and fully equilibrated is optically
transparent,
whereas an emulsion is generally opaque --ordinary milk is an emulsion, for
example. In
addition, if one takes the model of Friberg for the structure of an ordinary
emulsion to be true,
and this is generally recognized in the field, then the distinction at the
molecular scale can be
seen to be dramatic. According to that model, emulsion droplets can generally
be seen to be
stabilized by interfacial films which upon microscopic examination typically
prove to be
films of nanostructured liquid crystalline phase material; thus, these
emulsions have a
hierarchical structure in which a nanostructured phase plays the role of a
stabilizing layer
between the main building blocks, which are the emulsion droplets and the
continuous
medium. ~ur use of the term "nanostructured" instead of "microstructured" is
based on the
more precise and restricted nature of the term "nanostructured" and its
exclusion of other
liquid phases which fall into an entirely different realm, such as emulsions.
Clearly, simple
geometric considerations dictate that an emulsion which has droplets on the
order of 10
2 0 microns in size, and a stabilizing film which may be a liquid crystalline
layer, is not
appropriate as the interior of a microparticle of the present invention which
generally has a
size on the order of 1 micron.
Determination of nanostructured microemulsions. The methods and guidelines
discussed above for determination of nanostructured L1 phases carry over to
the
2 5 determination of nanostructured microemulsion phases, with the following
variations.
For microemulsions which do not clearly fall under either the L1 phase or the
L2
phase descriptions -- which is the remaining case to be treated here -- we
take note that many,
if not most, of these axe bicontinuous, and in the context of a single liquid
phase containing
oil, water and surfactant, bicontinuity provides strong proof that the phase
is nanostructured,
3 0 since emulsions and other common liquids are never bicontinuous. This
issue has been
addressed in "On the demonstration of bicontinuous structures in
microemulsions." Lindman.
-25-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
B., Shinoda, K., Olsson, U., Anderson, D. M., Karlstrom, G. and Wennerstrom,
H. (1989)
Colloids and Surfaces 38:205. The time-tested way to demonstrate bicontinuity
is to use
pulsed- gradient NMR and measure the effective self diffusion coefficients of
both oil and
water separately; generally it is best to measure also the self diffusion of
the surfactant.
Electrical conductivity can also be used to establish water continuity,
although this is prone to
problems associated with "hopping" processes. Fluoresence quenching has also
been used for
continuity determination. Sanchez-Rubio, M., Santos-Vidals, L.M., Rushforth,
D. S. and
Puig, J. E. (1985) J. Phys. Chem. 89:411. Small-angle neutron and x-ray
scattering analyses
have been used to examine bicontinuity. Auvray, L., Cotton, L R., Ober, R. and
Taupin. J.
(1984) J. Phys. Chem. 88:4586. Porod analysis of SAXS curves has been used to
deduce the
presence of interfaces, thus proving that a nanostructure is present. Martino,
A. and Kaler,
E.W. (1990) J. Phys. Chem. 94:1627. Freeze-fracture electron microscopy, with
extremely
fast rates of freezing, has been used to study microemulsions and is the
result of decades of
development on fixation methods for nanostructured liquids: a critical review
discussing the
methods and the reliability of the results has been given. Talmon, Y. in K.L.
Mirtal and P.
Bothorel (Eds), Vol. 6. Plenum Press, New York, 1986, p. 1581.
In the event that an oil-water-surfactant liquid phase is not clearly an L1 or
L2 phase,
and does not show strong evidence of bicontinuity, then the analysis to
demonstrate that it is
nanostructured can be fairly involved and no single technique will suffice. In
general, one
2 0 would apply the measurements discussed in this section, such as SANS or
SAXS. NMR self
diffusion, cryo EM, etc., to attempt to rationalize the data within the
context of a model
nanostrucrure.
L3 phase: L2-phase regions in phase diagrams sometimes exhibit "tongues"
sticking
out of them: long, thin protrusions unlike the normal appearance of a simple
L2 phase region.
2 5 This sometimes appears also with some L 1 regions, as described below.
When one examines
these closely, especially with X-ray and neutron scattering, they differ in a
fundamental way
from L2 phases. In an L2 phase, the surfactant film is generally in the form
of a monolayer
with oil (apolar solvent) on one side and water (polar solvent) on the other.
By contrast, in
this "L3 phase" as these phases are called, the surfactant is in the form of a
bilayer with water
3 0 (polar solvent) on both sides. The L3 phase is generally considered to be
bicontinuous and, in
fact, it shares another property with cubic phases: there are two distinct
aqueous networks
-26-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
interwoven but separated by the bilayer. So, the L3 phase is really very
similar to the cubic
phase but lacking the long-range order of the cubic phase. L3 phases stemming
from L2
phases and those stemming from Ll phases are given different names. "L3 phase"
is used for
those associated to L2 phases, and "L3 * phase" for those associated to L1
phases.
Determination of the nanostructured L3 phase. Determination of the L3 phase in
distinction to the other liquid phases discussed herein can be a sophisticated
problem,
requiring the combination of several analyses. The most important of these
techniques are
now discussed. In spite of its optical isotropy when acquiescent and the fact
that it is a liquid,
the L3 phase can have the interesting property that it can exhibit flow
birefringence. Often
this is associated with fairly high viscosity, viscosity that can be
considerably higher than that
observed in the L1 and L2 phases, and comparable to or higher than that in the
la.mellar
phase. These properties are of course a result of the continuous bilayer film,
which places
large constraints on the topology aald the geometry of the nanostructure.
Thus, shear can
result in the cooperative deformation (and resulting alignment) of large
portions of the bilayer
film, in contrast with, for example, a micellar L1 phase where independent
micellar units can
simply displace with shear, and in any case a monolayer is generally much more
deformable
under shear than a bilayer. Support for this interpretation comes from the
fact that the
viscosity of L3 phases is typically a linear function of the volume fraction
of surfactant.
Snabre. P. and Porte. G. (1990) Europhys. Len. 13:641.
Sophisticated light, neutron, and x-ray scattering methodologies have been
developed
for determination of nanostructured L3 phases. Safinya, C.R., Roux, D.,
Smith,. G.S., Sinha,
S.I~., Dimon, P., Clark, N.A. and Bellocq, A.M. (1986) Phys. Rev. Lett.
57:2718; Roux, D.
and Safinya, C.R. (1988) J. Phys. France 49:307; Nallet, F., Roux, D. and
Prost, J. (1989) J.
Phys. France 50:3147. The analysis of Roux, et al. in Roux, D., Gates, M.E.,
Olsson, U., Ball,
2 5 R.C., Nallet, F. and Bellocq, A.M., Europhys. Lett. purportedly is able to
determine that the
nanostructure has two aqueous networlcs, separated by the surfactant bilayer,
which gives rise
to a certain symmetry due to the equivalence of the two networks.
Fortunately, determination of the nanostructured nature of an L3 phase based
on phase
behavior can be more secure than in the case of typical L1, L2, or even
microemulsion
3 0 phases. This is first of all because the L3 phase is often obtained by
addition of a small
amount (a few percent) of oil or other compound to a lamellar or bicontinuous
cubic phase, or
-27-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
small increase of temperature to these same phases. Since these liquid
crystalline phases are
easy to demonstrate to be nanostructured (Bragg peaks in X-ray, in
particular), one can be
confident that the liquid phase is also nanostructured when it is so close in
composition to a
liquid crystalline phase. After all, it would be extremely unlikely that the
addition of a few
percent of oil to a nanostructured liquid crystalline phase would convert the
liquid crystal to a
structureless liquid. Indeed, pulsed-gradient NMR self diffusion measurements
in the Aerosol
OT - brine system show that the self diffusion behavior in the L3 phase
extrapolates very
clearly to those in the nearby reversed bicontinuous cubic phase. This same L3
phase has
been the subject of a combined SANS, self diffusion, and freeze-fracture-
electron microscopy
study. Strey, R., Jahn,. W., Skouri, M., Porte, G., Marisman,. J. and Olsson,.
U. in "Structure
and Dynamics of Supramolecular Aggregates- S.H. Chen, J.S. Huang and P.
Tartaglia, Eds.,
I~luwer Academic Publishers,. The Netherlands. Indeed, in SANS and SAXS
scattering
analysis of L3 phases, a broad interference peak is often observed at wave
vectors that
correspond to d-spacings that are the same order of magnitude as those in
bicontinuous cubic
phases that are nearby in the phase diagram, and the author has developed a
model for L3
phase nanostructure which is an extrapolation of known structures for
bicontinuous cubic
phases. Anderson, D.M., Wennerstrom, H. and Olsson, U. (1989) J. Phys. Chem.
93:4532.
The nanostructured liquid crystalline~hases of utility
As a component of the coated particle the nanostructured liquid crystalline
phase
2 0 material may be
a. a nanostructured normal or reversed cubic phase material,
b. a nanostructured normal or reversed hexagonal phase material,
c. a nanostructured normal or reversed intermediate phase material or
d. a nanostructured lamellar phase material.
2 5 The nanostructured liquid crystalline phases are characterized by domain
structures
composed of domains of at least a first type and a second type (and in some
cases three or
even more types of domains) having the following properties:
a) the chemical moieties in the first type domains are incompatible with those
in the
second type domains (and in general, each pair of different domain types are
mutually
3 0 incompatible) such that they do not mix under the given conditions but
rather remain as
-28-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
separate domains (for example, the first type domains could be composed
substantially of
polar moieties such as water and lipid head groups, while the second type
domains could be
composed substantially of apolar moieties such as hydrocarbon chains: or,
first type domains
could be polystyrene-rich, while second type domains are polyisoprene-rich,
and third type
domains are polyvinylpyrrolidone-rich);
b) the atomic ordering within each domain is liquid-like rather than solid-
like, lacking
lattice-ordering of the atoms; (this would be evidenced by an absence of sharp
Bragg peak-
reflections in wide-angle x-ray diffraction);
c) the smallest dimension (e.g., thickness in the case of layers, diameter in
the case of
cylinders or spheres) of substantially all domains is in the range of
nanometers (viz., from
about 1 to about 100 nm); and
d) the organization of the domains conforms to a lattice, which may be one-,
two-, or
three-dimensional amd which has a lattice parameter (or unit cell size) in the
nanometer range
(viz., from about 5 to about 200 run), the organization of domains thus
conforms to one of the
230 space groups tabulated in the International Tables of Crystallography and
would be
evidenced in a well-designed small-angle x-ray scattering (SAXS) measurement
by the
presence of sharp Bragg reflections with d-spacings of the lowest order
reflections being in
the range of 3-200 nm.
In the discussion of the identification of these liquid crystalline phases
using
2 0 deuterium NMR or self diffusion measurements, it is assumed that the
liquid crystal is not
polymerized. In the cases where it is polymerized, these measurements will be
strongly
affected by the polymerization and may not conform to the same rules that
apply for
unpolymerized liquid crystals. In particular, the self diffusion coefficients
of surfactants can
be dramatically reduced, as was reported by the present author in Strom, P.
and Anderson,
2 5 D.M. (1992) Langmuir 8:691. NMR spectra for polymerized cubic phases were
calculated
for certain conditions by the present author in Anderson, D.M. (1990)
Supplement to J. de
Phys. C7-1.
Lamellar phase: The lamellax phase is characterized by:
1. Small-angle x-ray shows peaks indexing as 1:2:3:4:5. . . in wave number.
3 0 2. To the unaided eye, the phase is either transparent or exhibits mild or
moderate
turbidity.
-29-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
3. In the polarizing optical microscope, the phase is birefringent, and the
well-
known textures have been well described by Rosevear and by Winsor (e.g., Chem.
Rev. 1968,
p. 1). The three most pronounced textures are the "Maltese crosses", the
"mosaic" pattern, and
the "oily streaks" patterns. The Maltese cross is a superposition of two dark
bands
(interference fringes) roughly perpendicular to each other, over a roughly
circular patch of
light (birefringence), forming a distinctive pattern reminiscent of the WWI
German military
symbol. The variations on this texture, as well as its source, is thoroughly
described in J.
Bellare, Ph.D. Thesis, Univ. of Minnesota, 1987. The "mosaic" texture can be
envisioned as
the result of tightly packing together a dense array of deformed Maltese
crosses, yielding dark
and bright patches randomly quilted together. The "oily streaks" pattern is
typically seen
when the (low viscosity) lamellar phase flows between glass and coverslip; in
this pattern,
long curved lines are seen, upon close inspection under magnification 400x),
to be composed
of tiny striations which run roughly perpendicular to the line of the curve,
as ties make up a
railroad track (to be contrasted with the hexagonal texture discussion below).
In some cases,
particularly if the phase is massaged gently between glass and coverslip for a
period of time,
the lamellar phase will align with its optic axis parallel to the line of
sight in the microscope,
resulting in a disappearance of the birefringence.
For lamellar phases in surfactant-water systems:
1. Viscosity is low enough so that the material flows (e.g. when a tube
2 0 containing the phase is tipped upside down),
2,. The self diffusion rates of all components are high comparable to their
values in bulk -- e.g., the effective self diffusion coefficient of water in
the lamellar phase is
comparable to that in pure water. Since the surfactants that form liquid
crystals are usually not
liquid at ambient temperatures, the reference point for the self diffusion
coefficient of the
2 5 surfactant is not clear-cut; and, in fact, the effective (measured) self
diffusion coefficient of
the surfactant in the lamellar phase is often taken to be the reference point
for interpreting
measurements in other phases.
3. If the surfactant is deuterated in the head group, and the ZH NMR bandshape
measured, one finds two spikes with the splitting between them twice what it
is in the
3 0 hexagonal phase.
4. In terms of phase behavior, the lamellar phase generally occurs at high
-30-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
surfactant concentrations in single-tailed surfactant / water systems,
typically above 70%
surfactant: in double-tailed surfactants, it often occurs at lower
concentrations, often
extending well below 50%. It generally extends to considerably higher
temperatures than do
any other liquid crystalline phases that happen to occur in the phase diagram.
For lamellax phases in single-component block copolymer systems:
1. Shear modulus is generally lower than other liquid crystalline phases in
the
same system.
2. In terms of phase behavior, the lasnellar phase generally occurs at volume
fractions of the two blocks is roughly 50:50.
Normal hexagonal phase: The normal hexagonal phase is characterized by:
1. Small-angle x-ray shows peaks indexing as 1:,/3:2:,7:3 . . . . in general,
~(h2 + hk - kz), where h and k are integers -- the Miller indices of the two-
dimensional
symmetry group,
2. To the unaided eye, the phase generally transparent when fully
equilibrated,
and thus often considerably clearer than any nearby lamellar phase.
3. In the polarizing optical microscope, the phase is birefringent, and the
well-
known textures have been well described by Rosevear and by Winsor (e.g., Chem.
Rev. 1968,
p. 1). The most distinctive of these is the "fan-like" texture. This texture
appears to be made
up of patches of birefringence, where within a given patch fine striations fan
out giving an
2 0 appearance reminiscent of an oriental fan. Fan directions in adjacent
patches axe randomly
oriented with respect to each other. A key difference distinguishing between
lamellar and
hexagonal patterns is that the striations in the hexagonal phase do not, upon
close
examination at high magnification, prove to be composed of finer striations
running
perpendicular to the direction of the larger striation, as they do in the
lamellar phase.
2 5 For normal hexagonal phases in surfactant-water systems:
1. Viscosity is moderate, more viscous than the lamellar phase but fax less
viscous than typical cubic phases (which have viscosities in the millions of
centipoise).
2. The self diffusion coefficient of the surfactant is slow compared to that
in
the lamellar phase: that of water is comparable to that in bulk water.
3 0 3. The zH NMR bandshape using deuterated surfactant shows a splitting,
wluch is one-half the splitting observed for the lamellar phase.
-31-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
4. In terms of phase behavior, the normal hexagonal phase generally occurs at
moderate surfactant concentrations in single-tailed surfactant water systems,
typically on the
order of 50°0° surfactant. Usually the normal hexagonal phase
region is adjacent to the
mieellar (L1) phase region, although non-bicontinuous cubic phases can
sometimes occur in
between. In double-tailed surfactants, it generally does not occur at all in
the binary
surfactant-water system.
For hexagonal phases in single-component block copolymer systems, the terms
"normal" and "reversed" do not generally apply (although in the case where one
block is polar
and the other apolar, these qualifiers could be applied in principle). The
shear modulus in
such a hexagonal phase is generally higher than a lamellar phase and lower a
bicontinuous
cubic phase, in the same system. In terms of phase behavior, the hexagonal
phases generally
occurs at volume fractions of the two blocks on the order of 35:65. Typically,
two hexagonal
phases will straddle the lamellar phase with, in each case, the minority
component being
inside the cylinders (this description replacing the 'normal/reversed'
nomenclature of
surfactant systems).
Reversed hexagonal phase: In surfactant-water systems, the identification of
the
reversed hexagonal phase differs from the above identification of the normal
hexagonal phase
in only two respects:
1. The viscosity of the reversed hexagonal phase is generally quite high,
higher
2 0 than a typical normal hexagonal phase, and approaching that of a reversed
cubic phase. And,
2. In terms of phase behavior, the reversed hexagonal phase generally occurs
at
high surfactant concentrations in double-tailed surfactant l water systems,
often extending to,
or close to, 100% surfactant. Usually the reversed hexagonal phase region is
adjacent to the
lamellar phase region which occurs at lower surfactant concentration, although
bicontinuous
2 5 reversed cubic phases often occur in between. The reversed hexagonal phase
does appear,
somewhat surprisingly, in a number of binary systems with single-tailed
surfactants, such as
those of many monoglycerides (include glycerol monooleate), and a number of
nonionic
PEG-based surfactants with low HLB.
As stated above in the discussion of normal hexagonal phases, the distinction
between
3 0 normal and 'reversed' hexagonal phases makes sense only in surfactant
systems, and generally
not in single-component block copolymer hexagonal phases.
-32-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
i
Normal bicontinuous cubic phase: The normal bicontinuous cubic phase is
characterized by:
1. Small-angle x-ray shows peaks indexing to a three-dimensional space group
with a cubic aspect. The most commonly encountered space groups, along with
their
indexings are: laid (#230), with indexing ~6:~8:,114:4... Pn3m (#224), with
indexing
,~2:,~3:2:J6:,~8: and lm3m (#229), with indexing ~2:~/4:,/6:,~8:J10...
2. To the unaided eye, the phase is generally transparent when fully
equilibrated, and thus often considerably clearer than any nearby lamellar
phase.
3. In the polarizing optical microscope, the phase is non-birefringent, and
therefore there are no optical textures.
For normal bicontinuous cubic phases in surfactant-water systems:
1. Viscosity is high, much more viscous than the lamellar phase and even more
viscous than typical normal hexagonal phases. Most cubic phase have
viscosities in the
millions of centipoise.
2. No splitting is observed in the NMR bandshape, only a single peak,
corresponding to isotropic motion.
3. In terms of phase behavior, the normal bicontinuous cubic phase generally
occurs at fairly high surfactant concentrations in single-tailed surfactant l
water systems
typically on the order of 70°!° surfactant with ionic
surfactants. Usually the normal
2 0 bicontinuous cubic phase region is between lamellar and normal hexagonal
phase regions,
which along with its high viscosity and non-birefringence make its
determination fairly
simple. In double-tailed surfactants, it generally does not occur at all in
the binary surfactant-
water system.
For bicontinuous cubic phases in single-component block copolymer systems, the
2 5 terms "normal" and "reversed" do not generally apply (although in the case
where one block
is polar and the other apolar, these qualifiers could be applied in
principle). The shear
modulus in such a bicontinuous cubic phase is generally much higher than a
lamellar phase,
and significantly than a hexagonal phase in the same system. In terms of phase
behavior, the
bicontinuous cubic phases generally occur at volume fractions of the two
blocks on the order
3 0 of 26:74. In some cases, two bicontinuous cubic phases will straddle the
lamellar phase with,
in each case, the minority component being inside the cylinders (this
description replacing the
-33-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
'normalJreversed' nomenclature of surfactant systems), and hexagonal phases
straddling the
cubic-lamellar-cubic progression.
Reversed bicontinuous cubic phase.- The reversed bicontinuous cubic phase is
characterized by:
In surfactant-water systems, the identification of the reversed bicontinuous
cubic
phase differs from the above identification of the normal bicontinuous cubic
phase in only
one respect. In terms of phase behavior, the reversed bicontinuous cubic phase
is found
between the lamellar phase and the reversed hexagonal phase, whereas the
normal is found
between the lamellar and normal hexagonal phases: one must therefore make
reference to the
discussion above for distinguishing normal hexagonal from reversed hexagonal.
A good rule
is that if the cubic phase lies to higher water concentrations than the
lamellar phase, then it is
normal, whereas if it lies to higher surfactant concentrations than the
lamellar then it is
reversed. The reversed cubic phase generally occurs at high surfactant
concentrations in
double-tailed stufactant / water systems, although this is often complicated
by the fact that the
reversed cubic phase may only be found in the presence of added hydrophobe
("oil") or
amphiphile. The reversed bicontinuous cubic phase does appear in a number of
binary
systems with single-tailed surfactants such as those of many monoglycerides
(include glycerol
monooleate) and a number of nonionic PEG-based surfactants with low HLB.
It should also be noted that in reversed bicontinuous cubic phases, though not
in
2 0 normal, the space group #212 has been observed. This phase is derived from
that of space
group #230. As stated above in the discussion of normal bicontinuous cubic
phases, the
distinction between 'normal' and 'reversed' bicontinuous cubic phases makes
sense only in
surfactant systems, and generally not in single-component block copolymer
bicontinuous
cubic phases.
~ 5 Normal discrete (non-bicontinuous) cubic phase: The normal non-
bicontinuous
cubic phase is characterized by:
1. Small-angle x-ray shows peaks indexing to a three-dimensional space group
with a cubic aspect. The most commonly encountered space group in surfactant
systems is
Pm3n (#223) with indexing ~12:J4:J5: .... In single-component block
copolymers, the
3 0 commonly observed space group is Im3m, corresponding to body-centered
sphere-packings
with indexing J2 :J4:~6: J8: ....
-34-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
2. To the unaided eye, the phase is generally transparent when fully
equilibrated, and thus often considerably clearer than any associated lamellar
phase.
3. In the polarizing optical microscope, the phase is non-birefringent and
therefore there are no optical textures.
For normal discrete cubic phases in surfactant-water systems:
1. Viscosity is high, much more viscous than the lamellar phase and even more
viscous than typical normal hexagonal phases. Most cubic phase have
viscosities in the
millions of centipoise, whether discrete or bicontinuous.
2. Also in common with the bicontinuous cubic phases, there is no splitting in
the NMR bandshape, only a single isotropic peak.
3. In terms of phase behavior, the normal discrete cubic phase generally
occurs
at fairly low surfactant concentrations in single-tailed surfactant water
systems, typically on
the order of 40% surfactant with ionic surfactants. Usually the normal
discrete cubic phase
region is between normal micellar and normal hexagonal phase regions, which
along with its
high viscosity and non-birefringence make its determination fairly simple. In
double-tailed
surfactants, it generally does not occur at all in the binary surfactant -
water system.
For discrete cubic phases in single-component block copolymer systems, the
terms "normal"
and "reversed" do not generally apply (although in the case where one block is
polar and the
other apolar, these qualifiers could be applied in principle). The shear
modulus in such a
2 0 discrete cubic phase is generally dependent almost entirely on the shear
modulus of the
polymer that forms the blocks in the continuous phase. In terms of phase
behavior, the
discrete cubic phases generally occur at very low volume fractions of one or
other of the two
blocks, on the order of 20% or less.
Reversed discrete cubic phase: The reversed discrete cubic phase is
characterized
2 5 by:
In surfactant-water systems, the identification of the reversed discrete cubic
phase
differs from the above identification of the normal discrete cubic phase in
three respects:
1. In terms of phase behavior, the reversed discrete cubic phase is found
between the
lamellar phase and the reversed hexagonal phase, whereas the normal is found
between the
3 0 lamellar and normal hexagonal phases: one must therefore make reference to
the discussion
above for distinguishing normal hexagonal from reversed hexagonal. A good rule
is that if the
-35-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
cubic phase lies to higher water concentrations than the lamellar phase, then
it is normal,
whereas if it lies to higher surfactant concentrations than the lamellar then
it is reversed. The
reversed cubic phase generally occurs at high surfactant concentrations in
double-tailed
surfactant / water systems, although this is often complicated by the fact
that the reversed
cubic phase may only be found in the presence of added hydrophobe ('oil') or
amphiphile. The
reversed discrete cubic phase does appear in a number of binary systems with
single-tailed
surfactants, such as those of many monoglycerides (include glycerol
monooleate), and a
number of nonionic PEG-based surfactants with low HLB.
2. The space group observed is usually Fd3m. #227.
3. The self diffusion of the water is very low, while that of any hydrophobe
present is
high; that of the surfactant is generally fairly high, comparable to that in
the lamellar phase.
As stated above in the discussion of normal discrete cubic phases, the
distinction between
'normal' and 'reversed' discrete cubic phases makes sense only in surfactant
systems, and
generally not in single-component block copolymer discrete cubic phases.
Intermediate phases: The intermediate phase is characterized by:
These phases occur quite rarely, and when they are found they generally occupy
very
narrow regions in the phase diagram. Presently the structures of many of these
are unknown
or under debate. The intermediate phases can be classified as follows:
Normal int(1) phases occur at lower surfactant concentration than the normal
2 0 bicontinuous cubic phase, adjacent to the hexagonal phase. Viscosity is
generally
low or moderately low, no higher than that of the normal hexagonal phase. The
phase is
birefringent, with textures typically similar to those of the hexagonal phase.
Self diffusion of
the components is very similar to those in the hexagonal phase. Small-angle x-
ray shows a
lower-symmetry space group than the cubic phases, typically monoclinic. Fairly
sophisticated
2 5 NMR bandshape and SAXS analyses can be used to distinguish this phase from
the normal
hexagonal phase. See Henriksson, U., Blackmore, E. S., Tiddy, G.J.T. and
Soderman, 0.
(1992) J. Phys. Chem. 96:389. Typically bandshape splittings will be
intermediate between
those of hexagonal and the zero splitting of the isotropic phase, which
provides good
evidence of an intermediate phase.
3 0 Normal int(2) is found at higher concentrations than the normal
bicontinuous cubic
phase, adjacent to the lamellar phase. These bear close resemblance, both in
terms of properly
-36-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
and probably also in terms of structure, to the normal bicontinuous cubic
phases, except that
they are birefringent and show differences in NMR bandshape and SAXS analyses.
Optical
textures are somewhat unusual, in some cases resembling lamellar textures and
in some
resembling hexagonal, but these can be considerably coarser than either of the
more common
phases. As in the int(1) phases, the space group is of lower symmetry,
typically rhombohedral
or tetragonal, requiring two unit cell parameters for characterization and
making SAXS
analysis difficult. In general, if the squares of the d-spacing ratios cannot
be fit to a simple
integral scheme, then an intermediate phase structure is suspect.
Reversed int(2) is found at lower concentrations than the reversed
bicontinuous cubic
phase, adjacent to the lamellar phase. These are birefringent and show unusual
in NMR
bandshape and SAXS analyses. As in the int(1) and int(2) phases, the space
group is of lower
symmetry, typically rhombohedral or tetragonal, requiring two unit cell
parameters for
characterization and making SAXS analysis difficult. SAXS analysis difficult,
though the
presence of Bragg peaks in the SAXS spectrum which do not index to a cubic or
hexagonal
lattice (which have only one lattice parameter) is, together with optical
birefringence,
indication of an intermediate phase. Space groups which axe likely for
bicontinuous
intermediate phases have been discussed in a publication by the present
author. D. M.
Anderson, Supplement to J. Physique, Proceedings of Workshop on Geometry, and
Interfaces, Aussois, France, Sept. 1990. C7-1 - C7-18.
2 0 At the time that the coated particle 10 is being formed and the exterior
coating 20 is
not yet formed, it is highly desirable that the nanostructured liquid phase
material or the
nanostructured liquid crystalline phase material or the combination be one
that is in
equilibrium with water (polar solvent) or, more precisely, with a dilute
aqueous solution.
Once the coated particle 10 has its exterior coating 20, the foregoing
nanostructured material
2 5 need not be one that is in equilibrium with water. The liquid phases that
can be in equilibrium
with water are:
L2 phase (a.lc.a. reversed micelles),
microemulsion, and
L3 phase (but not the L3 * phase).
3 0 These supplement the liquid crystalline phases that can be in equilibrium
with water:
reversed cubic phase,
-37-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
I
reversed hexagonal phase,
reversed intermediate phase, and
lamellar phase.
The phases that can be in equilibrium with water are preferred from the point
of view of
making coated particles of the present invention. Preferably, in using the
process described
herein to disperse a given phase as the matrix, it is desirable that the phase
be insoluble in
water, or whatever solvent the particles are dispersed in. Furthermore, when
the interior phase
has the additional property that it is in equilibrium with excess aqueous
solution during
formation of the particles, then concerns of phase transformation are
minimized. Similarly
when the interior phase is in equilibrium with excess aqueous solution under
the conditions
encountered when and after the particle coating is released, then the concerns
of phase
changes are likewise minimized, and in some applications this may be
advantageous.
Whereas insolubility in water (external solvent, in general) is preferred for
the matrix
at the instant of particle formation, and frequently also at the time of
application, there are
applications where solubility in water at the time of application is
advantageous, and this can
be accomplished with the instant invention. For example, consider a matrix
composed of 20%
C12E5 (pentaethylene glycol dodecyl ether) in water. At 75°C, this
composition produces an
L3 phase which is in equilibrium with excess water (dilute solution) and thus
this
composition would be readily dispersible at 75°C. If the application
temperature were
2 0 between 0 and 25°C, however, then this interior composition would
be soluble in water, and
in fact the C 12E5 acts as an ordinary water-soluble surfactant at room
temperature. This
could be advantageous if a non greasy, non-comedogenic -- and even cleansing --
final
product is desired after release of the particle coating.
The nanostructured liquid phase material may be formed from:
2 5 a. a polar solvent and a surfactant or
b. a polar solvent, a surfactant and an amphiphile or hydrophobe or
c. a block copolymer or
d. a block copolymer and a solvent.
The nanostructured liquid crystalline phase material may be formed from:
3 0 a. a polar solvent and a surfactant.
b. a polar solvent, a surfactant and an amphiphile or hydrophobe, or
-38-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
c. a block copolymer or
d. a block copolymer and a solvent.
Above under the heading Chemical Criteria, criteria were discussed which could
be
used to select operative polar and apolar groups in order to make an operative
surfactant.
Thus, suitable surfactants include those compounds which contain two chemical
moieties,
one being an operative polar group chosen from those described in that
discussion of polar
groups, and the other being an operative apolar group chosen from those
described in that
discussion of apolar groups.
Surfactants of utility.
Suitable surfactants or block copolymer components (or mixtures thereof) may
include: a. cationic surfactant
b. anionic surfactant
c, semipolar surfactant
d. zwitterionic surfactant
i . in particular, a phospholipid
ii . a lipid mixture containing phospholipids, designed to match the
physico-chemical characteristics of a biomembrane
e. monoglyceride
~ PEGylated surfactant
2 0 g. one of the above but with aromatic ring
h. block copolymer
i. with both blocks hydrophobic, but mutually immiscible
ii. with both blocks hydrophilic, but mutually immiscible,
iii. with one block hydrophilic and the other hydrophobic, i.e.,
2 5 amphiphilic)
i. a mixture of two or more of the above.
Suitable lipids include phospholipids (such as phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, or sphingomyelin), or
glycolipids (such as
MGDG, diacylglucopyranosyl glycerols, and Lipid A). Other suitable lipids are
phospholipids
3 0 (including phosphatidylcholines, phosphatidylinositols,
phosphatidylglycerols, phosphatidic
acids, phosphatidylserines, phosphatidylethanolamines, etc.), sphingolipids
(including
-39-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
splungomyelins), glycolipids (such as galactolipids such as MGDG and DGDG,
diacylglucopyranosyl glycerols, and Lipid A), salts of cholic acids and
related acids such as
deoxycholic acid, glycocholic acid, taurocholic acid, etc., gentiobiosyls,
isoprenoids,
ceramides, plasmologens, cerebrosides (including sulphatides), gangliosides,
cyclopentatriol
lipids, dimethylaminopropane lipids, and lysolecithins and other lysolipids
which are derived
from the above by removal of one acyl chain.
Other suitable types of surfactants include anionic, cationic, zwittenionic,
semipolar,
PEGylated, amine oxide and aminolipids. Preferred surfactants are:
anionic -- sodium oleate, sodium dodecyl sulfate, sodium diethylhexyl
sulfosuccinate,
sodium dimethylhexyl sulfosuccinate, sodium di-2-ethylacetate, sodium 2-
ethylhexyl sulfate, sodium undecane-3-sulfate, sodium
ethylphenylundecanoate, carboxylate soaps of the form IC~, where the chain
length n is between 8 and 20 and I is a monovalent counterion such as lithium,
sodiiun, potassium, rubidium, etc.,
cationic -- dimethylammonium and trimethylammonium surfactants of chain length
from 8 to 20 and with chloride, bromide or sulfate counterion, myristyl-
gammapicolinium chloride and relatives with alkyl chain lengths from 8 to 18,
benzalkonium benzoate, double-tailed quaternary ammonium surfactants with
chain lengths between 8 and 18 carbons and bromide, chloride or sulfate
2 0 counterions,
nonionic PEGylated surfactants of the form C"Em where the alka.ne chain length
n is
from 6 to 20 carbons and the average number of ethylene oxide groups m is from
2 to
80, ethoxylated cholesterol;
zwitterionics and semipolars -- N,N,N-trimethylaminodecanoimide, amine oxide
2 5 surfactants with alkyl chain length from 8 to 18 carbons;
dodecyldimethylammoniopropane-1-sulfate,
dodecyldimethylammoniobutyrate, dodecyltrimethylene di(ammonium
chloride); decylmethylsulfonediimine; dimethyleicosylammoniohexanoate and
relatives of these zwitterionics and semipolars with alkyl chain lengths from
8
3 0 to 20.
Preferred surfactants which are FDA-approved as injectables include
benzalkonium
-40-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
chloride, sodium deoxycholate, myristyl-gamma-picolinium chloride, Poloxamer
188,
polyoxyl castor oil and related PEGylated castor oil derivatives such as
Cremophor EL,
Arlatone G, sorbitan monopalmitate, Pluronic 123, and sodium 2-ethylhexanoic
acid. Other
low-toxicity surfactants and lipids, which are of at least relatively low
solubility in water, that
are preferred for the present invention for products intended for a number of
routes of
administration, include: acetylated monoglycerides, aluminum monostearate,
ascorbyl
palmitate free acid and divalent salts, calcium stearoyl lactylate, ceteth-2,
choleth,
deoxycholic acid and divalent salts, dimethyldioctadecylammonium bentonite,
docusate
calcium, glyceryl stearate, stearamidoethyl diethylamine, ammoniated
glycyrrhizin, lanolin
nonionic derivatives, lauric myristic diethanolamide, magnesium stearate,
methyl gluceth-120
dioleate, monoglyceride citrate, octoxynol-1, oleth-2, oleth-5, peg vegetable
oil, peglicol-5-
oleate, pegoxol 7 stearate, poloxamer 331, polyglyceryl-10 tetralinoleate,
polyoxyethylene
fatty acid esters, polyoxyl castor oil, polyoxyl distearate, polyoxyl glyceryl
stearate, polyoxyl
lanolin, polyoxyl-8 stearate, polyoxyl 150 distearate, polyoxyl 2 stearate,
polyoxyl 35 castor
oil, polyoxyl 8 stearate, polyoxy160 castor oil, polyoxyl 75 lanolin,
polysorbate 85, sodium
stearoyl lactylate, sorbitan sesquioleate, sorbitan trioleate, stear-o-wet c,
stear-o-wet m,
stearalkonium chloride, stearamidoethyl diethylamine (vaginal), steareth-2,
steareth-10,
stearic acid, stearyl citrate, sodium stearyl fumarate or divalent salt,
trideceth 10, trilaneth-4
phosphate, Detaine PB, JBR-99 rhamnolipid (from Jeneil Biosurfactant),
glycocholic acid and
2 0 its salts, taurochenodeoxycholic acid (particularly combined with vitamin
E), tocopheryl
dimethylaminoacetate hydrochloride, tocopheryl phosphonate, tocopheryl peg
1000 succinate,
cytofectin gs, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane,
cholesterol linked to
lysinamide or ornithinamide, dimethyldioctadecyl ammonium bromide, 1,2-
dioleoyl-sn-3-
ethylphosphocholine and other double-chained lipids with a cationic charge
carried by a
2 5 phosphorus or arsenic atom, trimethyl aminoethane carbamoyl cholesterol
iodide, lipoic acid,
O,O'-ditetradecanoyl-N-(alpha-trimethyl ammonioacetyl) diethanolamine chloride
(DC-6-14),
N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride, N-methyl-4-
(dioleyl)methylpyridinium chloride (saint-2), lipidic glycosides with amino
alkyl pendent
groups, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide, bis[2-
(11-
3 0 phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-10-[O-(4
acetoxy)-phenylundecanoate]ethyl-dimethylammonium bromide, bis[2-(11
-41-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
butyloxyundecanoate)ethyl]dimethylammonium bromide, 3-beta-[N-(N', N'-
dimethylaminoethane)-carbamoyl] cholesterol, vaxfectin, cardiolipin, dodecyl-
N,N-
dimethylglycine, and lung surfactant (Exosurf, Survanta).
Suitable block copolymers are those composed of two or more mutually
immiscible
blocks from the following classes of polymers: polydienes, polyallenes,
polyacrylics and
polymethacrylics (including polyacrylic acids, polymethacrylic acids,
polyacrylates,
polymethacrylates, polydisubstituted esters, polyacrylamides,
polymethacrylamides, etc.),
polyvinyl ethers, polyvinyl alcohols, polyacetals, polyvinyl ketones,
polyvinylhalides,
polyvinyl nitrites, polyvinyl esters, polystyrenes, polyphenylenes,
polyoxides, polycarbonates,
polyesters, polyanhydrides, polyurethanes, polysulfonates, polysiloxane,
polysulfides,
polysulfones, polyamides, polyhydrazides, polyureas, polycarbadiimides,
polyphosphazenes,
polysilanes, polysilazanes, polybenzoxazoles, polyoxadiazoles,
polyoxadiazoiidines,
polythiazoles, polybenzothiazoles, polypyromellitimides, polyquinoxalines,
polybenzimidazoles, polypiperazines, cellulose derivatives, alginic acid and
its salts, chitin,
chitosan, glycogen, heparin, pectin, polyphosphorus nitrite chloride, polytri-
n-butyl tin
fluoride, polyphosphoryldimethylamide, poly.-2,5-selenienylene, poly-4-n-
butylpyridinium
bromide, poly-2-N-methylpyridinium iodide, polyallylammonium chloride, and
polysodium-
sulfonate-trimethylene oxyethylene. Preferred polymer blocks are polyethylene
oxide,
polypropylene oxide, polybutadiene, polyisoprene, polychlorobutadiene,
polyacetylene,
2 0 polyacrylic acid and its salts, polymethacrylic acid and its salts,
polyitaconic acid and its salts,
polymethylacrylate, polvethylacrylate, polybutylacrylate,
polymethylmethacrylate,
polypropylmethacrylate, poly-N-vinyl carbazole, polyacrylamide,
polyisopropylacrylamide,
polymethacrylamide, polyacrylonitrile, polyvinyl acetate, polyvinyl caprylate,
polystyrene,
poly-alpha-methylstyrene, polystyrene sulfonic acid and its salts,
polybromostyrene,
2 5 polybutyleneoxide, polyacrolein, polydimethylsiloxane, polyvinyl pyridine,
polyvinyl
pyrrolidone, polyoxy-tetramethylene, polydimethylfulvene,
polymethylphenylsiloxane,
polycyclopentadienylene vinylene, polyalkylthiophene, polyalkyl-p-phenylene,
polyethylene-
altpropylene, polynorbomene, poly-5-((trimethylsiloxy)methyl)norbomene,
polythiophenylene, heparin, pectin, chitin, chitosan, and alginic acid and its
salts. Especially
3 0 preferred block copolymers are polystyrene-b-butadiene, polystyrene-b-
isoprene, polystyrene-
b-styrenesulfonic acid, polyethyleneoxide-b-propyleneoxide, polystyrene-b-
dimethylsiloxane,
-42-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
polyethyleneoxide-b-styrene, polynorborene-b-5-
((trimethylsiloxy)methyl)norbornene,
polyacetylene-b-5((trimethylsiloxv)methyl)norbornene, polyacetylene-b-
norbornene,
polyethyleneoxide-b-norbornene, polybutyleneoxide-b-ethyleneoxide,
polyethyleneoxide-b-
siloxane, and the triblock copolymer polyisoprene-b-styrene-b-2-vinylpyridine.
Third component: h~phobe or non-surfactant amphiphile.
This component can serve multiple functions in a matrix of the present
invention,
including modulation of phase behavior, tuning of poresize, solubilization of
an active,
modulation of release properties, etc. Choices appropriate for this invention
include:
a. alkane or allcene, other long-chain aliphatic compound
b. aromatic compound, such as toluene
c. long-chain alcohol
d. a glyceride (diglyceride or triglyceride)
e. an acylated sorbitan, such as a sorbitan triester (e.g., sorbitan
trioleate), or
sesquioleate, or mixture of sorbitans with different numbers of acyl chains
between 2 and 6
f. other hydrophobe or non-surfactant amphiphile or mixture with one or more
of the above,
g. none.
Suitable third components (hydrophobes or non-surfactant amphiphiles),
include: n-
2 0 alkane, where n is from 6 to 2,0, including branched, unsaturated, and
substituted variants
(alkenes, chloroalkanes, etc.), cholesterol and related compounds, terpenes,
diterpenes,
triterpenes, fatty alcohols, fatty acids, aromatics, cyclohexanes, bicyclics
such as naphthalenes
and naphthol, quinolines and benzoquinolines, etc., tricyclics such as
carbazole,
phenothiazine, etc., pigments, chlorophyll, sterols, triglycerides, sucrose
fatty acid esters
2 5 (such as OlestraTM), natural oil extracts (such as clove oil, anise oil,
cinnamon oil, coriander
oil, eucalyptus oil, peppermint oil), wax, bilirubin, bromine, iodine,
hydrophobic and
asnphiphilic proteins and polypeptides (including gramicidin, casein, receptor
proteins, lipid-
anchored proteins, etc.), local anesthetics (such as butacaine, ecgonine,
procaine, etc.), and
low-molecular weight hydrophobic polymers (see listing of polymers above).
Especially
3 0 preferred third components are: anise oil, clove oil, coriander oil,
cinnamon oil, eucalyptus
oil, peppermint oil, beeswax, benzoin, benzyl alcohol, benzyl benzoate,
naphthol, capsaicin,
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
cetearyl alcohol, cetyl alcohol, cinnamaldehyde, cocoa butter, coconut oil,
cottonseed oil
(hydrogenated), cyclohexane, cyclomethicone, dibutyl phthalate, dibutyl
sebacate, diocryl
phthalate, DIPAC, ethyl phthalate, ethyl vanillin, eugenol, fumaric acid,
glyceryl distearate,
menthol, methyl acrylate, methyl salicylate, myristyl alcohol, oleic acid,
oleyl alcohol, benzyI
chloride, paraffin, peanut oil, piperonal, rapeseed oil, rosin, sesame oil,
sorbitan fatty acid
esters, squalane, squalene, stearic acid, triacetin, trimyristin, vanillin,
and vitamin E.
Polar solvent.
The polar solvent (or in the case of a block copolymer, the~preferential
solvent) can
similarly serve multiple functions, including modulation of phase behavior
(indeed, making
nanostructured phases possible at all, in many surfactant systems),
solubilization of the active,
providing a polar environment for portions of the active molecule such as fox
example the
polar regions of a protein, etc. The choice of a non-volatile polar solvent
like glycerol can be
important in processes such as spray-drying. The polar solvent may be:
a. water
b. glycerol
c. formamide, N-methyl formamide, or dimethylformamide
d. ethylene glycol or other polyhydric alcohol
e. ethylammonium iutrate
~ other non-aqueous polar solvents such as N-methyl sydnone, N-methyl
2 0 acetamide, pyridinium chloride, etc.;
g. a mixture of two or more of the above.
Desirable polar solvents are water, glycerol, ethylene glycol, formamide, N-
methyl
formamide, dimethylformamide, ethylammonium nitrate, and polyethylene glycol.
It can be advantageous in certain circumstances to use, as the interior
matrix, a
2 5 composition that yields a nanostructured liquid or liquid crystalline
phase upon contact with
water (or more rarely, other polar solvent)-whether or not this dehydrated
composition itself
is a nanostructured liquid or liquid crystalline phase. In particular, this
contact with water or
a water-containing mixture could be either during a reconstitution step, or
more preferably,
during the application of the particle, most preferably after the coating
releases, and the de-
3 0 coated particle contacts an aqueous solution such as blood, extracellular
fluid, intracellular
fluid, mucous, intestinal fluid, etc. There are several reasons why this may
be advantageous:
-44-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
to protect hydrolytically unstable actives or excipients; to limit premature
release of water-
soluble actives; and as a natural result of a production process such as spray-
drying or freeze-
drying that can induce dehydration. Removal of most, or all, of the water from
a
nanostructured liquid or liquid crystalline phase will often yield another
nanostructured liquid
or liquid crystalline phase, but can sometimes yield a structureless solution,
precipitate, or a "
mixture of these with one or more nanostructured liquid or liquid crystalline
phases. In any
case, for many applications, it is the hydrated form that is important in the
application of the
particles, and thus if this hydrated form is a nanostructured liquid or liquid
crystalline phase,
then the composition of matter falls within the scope of the current
invention.
Coatings.
As previously stated, the exterior coating 20 may be formed of a nonlamellar
material.
The term "nonlamellar"as applied to crystal structure herein should be taken
in the following
context. Lamellar crystalline materials, which are distinct from lamellar
liquid crystalline
phases, occur in organic compounds (typically polar lipids), inorganic
compounds, and
organometallics. Although these materials can be true crystalline materials
and can thus
exhibit long range three-dimensional lattice ordering of the constituent atoms
(or molecules,
in the case of an organic crystalline material) in space, the forces and
interactions between
atoms-which can include covalent bonding, ionic bonding, hydrogen bonding,
steric
interactions, hydrophobic interactions, dispersion forces, etc. are much
stronger amongst the
2 0 constituent atoms or molecules than within the plane of a lamella than
across distinct
lamellae. For example, in the case of the layered structure of graphite, the
atoms within a
layer are covalently bonded with each other into a two-dimensional network,
whereas
between distinct layers there is no bonding, only the weaker dispersion forces
and steric
interactions. This absence of strong local interlamellar interactions gives
rise to a number of
2 5 physicochemical properties which make them undesirable as coating
materials in the present
invention.
To begin with, the physical integrity of lamellar crystals is inherently
compromised by
the weak local interactions between layers. This is dramatically evidenced by
the comparison
between graphite (a layered crystalline form of carbon) with diamond (a
crystalline form of
3 0 carbon that has three-dimensional bonding). Indeed, the fact that graphite
is an important
-45-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
ingredient in certain lubricants, due to the ease with which the layers slide
over each other,
whereas diamond is an abrasive, illustrates the "liquid-like" (or "liquid
crystal-like") character
of layered crystalline structures in terms of their response to shear. This
same inter-lamellar
sliding effect is, in fact, the same effect that gives rise to the much lower
viscosity of the
lamellar liquid crystalline phase compared with that of other liquid
crystalline phases, in
particular compared with the very high viscoelasticity of the bicontinuous
cubic phase. As a
fizrther indication of this liquid-like nature, the Moh's hardness of graphite
is 1.0, whereas that
of diamond is 10. The loss of integrity with shear in the case of graphite is
seen in everyday
life with "lead" pencils, which are graphite.
The detrimental effects associated with layered crystalline structures can be
seen in
every day life even in situations where macroscopic shear is not involved.
According to a
widely-accepted model of the structure of emulsions advanced by Stig Friberg,
as reviewed
by Larsson,. K. and S. Friberag, Eds. 1990, Food Emulsions 2" Edition, Marvel
Dekker, Inc.
NY, lamellar liquid crystalline or, commonly, lamellar crystalline coatings
stabilize the oil
droplets in an oil-and-water emulsion, and the water droplets in a water-in-
oil emulsion. In
commonly encountered emulsions such as milk, ice cream, mayonnaise, etc., the
instabilities
that are wellknown to the lay person -- and in the field referred to as
"breaking" of emulsions
-- are due in large part to the fluidity of these layered coating materials.
Even in a quiescent
emulsion, these layered coatings undergo continual disruption, streaming and
coalescence,
2 0 and with time any emulsion must ultimately succumb to the destabilizing
effect of these
disruptions.
And at yet another level, layered crystalline materials exhibit chemical
instabilities of
the type that would prevent their application as coatings in embodiments of
the current
invention. Consider the case of the Werner complexes isomorphous to nickel
dithiocyanate
2 5 tetra(4methylpyridine) that form clathrate compounds With a host lattice
containing embedded
guest molecules, in most cases yielding permanent pores upon removal of the
guest. One such
Wemer complex was used as the coating in a particle in Example 22, thus
illustrating the use
of the present invention in creating particles with coatings possessing fixed,
controlled-size
and highselectivity pores. According to J. Lipkowski, Inclusion Compounds 1,
Academic
3 0 Press, London (1984), p. 59, "Layered structures of Ni(NCS)2(4-MePy)4 are
stable only in the
presence of guest molecules while the beta-phases preserve their porosity even
in the absence
-46-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
of guest molecules." The three-dimensional, non-layered structure of the beta-
phases is
discussed in detail in the same publication, such as: "... beta-phases... have
a three-
dimensional system of cavities interconnected through channels of molecular
size".
Nonlamellar amorphous and semi-crystalline materials are materials comprising
non-
crystalline domains (or lacking crystallinity altogether) in which strong
atomic interactions
exist in all three dimensions. In the amorphous trehalose that provides the
coating in
Example 40, for example, the packing of these sugar molecules and the multiple
hydrogen
bonds that each individual molecule can participate in make this a compound
that exhibits
strong interactions in all three dimensions (and the amorphous property rules
out any
lamellas-type structure). Similarly amorphous PLGA has strong interactions
between the
carboxyl groups across neighboring polymer chains which, since the material is
optically
isotropic, are not limited to two dimensions. The release of a coating in a
PLGA-coated
particle will be chosen to be based on its hydrolysis rate in the body, as is
well-known in the
art, and not by mechanical shear or deformation as could occur in a particle
coated with a
lamellas coating. Since most production protocols used in industrial or
pharmaceutical
practice involve shear, release upon the application of such shear rates to a
lamellas-coated
paxticle system could be detrimental or disastrous in the context of such a
process.
As is well-known in the art, in the case of polymers, polymers universally
have
amorphous domains: no polymer is ever 100% crystalline, and thus even high-
crystallinity
2 0 polymers are semi-crystalline and possess a finite fraction of amorphous
domains. Often this
is in the range of about 1-50%. The glass transition temperature of these
amorphous domains
can usually be detected by thermodynamic (e.g., DSC) techniques or rheometric
measurements, though in certain very high-crystallinity polymers (greater than
about 98%),
this may be a difficult undertaking. Nevertheless, even in these high-
crystallinity cases the
2 5 amorphous domains can play important roles: they can mitigate structural
problems
associated with microcrystallite boundaries, thus conferring greater
homogeneity and
cohesiveness to microcrystalline polymers; this in turn can have strong
effects on rheological
properties and behavior as diffusional barriers; according to the fringed
micelle model, an
amorphous domain can provide a medium that allows for a single chain to extend
through
3 0 several microcrystallites, yielding a physical crosslinking (analogous to
the physical
crosslinking that occurs in thermoplastic elastomers); and their presence may
in fact allow for
-47-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
crystallinity in high-MW polymers where the amorphous domains are the
necessary result of
chain folding. Being amorphous, these domains are non-lamellar regions in the
polymer that
are distinct from the crystalline regions but nonetheless actually play
crucial roles in the
crystallization of polymers and in determining their overall properties.
The exterior coating 20 can protect the internal core 10 and any active
agents) or
components) disposed therein, for example, against oxidation, hydrolysis,
premature release,
precipitation, shear, vacuum, enzymatic attack, degradation from other
components of the
preparation, and/or conditions external to the coated particles, for example,
in their
preparation such as pH, ionic strength, or the presence of bioactive
impurities such as
proteases or nucleases. Examples of each of these are:
oxidation: e.g. for antioxidants such as vitamin C, which are by their very
nature
sensitive to oxidation, or unsaturated lipids:
hydrolysis: e.g., for a drug with a labile ester bond:
premature release: during storage:
precipitation: e.g., for a drug in the protonated (hydrochloride) form that
would
deprotonate at the body pH and thereby become insoluble;
shear: e.g., in cases where processing after encapsulation endangers shear-
sensitive
compounds, such as proteins;
vacuum: e.g., in cases where processing involves vacuum-drying;
2 0 enzymatic attack: a peptide hormone, such as somatostatin, which is
normally
quickly digested by enzymes in the body, can be held active in circulation
until
reaching the site of release and action:
degradation from other components: e.g., where even a slight reactivity
between an
component disposed in the internal core and an exterior one could, over a
shelf life of
2 5 months or years, pose a problem:
external pH: e.g., a drug in protonated form could be encapsulated at low
internal pH
to ensure solubility, but without requiring a low pH of the exterior liquid
which would
otherwise upset the stomach,
external ionic strength: e.g., where a protein is encapsulated to avoid
salting-out and
3 0 denaturation;
external impurities such as proteases, nucleases, etc.: e.g., when the
exterior
-48-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
contains a bioreactor-derived product from which removal of proteases might be
prohibitively expensive.
Examples of suitable nonlamellar coating materials, namely, compounds which
occur
in nonlamellar form over useful temperature ranges, and which are in most
cases of low
toxicity and environmental impact are: ascorbic acid; ascorbic palmitate;
aspartic acid;
benzoin; beta-naphthol; bismuth subcarbonate; butylated hydroxytoluene;
butylparaben;
calcium acetate; calcium ascorbate; calcium carbonate; calcium chloride;
calcium citrate;
calcium hydroxide; calcium phosphate, dibasic; calcium phosphate, tribasic;
calcium
pyrophosphate; calcium salicyiate,;calcium silicate; calcium sulfate; carmine;
cetearyl
alcohol; cetyl alcohol; cinnamaldehyde; citric acid; cysteine hydrochloride;
dibutyl sebacate;
esculin; ferric oxide; ferric citrate; ferrosoferric oxide; gentisic acid;
glutamic acid; glycine;
gold; histidine; hydrochlorothiazide; iodine; iron oxide; lauryl sulfate;
leucine; magnesium;
magnesium aluminum silicate; magnesium carbonate; magnesium hydroxide;
magnesium
oxide; magnesium silicate; magnesium sulfate; magnesium trisilicate; malefic
acid; malic
acid; DL- methyl salicylate; methylparaben; monosodium glutamate; propyl
gallate;
propylparaben; silica; silicon; silicon dioxide; sodium aluminosilicate;
sodium
aminobenzoate; sodium benzoate; sodium bicarbonate; sodium bisulfate; sodium
bisulfate;
sodium carbonate; sodium chloride; sodium citrate; sodium metabisulfite;
sodium nitrate;
sodium phosphate, dibasic; sodium propionate; sodium salicylate; sodium
stannate; sodium
2 0 succinate; sodium sulfate; sodium sulfate; sodium thiosulfate; sodium
thiosulfate; succinic
acid; talc; talc triturate; tartaric acid; tartaric acid; DL- tartrazine;
tellurium; titanium dioxide;
triacetin; triethyl citrate; trichloromonofluorethane; tromethamine and 2-
hydroxy-n-
cyclopropylmethyl morphinan hydrochloride; zinc oxide.
Calcium phosphate coatings are of interest in biomedical and pharmaceutical
2 5 applications, since calcium phosphates are a major component of bone,
teeth, and other
structural components. For example, in the treatment of osteoporosis, the
release of the
appropriate pharmaceutical compound could be triggered by physiological
conditions that
induce dissolution of bone (and thus of the particle coating).
Potassium nitrate coatings are of interest in agricultural applications since
the coating
3 0 also act as plant fertilizers.
Iodine, aspartic acid, benzoic acid, butylated hydroxytoluene, calcium edetate
-49-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
disodium, gentisic acid, histidine, propyl gallate and zinc oxide can be
particularly useful as
coatings in potential pharmaceutical applications because they have relatively
low water
solubility (generally less than 5%) and are on the FDA list of approved
inactive ingredients
for injectable formulations.
Of particular interest as coating materials are clathrates. Examples of such
materials
are as follows:
1. Clathrates and inclusion compounds (some of which retain permanent porosity
upon
removal of the guest molecules): Werner complexes of the form MXZAø where M is
a divalent
cation (Fe, Co, Ni, Cu, Zn, Cd, Mn, Hg, Cr), X is an anionic ligand (NCS-, NCO-
, CN-, N03-,
Cl-, Br-, I-), and A is an electrically neutral ligand-substituted pyridine,
alpha-arylalkylamine
or isoquinoline, examples of A include 4-methylpyridine, 3,5-dimethylpyridine,
4-
phenylpyridine, and 4-vinylpyridine. A wide range of guest molecules can be
included in
these complexes, examples being benzene, toluene, xylene, dichlorobenzene,
nitrotoluene,
methanol, chloromethane, argon, krypton, xenon, oxygen, nitrogen, carbon
dioxide, carbon
disulfide, etc.;
reversible oxygen-carrying chelates such as bis-salicyladehyde-
ethylenediiminecobalt
and other bis salicyladehyde iminecobalt derivatives, cobalt(II) dihistidine
and related
cobalt(II) amino acid complexes, iron(II) dimethylglyoxime and nickel(II)
dimethylglyoxime;
and
2 0 complexes of the form KZZn3[Fe(CN)6]z.xH,O, where certain values of the
variable x
correspond to complexes which yield permanent pores upon removal of the water.
2. Zeolites:
faujasite-type NaX zeolite;
faujasite-type NaY zeolite; and
~ 5 VPI-5 zeolite.
Amorphous and semi-crystalline nonlamellar materials.
In some embodiments of the present invention, the exterior coating of the
particles of
the present invention comprises nonlamellar materials which are not entirely
in crystalline
form. Such non-crystalline materials may be amorphous or semicrystalline. In
the art, the
-50-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
term "amorphous" as applied to materials means lacking long-range order; this
is in direct
contrast to the case of a crystalline material, in which there is long-range
order in the
positions of atoms, such that their positions conform to a lattice with its
associated
periodicity. The x-ray diffraction pattern of an amorphous material will be
absent of any
Bragg reflections, and any short-range correlations can at most give rise to
broad maxima in
the diffraction pattern, maxima which exhibit neither the sharpness nor the
functional form of
a true Bragg reflection. By "semi-crystalline" is meant a material which has a
mixture of
crystalline domains and amorphous domains.
Those of skill in the art will recognize that many materials can exist in a
crystalline,
an amorphous, or a semicrystalline form, depending on the preparation of the
material. For
example, many materials which otherwise occur in crystalline form instead
occur in
amorphous form when spray-dried, freeze-dried (as exemplified in Example 40,
below), or
prepared in other methods that are of central importance in the food,
cosmetic, and
pharmaceutical industries.
Amorphous materials have a number of properties which make them advantageous
for
certain embodiments of the current invention. For example, one property of
amorphous
materials is that they are generally faster-dissolving than a corresponding
(or comparable)
material in crystalline form, and this can be advantageous in cases where fast
dissolution of
the exterior coating is desirable. Further, amorphous materials can be
superior to their
2 0 corresponding crystalline forms in certain material properties. For
example, amorphous
materials tend to exhibit higher ductility, and thus allow the adsorption of
stress without
cracking.
In general, small-molecule amorphous materials tend to exhibit lessor
stability over
time than their corresponding crystalline materials. In particular, a small-
molecule amorphous
2 5 material will often show a tendency to revert to a crystalline form over a
period of time that is
comparable to, or shorter than, timescales that are relevant for the storage
and use of a
product. In the case of high-MW polymers, even though the true equilibrium
condition may
be a crystal, kinetics of rearrangement can be so slow that the timescale
required for
attainment of this equilibrium is for all intents and purposes infinite, so
that the material can
3 0 be locked into an amorphous or semi-crystalline state. For certain
applications, this may be
highly desirable. For example, many of the well-known elastomers and plastics,
such as
-51-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
natural rubber (an example of an elastomer) or polymethylmethacrylate (PMMA,
also known
as Plexiglass, an example of a thermoplastic), are amorphous materials.
Semi-crystalline materials can in certain ways offer significant advantages,
though
their occurrence as long-lasting states is largely limited to high-MW
polymers. A semi-
s crystalline polymer with high crystallinity can offer high modulus due to
the preponderance
of crystalline domains, but a certain amount of ductility due to the presence
of amorphous
domains, which can absorb stress without cracking. A number of the most
important
polymers, both cormnodity and engineering plastics, are semi-crystalline.
Examples of materials which occur in an amorphous or semi-crystalline form
that may
be utilized in the practice of the present invention include: polydienes,
polyallenes,
polyacrylics and polymethacrylics (including polyacrylic acids,
polymethacrylic acids,
polyacrylates, polymethacrylates, polydisubstituted esters, polyacrylamides,
polymethacrylamides, etc.), polyvinyl ethers, polyvinyl alcohols, polyacetals,
polyvinyl
ketones, polyvinylhalides, polyvinyl nitrites, polyvinyl esters, polystyrenes,
polyphenylenes,
polyoxides, polycarbonates, polyesters, polyanlzydrides, polyurethanes,
polysulfonates,
polysiloxane, polysulfides, polysulfones, polyamides, polyhydrazides,
polyureas,
polycarbodiimides, polyphosphazenes, polysilanes, polysilazanes,
polybenzoxazoles,
polyoxadiazoles, polyoxadiazoiidines, polythiazoles, polybenzothiazoles,
polypyromellitimides, polyquinoxalines, polybenzimidazoles, polypiperazines,
cellulose
2 0 derivatives, alginic acid and its salts, gum arabic and its salts,
gelatin, PVP, tragacanth, agar,
agarose, guar gum, carboxymethylcellulose, arabinogalactan, Carbopol, chitin,
chitosan,
Eudragits, glycogen, heparin, pectin, sugars (such as trehalose, lactose,
maltose, and sucrose,
or mixtures of sugars with albumin) and more complex carbohydrates, as well as
amorphous
forms of the coating materials listed above in connection with crystalline
coating materials,
2 5 obtained by processes that hinder crystallization, such as spray-drying,
vitrification, etc.
In any case, taking the larger view, the availability of the full spectral
range of
amorphous, semi-crystalline and crystalline materials yields great power and
flexibility to the
technology of creating particles with nanostructured liquid and liquid
crystalline interiors.
The case of lactide-glycolide copolymers provides a particularly pertinent
example, because
3 0 these copolymers are amorphous over a range of lactide:glycolide ratios,
and crystalline over
other ranges. By adjusting this ratio, it is possible to alter the form of the
material and thus its
-52-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
properties, thereby "tuning" the rate of hydrolysis of the coating material.
This, in turn,
"tunes" the rate of release of active agents disposed in either the coating or
the particle
interior.
Proteins, and perhaps to a lessor extent polypeptides, can also provide
amorphous and
semi-crystalline coating materials with advantageous properties. Due to the
intimacy of
interactions that are well-known between proteins and lipid matrices, the
crystallization of a
protein in an aqueous dispersion of nanostructured liquid or liquid
crystalline particles,
preferably of the reversed bicontinuous cubic phase, could yield particles of
the instant
invention wherein the coating was composed of semi-crystalline protein.
Alternatively,
gelation or precipitation of a protein at the surface of a nanostructured
liquid or liquid
crystalline particle could yield a particle of the instant invention wherein
the coating was
composed of amorphous protein. The presence of protein in the coating of such
particles
could serve one or more important roles, including: targeting (that is, the
coating itself could
serve a dual role as a targeting compound); inhibition of unfavorable protein
adsorption (e.g.,
albumin binding); presentation of a biocompatible particle surface that would
minimize
uptake by the body's defenses (e.g., the RES) and yield long circulation
times; and functional
proteins that could perform metabolic functions at the site of delivery that
might yield
enhanced absorption, diminished drug degradation/metabolism, a.nd/or
regulation of cellular
processes in concert with the drug action. Furthermore, since the release of
the coating could
2 0 be in response to enzymatic degradation (by, e.g., proteases), this could
provide a means by
which to achieve slow release, or targeted release to sites of accelerated
metabolism.
Applications of the invention.
The coated particles 1 of the present have application in a variety of fields.
The coated
particles 1 are adapted to absorb one or more materials from a selected
environment, adsorb
2 5 one or more materials from a selected environment or release one or more
materials, such as
active agents, disposed in the matrix. With respect to absorption, the coated
particles may be
used to harvest products or scavenge waste, in biological or chemical reaction
processes, to
carry catalysts in those processes, to remove toxins, antigens or waste
products in medical
applications, to identify a few examples.
3 0 With respect to adsorption, the coated particles may be used as
chromatographic
-53-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
media and as adsorbents.
With respect to release, the coated particles may be used for the controlled
release of
pharmaceutical agents such as anticancer agents or photodynamic therapy
agents, or cosmetic
or cosmeceutical materials. An active agent may be disposed in the matrix for
release upon
the triggering of release. For example, a pharmaceutical or biologically
active material may
be disposed in the matrix, that is, it may be either dissolved, or dispersed,
or in some cases be
partially dissolved and the remainder dispersed.
In applications of these microparticles in drug-delivery or with embedded
proteins or
polypeptides (in particular receptor proteins), it can be highly advantageous
to have an
interior matrix which, although synthetic or semisynthetic, is designed to
simulate closely the
physiochemical properties of a natural biomembrane from a living cell. This
could be
important for the proper functioning of a receptor protein or other membrane
component, for
example, or for promoting assimilation of the interior matrix into the natural
biomembrane in
drug delivery, or especially in targeting of the microparticles.
Physiochemical properties that
can be important in such a context include the bilayer rigidity, (a measure of
the resistance to
bending), bilayer fluidity (a measure of the microviscosity of the bilayer
interior), the acyl
chain length and bilayer thickness, the order parameter as a function of
position on the lipid
acyl chains, the surface charge density, the presence or absence of segregated
lipid domains of
differing composition within the bilayer, bilayer curvature and monolayer
curvature (for a
2 0 discussion of the relationship between these two curvatures see H.
Wennerstrom and D.M.
Anderson, in Statistical Thermodynamics and Differential Geometry of
Microstructured
Materials, Eds. H.T. Davis and J.C.C. Nitsche, Springer-Verlag, 1992, p. 137),
cholesterol
content, carbohydrate content, and the lipid:protein ratio. By proper choice
of composition,
one can adjust these parameters to a large extent in an artificial system,
namely a
2 5 nanostructured liquid phase or liquid crystalline phase. For example, the
bilayer rigidity can
be reduced by the addition of amphiphiles, particularly aliphatic alcohols;
and bilayer charge
can be adjusted by adjusting the ratio between uncharged lipids (such as
phosphatidylcholine)
and charged lipids (such as phosphatidic acid). Also, the addition of
cholesterol is important
for the function of a number of membrane proteins. The lamellar phase, the
reversed
3 0 bicontinuous cubic phase, the L3 phase, and to a lesser extent the
reversed hexagonal phase
are in particular well suited for this approach. Thus, a particle of the
present invention, with
-54-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
the interior matrix being such a phase with tuned physiochemical
characteristics for the
functioning of incorporated proteins or other biomolecules, can be very
valuable in products
for pharmaceutics, clinical assays, biochemical research products, etc.
Membrane proteins are generally dependent on a bilayer milieu in order to
function properly and even to maintain proper conformation, and for such
proteins the present
invention - particularly with the bilayer properties tuned as described above -
could be an
excellent and very useful matrix. Examples of membrane proteins include, in
addition to
receptor proteins, such proteins as proteinase A, amyloglucosidase,
enlcephalinase, dipeptidyl
peptidase IV, gamma-glutamyl transferase, galactosidase, neuraminidase, alpha-
mannosidase,
cholinesterase, arylamidase, surfactin, ferrochelatase, spiralin, penicillin-
binding proteins,
microsomal glycotransferases, kinases, bacterial outer membrane proteins, and
histocompatibility antigens.
In view of the demanding requirements for the delivery of pharmaceuticals in
the
treatment of cancers, the advantages and flexibility of the present invention
make it
particularly attractive in the delivery and release of antineoplastic agents,
such as for example,
the following:
Alkylating Agents
Alkyl Sulfonates -Busulfan, Improsuflan, Piposulfan.
Azif iaines - Benzodepa, Carboquone, Meturedepa, Uredepa,
2 0 EtlZyleheimines and Methvlmelamihes - Altretamine, Triethylenemelamine,
Triethylenephosphoramide, Triethylenetluophosphoramide, Trimethylolmelamine,
Nitf°ogen lllusta~ds - Chlorambucil, Chloramphazine, Cyclophosphamide,
Estramustine,
Ifosfamide, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Melphalan,
Novembichin, Phenesterine, Prednimustine, Trofosfamide, Uracil, Mustard.
2 5 Nitf°osous°ea- Carmustine, Ghlorozotocin, Fotemustine,
Lomustine, Nimustine, Ranimustine,
Others - Dacarbazine, Mannomustine, Mitobronitol, Mitolactol, Pipobroman.
Antibiotics -Actacinomveins - Actinomycin FI, Anthramycin, Azaserine,
Bleomvyins,
Cactinomycin, Carubicin, Carzinophilin, Chromomycins, Dactinomycin,
Daunorubicin, 6-
Diazo-5-OXO-Leucine, Doxorubicin, Epirubicin, Mitomycins, Mycophenolic Acid,
3 0 Nogalamycin, Olivomycins, Peplomycin, Plicarmcin, Porfiromycin, Puromycin,
Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin.
-55-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Antimetabolites
Folic Acid Analogs - Denopterin, Methotrexate, Pteropterin, Trimetrexate.
PuriszeAhalogs - Fludarabine, 6-Mercaptopurine, Thiamiprine, Thioguanine,
Py~irnidihe Analogs -Ancitabine, Azacitidine, 6-Azauridine, Carmofur,
Cytarabine,
Doxifluridine, Enocitabine, Floxuridine, Fluorouracil, Tegafur.
Enzymes - L-Asparaginase, etc.
Others - Aceglatone, Amsacrine, Bestrabucil, Bisantrene, Carboplatin,
Cisplatin,
Defosfamide, Demecolcine, Diaziquone, Eflorithine, Elliptinium Acetate,
Etoglucid,
Etoposide, Gallium Nitrate, Hydroxyurea, Interferon-ot, Interferon-P,
Interferon-y,
Interleukin-2, Lentinan, Lonidamine, Mitoguazone, Mitoxantrone, Mopidamol,
Nitracrine,
Pentostatin, Phenamet, Pirarubicin, Podophyllinic Acid, 2-Ethylhydrazide,
Procarbazine,
PSK09, Razoxane, Sizofiran, Spirogermanium, Taxol, Teniposide, Tenuazonic
Acid,
Triaziquone, 2,2',2,1,1 -Trichlorotriethylamine, Urethan, Vinblastine,
Vincristine. Vindesine.
Androgens - Calusterone, Dromostanolone Propionate, Epitiostanol,
Mepitiostane.
Testolactone.
Antiadrenals -Aminoglutethimide, Mitotane, Trilostane.
Andandrogens - Flutamide, Nilutamide.
Antiestrogens - Tamoxifen, Toremifene.
Estrogens - Fosfestrol, Hexestrol, Polyestradiol Phosphate.
2 0 LH-RH Analogs - Buserelin, Goserelin, Leuprolide, Triptorelin.
Progestogens - Chlormadinone Acetate, Medroxyprogesterone, Megestrol Acetate,
Melengestrol.
Antineoplastic (Radiation Source) Americium, Cobalt, '3'I-Ethiodized Oil, Gold
(Radioactive, Colloidal), Radium, Radon, Sodium Iodide (Radioactive), Sodium
Phosphate
2 5 (Radioactive),
Antineoplastic Adjuncts
Folic Acid Replenisher - Folinic Acid,
Uroprotective - Mesna.
Other pharmaceutical compounds that are particularly well-suited for
encapsulation
3 0 according to the instant invention, and suffer from problems or
limitations in the currently
marketed formulations, include: Dacarbazine, Ifosfamide, Streptozocin,
Thiotepa,
-56-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Nandrolone decanoate, Fentanyl citrate, Testosterone, Albendazole, Esmolol,
Bleomycin
sulfate, Dactinomycin, Amikacin sulfate, Gentaxnicin, Netilmicin,
Streptomycin, Tobramycin,
Doxorubicin, Epirubicin, Idarubicin, Valrubicin, Bacitracin, Colistimethate,
Oxybutinin,
Antithrombin III Human, Heparin, Lepirudin, Adenosine phosphate, Amphotericin
B,
Enalaprilat, Cladribine, Cytarabine, Fludarabine phosphate, Gemcitabine,
Pentostatin,
Docetaxel, Paclitaxel, Vinblastine, Vincristine, Vinorelbine, Batimastat,
Rituximab,
Trastazumab, Abciximab, Eptifibatide, Tirofiban, Droperidol, Aurothioglucose,
Capreomycin
disulfide, Acyclovir, Cidofovir, Pentafuside, Saquinavir, Ganciclovir,
Cromolyn,
Aldesleukin, Denileukin, Edrophonium, Infliximab, Doxapram, SN-38
(Irinotecan),
Topotecan, Hemin, Daunorubicin, Teniposide, Trimetrexate, Octreotride,
Ganirelix acetate,
Histrelin acetate, Somatropin, Epoetin, Filgrastim, Oprelvekin, Leuprolide,
Basiliximab,
Daclizumab, Glatiramer acetate, Interferons, Muromonab-CD3, Clyclosporin A,
Milrinone
lactate, Buprenorphine, Nalbuphine, Urofollitropin, Desmopressin, Caxboplatin,
Cisplatin,
Mitoxantrone, Estradiol, Hydroxyprogesterone, L-Thyroxine, Etanercept,
Neostigmine,
Epoprostenol, Methoxaxnine, Versed, Bupivacaine, Heparin, Insulin, Antisense
compounds,
Ibuprofen, Ketoprofen, Alendronate, Etidronate, Zoledronate, Ibandronate,
Risedronate, and
Pamidronate. These compounds represent the following classes of drug:
Alkylating agent,
Anabolic steroid, Analgesic, Androgen, Anthelmintic, Antiadrenergic,
Antibiotic, Antibiotic,
aminoglycoside, Antibiotic, antineoplastic, Antibiotic, polypeptide,
Anticholinergic,
2 0 Anticoagulant, Anticonvulsant, Antifungal, Antihypertensive,
Antimetabolite, Antimitotic,
Antineoplastic, Antiplatelet, Antipsychotic, Anesthetic, Antirheumatic,
Antituberculosal,
Antiviral, Antiviral (HIV), Asthma anti-inflammatory, Biological response
modifier,
Cholinergic muscle stimulant, CNS stimulant, DNA topoisomerase inhibitor,
Enzyme
inhibitor, Epipodophyllotoxin, Folate antagonist, Gastric antisecretory, Gene
therapy agents,
2 5 Gonadotropin-releasing, Growth hormone, Hematopoietic, Hormone,
Immunologic agent,
Immunosuppressant, Inotropic agent, Local anesthetic, Narcotic
agonist/antagonist, Ovulation
stimulant, Pituitary hormone, Platinum complex, Sex hormone, Thyroid hormone,
TNF
inhibitor (arthritis), Urinary cholinergic, Vasodilator, and Vasopressor. We
note that the
current invention is also very well suited for the incorporation of functional
excipients, such
3 0 as gum benzoin or essential oils that improve absorption of poorly-
absorbed drugs, in some
cases by inhibiting drug efflux proteins. As discussed in more detail
elsewhere herein, there
-57-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
are a number of sites within, and at the surface of the particles, where
actives, excipients, and
functional excipients can be localized within the context of this invention.
Other examples of uses of coated particles of the present invention include:
1. Paints and inks. Including Microencapsulation of pigments; Cationic
charging of pigments
(where pH-dependence can be important); Fillers and texturizing agents for non-
aqueous
paints;
2. Paper. Including Microcapsular opacifiers (also in paints); Pressure-
sensitive ink
microcapsules for carbonless copying paper;
3. Non-wovens. Including Additives that adhere to fibers throughout
processing;
4. Agricultural. Including controlled release of pheromones (some of which are
otherwise
volatile or environmentally unstable if not encapsulated) for insect control;
Controlled release
of insect chemosterilants and growth regulators (many of which are otherwise
environmentally unstable): Controlled release of other pesticides (with
temperature
independence being important); Controlled release of herbicides; Encapsulation
of the plant
growth regulators ethylene and acetylene (that are otherwise volatile); Taste
modifiers to
deter mammalian pests (e.g. capsaicin), Nutrient and fertilizer release;
5. Environment and forestry. Including Controlled release of aquatic
herbicides for weed
control; Controlled release of other herbicides; Controlled release of
nutrients in agriculture;
Soil treatment and nutrient release; Encapsulation and release of chelating
agents (e.g., for
2 0 heavy metal contaminants): Control of deposition and environmental fate of
actives (viz.,
through targeted release of crystal coating and/or adhesive property of cubic
phase):
Encapsulation of hygroscopic or other (e.g., urea and sodium chloride)
"seeding" agents for
meteorological control;
6. Vaccines. Including HIV gag, gag-pol transfection of cells as an example;
Adjuvants for
2 5 the proper presentation of antigens or antibodies;
7. Nuclear medicine. Including Separation of two (otherwise mutually-
destructive)
radionuclides into separate particles for treatment of cancer;
8. Cosmetics. Including Antioxidant, Antiaging skin cream: Separation of two
components of
an antiacne medication; Suntan lotions with encapsulated prostaglandins and
vitamins;
3 0 Encapsulation of fat-soluble vitamins, oxidatively sensitive vitamins,
vitamin mixes;
Encapsulation of volatile perfumes and other odorants; Encapsulated volatile
perfumes for
-58-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
scratch and sniff advertisements, Encapsulation of volatile make-up removers
or other
cosmetics for sheet formation; Encapsulated solvents for nail polish removers
(or the polish
itself ); Aerosol particles containing encapsulated hair dye; Sanitary napkins
containing
encapsulated deodorant;
9 Veterinary. Including Controlled release of volatile anti-flea compounds;
Encapsulated feed
additives for ruminants; Encapsulation of anti-microbial and insecticides in
animal
husbandry;
10. Dental. Including Controlled-release dentifrice components, particularly
hydrolytically
unstable anti-calculus compounds; Delivery of oral anti-cancer compounds
(photophyrin);
11. Polymerization catalysts in one-pot (single-package) resin systems;
12. Household Products. Including controlled-release air fresheners, perfumes;
Controlled-
release insect repellents; Laundry detergents (e.g., encapsulated proteases);
Other detergency
applications; Softeners; Fluorescent brighteners;
13. Industrial. Including encapsulation of phosphine, ethylene dibromide, etc.
volatiles for
fumigating stored products; Catalytic particles; Activated charcoal
microparticles for sorption
and purification,
14. Polymer additives. Including polymer additives for protection of
wires,paper cartons etc,
from rodents; Impact modifiers; Colorants and opacifiers; Flame retardant and
smoke
suppressants; Stabilizers; Optical brighteners; Limitations in current polymer-
based
2 0 encapsulation of additives include low melting point (during processing,
polymer-polymer
incompatibility, particle size limitations, optical clarity, etc. Some polymer
additives used for
lubrication of the polymer are based on waxes, which suffer from low melting
point, except
for certain synthetic waxes which are expensive;
15. Food and beverage processing. Including Encapsulation of (volatile)
flavors, aromas, and
2 5 oils (e.g., coconut, peppermint); Encapsulation of vegetable fats in
cattle feeds; Encapsulated
enzymes for fermentation and purification (e.g., diacetyl reductase in beer
brewing);
Encapsulation as an alternative to blanching, for improved lifetime of frozen
foods;
Microencapsulated tobacco additives (flavorings); pH-triggered buffering
agents; Removal of
impurities and decolorization using activated charcoal encapsulated in a
porous material;
3 0 16. Photographics. Including Fine-grain film with dispersions of submicron
photoreactive
particles; Faster Film due to optical clarity (and thus higher transmission)
and shorter
-59-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
diffusion times of submicron dispersion; Microencapsulation of photoprocessing
agents;
17. Explosives and propellants. Including both liquid and solid propellants
and explosives are
used in encapsulated form; also, water is used in encapsulated form as a
temperature
moderator in solid propellants;
18. Research. Including Microcapsule-packed columns in extractions and
separations;
Biochemical assays, particularly, in pharmaceutical research and screening;
19. Diagnostics. Including encapsulated markers for angiography and
radiography and clinical
assays involving milieu-sensitive proteins and glycolipids; indeed, particles
incorporating
certain radiopaque or optically dense materials could themselves be used for
imaging, and
when coupled to targeting compounds as described herein could target specific
sites in the
body and allow their visualization.
Desirable triggers for commencing the release of active agents, or
alternatively
commencing absorption, are:
I. Release is by dissolution or disruption of the coating
A. Intensive variable
1. pH
2. Ionic strength
3. Pressure
4. Temperature
B. Extensive variable or other
1. Dilution
2. Surfactant action
3. Enzymatic activity
4. Chemical reaction (non-enzymatic)
2 5 5. Complexation with target compound
6. Electric current
7. Irradiation
8. Time (i.e. slow dissolution)
9. Shear (critical shear rate effective)
3 0 II. Release or absorption is via pores in the coating.,circumventing the
need for
dissolution or disruption of the coating
-60-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
1. Selective by pore size vs. compound size
2. Selective by pore wall polarity vs. compound polarity
3. Selective by pore wall ionicity vs. compound ionicity
4. Selective by pore shape vs. compound shape
5. Selective by virtue of the fact that some compounds or ions form porous
inclusion compounds with the coating, whereas others do not (although this is
generally a combination of the above 4 effects).
Methods for makin~particles of the invention.
In a preferred embodiment, the coated particle may be made by
1. providing a volume of the matrix that includes at least one chemical
species having
a moiety capable of forming a nonlamellar material upon reaction with a second
moiety and
2. contacting the volume with a fluid containing at least one chemical species
having
the second moiety under nonlamellar solid material-forming conditions so as to
react the first
moiety with the second moiety and subdividing the volume into particles by the
application of
energy to the volume, or performing this subdivision into particles before,
and/or after, the
chemical reaction.
Alternatively, the coated particle can be made by one of the following
processes:
providing a volume of the matrix that includes a material in solution in it
that
is capable of forming a nonlamellar material that is insoluble in the matrix
and causing the
2 0 aforesaid material to become insoluble in the matrix and subdividing the
volume into
particles by the application of energy to the volume;
dispersing particles of said matrix into a fluid that includes at least one
chemical species having a moiety capable of forming a nonlamellar material
upon reaction or
association with a second moiety and adding to said dispersion at least one
chemical species
2 5 having said second moiety to react said first moiety with said second
moiety;
dispersing particles of said matrix into a fluid that includes at least one
chemical species having a moiety capable of forming a nonlamellar material
upon reaction or
association with a second moiety, adding to said dispersion at least one
chemical species
having said second moiety to react said first moiety with said second moiety,
and subdividing
3 0 the resulting material into particles by the application of energy to said
material;
-61-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
dispersing a volume of said matrix in a form of said nonlamellar material
selected from the group consisting of liquefied form, solution, or fluid
precursor, and
solidifying said nonlamellar material by a techniques selected from the group
consisting of
cooling, evaporating a volatile solvent, or implementing a chemical reaction;
dispersing or dissolving a volume of said matrix in a liquid comprising said
nonlamellar material in solution or dispersed form and comprising also a
volatile solvent, and
spray-drying said solution or dispersion; or
applying spray-drying, electrospinning, or other comparable process to a
solution or dispersion that contains the components of both the matrix and the
coating.
Or, a combination of these processes can be used.
In a general method, a volume of the matrix is loaded with a compound A
capable of
forming a nonlamellar material on reaction with compound B, and a fluid
(typically an
aqueous solution, often referred to as the "upper solution") containing a
compound B is
overlaid on this, and the contact between compound A and compound B induces
precipitation
at the interior/exterior interface, which coupled with the application of
energy, such as
sonication, causes particles coated with the nonlamellar material to break off
into the fluid.
This method of the present invention is uniquely well-suited for producing
aqueous
dispersions of coated particles having coatings of materials with low water
solubilities, i.e.,
preferably less than about twenty (20) grams per liter of water and even more
preferably less
2 0 than about ten (10) grams per liter of water. It is highly advantageous in
these processes for
component A to be dissolved (not merely dispersed or suspended) in the matrix
before the
contact with B and sonication begins, in order to obtain a homogeneous
dispersion of
microparticles in the end. As discussed above, this is one reason (in addition
to requirements
for optimizing solublilization of actives, particularly biopharmaceuticals, in
the matrix) for
2 5 the importance of a nanostructured matrix having aqueous microdomains, in
order to allow
for the solubilization of compound A which in many cases is soluble only in
polar solvents. In
particular, reactions yielding nonlamellar organic precipitates are generally
performed most
conveniently and effectively in aqueous media, and reactions yielding
nonlamellar organic
precipitates from solubilized precursors are often most conveniently and
effectively selected
3 0 to be pH induced protonation or deprotonation reactions of soluble salt
forms of the desired
nonlamellar exterior coating material, where water (or an aqueous microdomain)
is an
-62-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
obvious medium.
Alternatively, a cool temperature, or a crystallization promoter, or electric
current
could be used to produce precipitation.
In addition to sonication, other standard emulsification methods could be used
as
energy inputs. These include microfluidization, valve homogenization
[Thornberg, E. and
Lundh, G., 1978) J. Food Sci. 43:1553] and blade stirring, etc. Desirably, a
water-soluble
surfactant, preferably an amphiphilic block copolymer of several thousand
Dalton molecular
weight, such as Pluronic F68, is added to the aqueous solution in order to
stabilize the coated
particles against aggregation as they form. If sonication is used to promote
particle formation,
this surfactant also serves to enhance the effect of sonication.
Many of the nonlamellar material-coated particles described in the Examples
were
made by a process in which two or more reactants react to form a precipitate
at the interface
between the external solution and the nanostructured liquid or liquid
crystalline phase, and
the precipitate forms the exterior coating. Another method which bears
important similarities,
as well as important differences, to this method is a general method in which
the material that
is to form the coating, call it material A, is dissolved in the liquid phase
material or liquid
crystalline phase material, with this dissolution being promoted by the change
of one or more
conditions in the material, such as an increase in temperature (but it could
be another chance
such as decrease in pressure, addition of a volatile solvent, etc.). This
change must be
2 0 reversible, so that upon reversal of the condition -- decrease of the
temperature, increase in
pressure, evaporation of solvent, etc. -- the system reverts to a two-phase
mixture of a
nanostructured liquid or liquid crystalline phase material and a nonlamellar
material A.
Energy input is applied, sometimes before the nonlamellar material A has the
time to coarsen
into large precipitates, where this may be through the application of
ultrasound, or other
2 5 emulsification methodology. This causes the breaking off of particles
coated with
nonlamellar material A.
For the case where temperature is used, the solubility of compound A in the
nanostructured liquid phase material or nanostructured liquid crystalline
phase material must
change with temperature, and the higher the magnitude of the slope of the
solubility versus
3 0 temperature plot, the smaller the temperature increment needed to perform
this process. For
example, the solubility of potassium nitrate in water is a very strong
function of temperature.
-63-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
A fundamental difference between the precipitation reaction process and this
type of process
is that in this type of process, only one compound (A) is needed in addition
to the
nanostructured interior matrix. In the precipitation reaction method, at least
two compounds
are needed, component A which is in the nanostructured phase, and component B
which starts
out in the exterior phase ("upper solution") which is overlaid on top of the
nanostructured
phase. Component B in that case is often simply a suitably chosen acidic or
basic component.
This serves to point out a similarity between the two processes, in that the
presence of
component B in the exterior phase can alternatively be thought of as a
"condition" (in
particular, pH in the acid/base case) which causes the precipitation of A,
that is, A may be
solubilized by the use of basic pH, and this is reversible by the use of
acidic pH conditions,
which are applied by the presence of the exterior phase. Probably the most
important
distinction between the two methods is whether the change in conditions that
causes the
precipitation of A occurs only when and where the exterior phase ("upper
solution") contacts
the nanostructured phase, as in the A/B precipitation reaction, or whether it
is occurring
simultaneously throughout the bulls of the nanostructured phase, as in the
temperature-
induced precipitation.
It is also possible to use a process which is a combination of the AlB
reaction process
and the temperature process described above. Typically in such a scheme, the
compound
desired as the particle coating would be added to the matrix in two chemical
forms. The first
2 0 would be the chemical fornl of the final coating, typically the free acid
(free base) form of a
compound, which would be soluble only at elevated temperature and insoluble in
the matrix
at the temperature of particular formation. The second would be a precursor
form, typically
the salt form made by reacting the free acid with a base such as sodium
hydroxide (or reacting
the free base form with an acid such as hydrochloric acid), where this
precursor form would
2 5 be soluble in the matrix even at the temperature of particle formation.
For example, for the
case of a benzoic acid particle coating, both benzoic acid and sodium benzoate
would be
added to the matrix, where the matrix is such that is does not dissolve
benzoic acid at ambient
temperature, but does at a higher temperature. The upper solution would
contain the
necessary components) to convert the precursor form to the final coating form,
such as
3 0 hydrochloric acid in the case of sodium benzoate. Upon heating (so that
both forms are
substantially dissolved), and then cooling, overlaying the upper solution, and
sonicating or
-64-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
otherwise adding energy to the system, the formation of coated particles would
involve the
two methods of cooling-induced precipitation and reaction- mediated formation
and
precipitation of coating. This could have advantages, in terms of providing
two sources of
coating material that could result in particle coverage at an earlier stage
than with either
method separately, thus providing added protection against particle fusion
(and possibly
leading to a more uniform particle size distribution), and more efficient
particle formation
with less energy input requirement, etc. Other methods that may be used for
making coated
particles of the present invention are: A. Electrocrystallization, B. Seeding
(with
supersaturated solution in matrix, seed in exterior phase), C. Promotion (with
supersaturated
solution in one phase, crystallization promoter in the other phase), D.
Inhibition removal
(with supersaturated solution in one phase and seed in the other phase), or E.
Time method
(precipitate grows slowly from supersaturated solution in interior phase).
In order to form many of the desired exterior coatings, including nearly all
of the
inorganic ones, one of the reactants (and usually both) will inevitably be
soluble only in water
or other polar solvent. In particular, most of the salts that are used in
these precipitation
reactions will dissolve only in highly polar solvents. At the same time, in
order for a matrix
material to be dispersible in water in accordance with the present invention,
it appears to be a
highly desirable, if not an absolute, condition that the interior phase
material not be of
substantial solubility in water, otherwise some or all of the material will
dissolve in the upper
2 0 solution rather than be dispersed in it. Therefore, in order to form these
coatings, the matrix
must satisfy two conditions:
condition l: it must contain aqueous (or other polar solvent) domains: and
condition 2: it must be of low solubility in water, i.e., sufficiently low (or
with
sufficiently slow dissolution kinetics) that substantial dissolution of the
phase does not occur
2 5 during the process of particle production from the phase (typically 5 to
100 minutes to
disperse the entire material into particles), since this would substantially
reduce the yield
efficiency and could thus diminish the overall attractiveness of the method.
These two conditions are working in nearly opposite directions, and very few
systems can be
found that will satisfy both. Nanostructured liquid phase materials and liquid
crystalline phase
3 0 materials of the reversed type or the lamellar type, are several of these
very few systems. In
some cases, it will be advantageous to incorporate into the upper solution one
or more of the
-65-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
components that are in the nanostructured liquid or liquid crystalline phase,
and at times in
appreciable amounts. Indeed, there are instances when it may be advantageous
to have a
nanostructured surfactant-rich liquid phase for the upper solution. In
particular, this could
occur when the matrix phase is not in equilibrium with water (or a dilute
aqueous solution)
but is in equilibrium with another liquid or liquid crystalline phase, such as
a micellar phase
or even a low-viscosity lamellar phase. Thus, as the "fluid" referred to in
the general
description of the process given above, one could use such a phase, or such a
phase to which
additional components, such as reactant B and/or amphiphilic block copolymer
stabilizer
have been added. In this case there might well be no complications by any
incorporation of
this upper phase into the microparticles, should that occur, since the upper
phase could be
(and generally would be) chosen so as to be in equilibrium with the matrix
phase (except
possibly for exchange of the active ingredient between the two materials,
which would have
some consequences but these would often be relatively unimportant). After
formation of the
coated particles, which would originally be dispersed in this upper
"solution", through the use
of filtration or dialysis the continuous outer phase could be changed from
this to another
medium, such as water, saline, buffer, etc.
In other embodiments of the present invention, advantages can be obtained by
using a
precursor to the coating material that localizes preferentially the surface of
particles of the
nanostructured liquid or liquid crystalline matrix, and dispersing the
nanostructured liquid or
2 0 liquid crystalline phase-often with the aid of this surface-localized
precursor-prior to
converting this precursor to the actual coating material. This is especially
preferred in the
case where a surface-active precursor can be found, or when the precursor can
otherwise be
substantially localized near the surface of the dispersed particles, by a
favorable interaction
with another component (ionic pairing, hydrogen bonding, etc.), or by a non-
specific effect
2 5 such as the hydrophobic effect, or by selecting a precursor or precursor-
containing solution
with the proper surface energy. When this can be achieved, as it is in Example
41 below,
then the localization of the precursor at the particle surface can be
maintained throughout its
conversion to the coating resulting in good intimacy between the particle and
coating and
efficient use of the coating material. In Example 41, the sodium salt of N-
acetyltryptophan,
3 0 which is a surface-active compound (due to the hydrophobicity of N-
acetyltryptophan,
augmented by the polarity of the ionized carboxylate group at one end), is
used to disperse a
-66-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
cubic phase into microparticles with a particle surface that is rich in this
precursor to the final
coating material, which in this case is the zinc salt of N-acetyltryptophan.
This is a very
general approach, for example since the most useful coating materials are of
course of low
solubility in water, and thus each possesses at least one dominant hydrophobic
group, but also
has at least one polar group that allows it to have sufficient solubility or
interaction with
water in some precursor state; this is in fact tantamount to saying that it is
an amphiphile, or
even a surface-active compound, in this precursor state (or that such a state
can be found).
Other approaches for localizing the precursor at the particle surface include:
ion-pairing the
precursor to an oppositely-charged molecule that partitions strongly into the
cubic phase;
using a melted or solubilized form of the precursor such that the surface
energy of the melted
precursor or precursor solution favors its localization in between the
nanostructured phase
and the exterior phase in which the nanostructured phase is dispersed;
choosing a precursor
that has favorable interactions such as extensive hydrogen bonding with the
nanostructured
phase surface, particularly in the case where the precursor (and coating) is a
polymer, so that
it is by virtue of its high MW excluded from the interior of the
nanostructured phase particle;
invoking specific interactions such as antibody-antigen or receptor-ligand
interactions; and
using a precursor, preferably a polymer or biomacromolecule (protein, nucleic
acid,
polysaccharide, etc.) that is substantially insoluble in the nanostructured
phase but contains
hydrophobic anchor groups that partition into the nanostructured phase, where
such
2 0 hydrophobic anchors are known in the art and typically are alkanes or
cholesterol-derivatives
that are grafted onto the polymer or biomacromolecule.
As exemplified in Example 42, a related approach is one in which the matrix is
dispersed in the precursor itself. That is, the precursor forms the continuous
(exterior) phase
of a dispersion of microparticles of the matrix. Then, this precursor is
converted to the
2 5 coating material, entrapping the microparticles (if and when they remain
as nanostructured
liquid or liquid crystalline bodies) within the coating material.
In this type of approach, there is first the step of dispersing the matrix
material, with
in many cases the precursor playing a central role as a dispersant or matrix,
followed by the
step of converting the precursor to the coating material, be it by chemical
reaction (often as
3 0 simple as an acid-base reaction or formation of a complex by the
introduction of multivalent
ions, as in Example 41), cooling, evaporating a volatile solvent, or other
method. This series
-67-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
of actions can result either in a dispersion of coated microparticles, or
conglomerates of such
particles which one may want to separate by a second dispersing step (or it
can yield a
combination of conglomerates and dispersed microparticles). W both Examples 41
and 42,
macroscopic particles were the result of these two steps, and a second
dispersing step is
required if the resulting contiguous solid is to be reduced to microparticles,
as was performed
in Example 41, where the final result was a dispersion of submicron-sized,
coated
microparticles.
As discussed above, in certain embodiments of this invention the interior
matrix will
be a dehydrated variant of the desired phase, that will form the desired
nanostructured liquid
or liquid crystalline phase upon contact with a water-containing fluid. There
are three general
ways in which such a particle can be produced. One is to use a process similar
to that used in
Example 42, where a matrix or, in this case dehydrated matrix, is dispersed in
a non-aqueous
solution or melt that is, or contains, a precursor of the coating material;
upon cooling or
otherwise converting this precursor to the coating, the dehydrated matrix
would then be the
encapsulated entity. A second general method is to apply a drying process,
such as freeze-
drying, electrospinning, or preferably spray-drying, to a water-containing
dispersion of the
particles in which the coating material (or a precursor thereof) has been
dissolved or very
finely dispersed. And a third general method is to dissolve or disperse all
the components of
the coating and of the matrix, either including or excluding the water, in a
volatile solvent and
2 0 applying a drying process, again preferably spray-drying. Several of these
methods can avoid
the use of water completely, which would be important in the case of actives
(or special
excipients) that should not contact water even during production.
Incorporation of tar~eting_ roups and bioactive compounds.
The utilization of amorphous and semi-crystalline materials as exterior
coatings in the
2 5 instant invention makes it all the more practical to incorporate, in a
number of different ways,
chemicals or chemical groups that can be invoked to target particles
temporally and spatially,
for example, to target particles to specific sites in the body. Similarly,
other bioactive
compounds incorporated on or in the coating could serve important functions,
such as:
absorption enhancers such as menthol could be present so as to increase
permeability of
3 0 absorption barriers (lipid bilayers, gap junctions) prior to or
concomitant with the release of
-60-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
drug; proteins or other adsorption-modulating materials could be incorporated
that would
inhibit unfavorable binding of endogenous proteins such as albumin; adjuvants
could be
incorporated that would enhance the effect of vaccine components or other
immune
modulating materials. In general, an amorphous or semi-crystalline material
can, for
example, incorporate molecules or even submicron solids as embedded materials,
more
readily and efficiently than with crystalline materials which tend to exclude
other materials
during their crystallization--particularly when the crystallization is
performed in accordance
with the tight regulations that govern the pharmaceutical industry.
Furthermore, covalent or
ionic attachment of organic groups to polymers at their surfaces is a well-
developed art. U.S.
patents 6,344,050 and 5,484,584 (incorporated herein by reference in their
entirety) are
examples of methods known in the art for attaching molecular targets to
polymers and
microparticle coatings in particular. Antibodies, steroids, hormones, oligo-
or
polysaccharides, nucleic acids, vitamins, immunogens, and even nanoprobes are
all examples
. of a wide range of materials that could be attached to particles of the
instant invention with an
exterior phase of amorphous, semi-crystalline, or less likely crystalline,
material.
It is also within the scope of this invention to use pharmaceutical actives
themselves
as coatings, with the nanostructured interior playing one or more of several
roles: enhancing
absorption by virtue of surfactancy and/or interactions with biomembranes;
solubilizing and
then releasing absorption enhancers (e.g., gum benzoin), acids, bases,
buffers, specific ions
2 0 (e.g., manganese in the case where lectin binding is important),
modulators of protein binding
or activity, or other bioactive materials; and providing a matrix ensuring the
proper
presentation of molecular recognition sites.
While it is not always crucial for a given application to know the exact
localization (or
more precisely, the spatial probability distribution) of a targeting moiety
within or in
2 5 association with a particle, this may be an important consideration in the
design of a particle-
targeting moiety combination, and the instant invention lends itself to a good
deal of
flexibility and power in this respect. Typically, targeting moieties could be
substantially
localized at one or more of the following sites in reference to the coated
microparticle:
1) in the interior of the particle, i.e., dissolved or dispersed in the
nanostructured liquid or
3 0 liquid crystalline phase interior; this locality can offer the distinct
advantage of providing a
"biomimetic" milieu for the targeting moiety, a milieu which can comprise a
lipid bilayer as
-69-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
well as hydrophilic domains each of which can be tuned to optimize the
environment;
2) at the outer surface of the interior-particularly if there is a distinct
phase in between the
interior phase and exterior coating, such as an aqueous layer; such a location
could be
particularly advantageous for a particle that would present its targeting
moiety at the new
outer surface after release of the exterior coating;
3) adsorbed to the inner surface of the exterior coating; in this location, as
well as in the other
locations listed here, there may be a synergy between the solid shell and the
targeting moiety,
in that certain solid materials (such as aluminum-based compounds, for
example) can
sometimes act as adjuvants, to increase the effectiveness of molecules in the
body particularly
if they are meant to interact with the body's immune system;
4) embedded in the exterior coating, which as discussed above is most likely
to be achievable
if the coating is amorphous or at least semi-crystalline;
5) at the surface of the exterior coating, either adsorbed or bound via, e.g.,
covalent, ionic,
hydrogen bonding, and/or hydrophobic interactions;
6) attached to, but at a distance from, the surface of the exterior coating,
through attachment
via a flexible spacer, e.g., a polymer that is attached (e.g. by covalently
bonding) at one end to
a component of the particle (interior or exterior) and at the other end to the
targeting moiety.
Experience with other types of microparticles in the art has shown that this
is generally an
excellent approach for achieving good targeting because it preserves important
2 0 conformational and diffusional degrees of freedom that are sometimes
required for good
doclcing of a targeting moiety with a receptor or target.
It should be noted that in the important case wherein a flexible spacer
extends
between the targeting moiety and the ifztef ion nanostructured phase, it may
be possible to reap
the advantages inherent in both locations, namely, before dissolution of the
coating the
2 5 moiety would be in a biomimetic environment provided by the nanostructured
interior phase,
and then after dissolution of the coating the moiety would be tethered to the
(now uncoated)
nanostructured phase and thus relatively free from hindrance in its
interactions with receptors.
Beyond that fact that the interior phases of the instant invention are well-
suited for
solubilization of targeting moieties such as proteins, peptides, nucleic
acids, polysaccharides,
3 0 arid maintenance of their conformation, it is also important that many of
the lipids,
surfactants, and block copolymers which form the basis of many of the
embodiments in the
-70-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
instant invention lend themselves in a very natural way to modulating the
properties of these
moieties and their interactions with receptors in the body. For example, it is
known in the art
that close association between polyethylene glycol (PEG) chains and proteins
or peptides can
have a dramatic effect on stabilizing these peptides, as well as reducing
their degradation by
enzymes in the body, in many cases without negating their ability to interact
with receptors.
U.S. Patent 6,214,966 (the contents of which are hereby incorporated by
reference in entirety)
provides examples wherein PEGylation of polypeptides can enhance their
performance in the
body, including reduced immunogenicity and slower clearance. Furthermore, this
effect can
be even more dramatic when the peptide is associated with a hydrophobic chain
(or
cholesterol-like group) in conjunction with the PEG chain. U.S. Patent
6,309,633 (the
contents of which are hereby incorporated by reference in entirety) provides
examples of
peptides that show greatly increased stability, resistance to enzymes, and
oral absorption
when coupled to PEGylated hydrophobic chains or ring systems. Many of the
surfactants and
lipids referred to in this specification are PEGylated, or contain other
oligomeric or polymeric
chains that can substantially modify the fate of drugs in the body-or of
targeting moieties, as
is suggested here.
A number of compounds could potentially be used as targeting moieties in a
pharmaceutical application of particles of the instant invention. To begin
with certain lipids,
such as Lipid A, have very specific interactions with components of the immune
system, for
2 0 example, and can be incorporated into the interior phase or in association
with the coating.
Similarly, block copolymers in which one of the blocks could have targeting
potential, such
as glycogen and heparin, may be utilized. Small molecules that could be
present either in the
interior or exterior to achieve a degree of targeting include sterols, fatty
acids, gramicidin,
fragments or simulants of appropriate protein epitopes, and amino acids
including aspartic
2 5 acid, cysteine, tryptophan, leucine and others. Leucine is an example of a
compound that is
recognized and bound by a specific protein in the body (the branched-chain
amino acid
transporter). Several Examples below describe the production of particles
coated with
leucine.
The ability of the interior phases of the instant invention to provide for
solubilization
3 0 and stabilization of biomolecules, such as the targeting moieties of focus
here, has been
described above, where a number of examples of membrane proteins are given
(receptor
-71-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
proteins, such proteins as proteinase A, amyloglucosidase, enkephalinase,
dipeptidyl
peptidase IV, gamma-glutamyl transferase, galactosidase, neuraminidase, alpha-
mannosidase,
cholinesterase, arylamidase, surfactin, ferrochelatase, spiralin, penicillin-
binding proteins,
microsomal glycotransferases, kinases, bacterial outer membrane proteins, and
histocompatibility antigens), many of which could serve a targeting role if
incorporated in
particles of the instant invention. Examples of polymeric components adsorbed
to the
exterior coating that could serve as attachment points for targeting moieties,
include, for
example, stabilizing layers on the exterior, i.e., outside the exterior
coating 20 such as
polyelectrolytes or surfactant monolayers (as discussed above). The Pluronic F-
68 that is used
in a number of the Examples is one such polymeric surfactant.
In yet another embodiment of the invention, "externally-directed targeting" of
the
coated particles may be achieved. This may be accomplished by directing
particles coated
with certain magnetically responsive materials discussed above (e.g. ferric
oxide) through the
application of magnetic fields.
Antibodies are broadly useful for targeting to specific sites or molecules in
the body or
other environments, and can be incorporated at various sites in a particle as
discussed above.
In particular, intact antibodies with their more hydrophobic Fc fragment are
prone to
partitioning into matrices of the type used in this invention, and furthermore
it is well known
that antibodies can be adsorbed or attached (including covalently) to solid
surfaces with
retention of binding and binding specificity. Commercial sources supply
antibodies to, for
example, each of the following:
8-hydroxy-guanosine, AAV (adeno virus), ACHE (acetylcholinesterase), ACHER
(acetylcholine and NMDA receptor), acid phosphatase, ACTH, Actin (cardiac,
smooth
muscle, and skeletal), Actinin, Adeno-associated virus, adenosine deaminase,
Adipophilin
2 5 (adipocy differentiation related peptide), Adrenomedulin 1-6, Advanced
glycation end-
products (AGE), alanine transaminase, albumin, alcohol dehydrogenase, aldehyde
dehydrogenase, aldolase, Alfentanil AB, Alkaline Phosphatase, alpha Actinin,
Alpha-1-anti-
chymotrypsin, alpha-1-antitrypsin, alpha-2-macroglobulin, alpha-catenin, beta-
catenin and
gamma cateinin, Alpha-Fetoprotein, Alpha-fetoprotein receptor, Alpha-
Synuclein, Alzheimer
3 0 Precursor Protein 643-695(Jonas), Alz-90, Precursor Protein A4, amino acid
oxidase,
Amphetamine, amphiphysin, amylase, amylin, Amylin Peptide, Amyloid A and P,
Amyloid
_7
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
precursor protein, ANCA (Proteinase PR3), androgen receptor, Angiogenin,
Angiopoietin-1
and Angiopoietin-2 (ang-1/Ang-2), Angiotensin Converting Enzyme, Angiotensin
II Receptor
Atl and At2, Ankyrin, Apolipoprotein D, Apolipoprotein E, arginase I, B
Arrestin 1 and B
Arrestin 2, ascorbate oxidase, asparaginase, aspartate transaminase, Atpase
(p97), atrial
Natriuretic Peptide, AU1 and AUS, Bacillus Antracis (Anthrax) and Bacill,
antracis lethal
factor, Bad, BAFF, Bag-1, BAX, bcl-2, BCL-Xl, B Nerve Growth Factor, beta
Catenin,
Benzoylecognine (cocaine), beta-2 microglobulin, beta Amyloid, Galactosidase,
beta
Glucuronidase, Blood Group antigens (RhoD, Al,A2 A1,A2,A3, B, A, Rh(0)D, RhoC,
B M,
N), Blood Group H antigen, bombesin and bombesin/gastrin releasing peptide,
Bone
Morphogenetic Protein (BMP), Bone marrow stromal cell antigen, BST-3, Borrelia
burgdorferi garinii, borrelia burgdorferi sensustricto, Bovine Serum,
Bradykinin Receptor B2,
Brain derived neutrophic factor, Bromodeoxyuridine, CA 19-9, CA 125, CA 242,
CA 15-3,
CEA, Ca+ ATPase, Calbindin D-28K (Calcium binding protein), Calgranulin A,
Cadherin,
CD 144, Calcineurin, Calcitonin, Calcitonin gene related peptide, Calcium
Channel,
Caldesmon, Calmodulin, Calnexin, Calpactin light chain, Calpain, Calpastatin,
Calreticulin,
Calretinin, Calsequestrin, Cam Kinase II, Canine Distemper virus, carbonic
anhydrase I and
II, Carboxypeptidase A, B and E, Carboxypeptidase Y, Cardi, Troponin C and T,
cardiotrophin-1, Caspase 3 (CPP32), Catalase, Catenins, Caveolin 1, 2 a and 3,
CCR, CD44
(HCAM), CD56 (NCAM), CDK2, CDK4 (Cyclin Dependent Kinase C), Carcinoembryonic
2 0 Antigen, Cellular antigens, CFTR (cystic fibrosis transmembrane
conductance protein),
chemokine receptors, chlamydia, CHO cell (Chinese Hamster Ovary Cell)
Proteins, cholera
toxin, choline oxidase, Chondroitin, Chloramphenic, Acetyltransferase(CAT),
Chromogranin
A, B and C (Secreogranin III), cholesterol oxidase, Chymotrypsin, Cingulin,
Citrate
Synthethetase, C-kit/stem cell factor receptor, CK-MB, Clathrin Antigen,
Clostridium
Botulinum D Toxoid, Clusterin, C-MYC, CNS Glycoprotein 130kD, Collagen Type IV
and
Type VII, Complement Sb neoepitope, Complement C3a, C3b, CS and C9, complexin
2,
Corticoliberin (CRF), C-peptide, CRF (Corticotropin Releasing Factor),
Corticotropin
releasing factor receptor, COX-1 and Cox-2, CPP32 (also known as Caspase 3,
apopain or
Yama), Creatine transporter, C-Reactive Protein (CRP), Cryptosporidium, CXCR-
5, Cyclin
3 0 A, Cyclin D1, D2 and D3, Cyclosporine A, Cylicin I, Cytochrome B5,
Cytochrome C,
Cytochrome oxidase,~ Cytochrome P450, Cytokeratin Types I and II,
Cytomegalovirus, DAP
-73-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
I~inase, Dendritic cells, Desmin, Desmocollin 1, 2 and 3, Desmoglein 1, 2 and
3,
Desmoplakin 1 and 2, Dextranase, DHT (Dihydrotestosterone), Dihydrofolate
Reductase
(DHFR), Dioxin, Diptheria toxin, Distemper, DJ-l, DNA single-stranded, DNA
double
stranded, DNA Topoisomerase II and Phospho-topoisomerase IIa + II alpha/beta,
Dopamine,
Dopamine Beta-Hydroxylase, Dopamine Receptor, Dopamine Transporter, Drebrin,
Dysferlin, Dystrobrevin, E.Coli expression plasmid, Elastase, Elastin,
Endocrine Granu,
Constituent (EGC), Endorphin, Endothelial cell, Endothelin, Endothelin
Receptor,
Enkephalin, enterotoxin Staphylococcus aureus, Eosinophil Peroxidase,
Eosinophil derived
neurotox, (EDN), Eotaxin, Eotaxin-2, Epidermal Growth Factor, Epidermal Growth
Factor 2,
epidermal growth factor receptor, testostosterone, Epithelial Proliferating
antigen, Epithelium
Specific Antigen, c-MYC, HA.1, VSV-G Tag, Glu-Glu, EEEYMPME, Thioredoxine
(trx),
Epstein Barr virus and Epstein Barr Virus capsid antigen gp120, ERK (ERK1,
ERK2, ERK3,
pan ERIC also called MAP kinase), Erythrocytes, Erythropoietin (EPO),
Esterase, Estradiol,
Estriol, Estrogen Receptor, Estrone, Ets-1 transcription, F1 antigen Yersina
pestis, Factor 5,
Factor VII, Factor VIII, Factor 9, Factor 10, Factor 1 l, Factor 12, Factor
XIII, FAK (Focal
Adhesion Kinase), FAS (CD95), FAS-L (CD 178), Fascin, Fatty Acid Binding
Protein,
Ferritin, Fetal Hemoglobin, Fibrillin-1, Fibrinogen, Fibroblasts, Fibroblast
Growth Factor,
FGF-9, Fibronectin, Filamin, FI~BP51, FKBP65, FI~506, FLI~1, flt-1 FLt-4 and
FLT-3/FLK-
2, FLT 3 Ligand, Fluorescein (FITC), FODR1N, Folate, Folate Binding Protein,
fractalkine,
2 0 frequenin, Frizzled, Fructose-6-p-kina, FSH, Fusin (CXCR4), GABA A and
GABA B
Receptor, Galectin, galanin, gastrin, GAP-43, G-CSF, G-CSF receptor, gelsolin,
GIP (gastric
inhibitory peptide), GO-protein (bovine), GDNF, GDNF-Receptor, Giardia
intestinalis, Glial
fibrillary acidic Protein, Glial filament protein, Glucagon/Glycentin, Glucose
oxidase,
Glucose 6 Phosphate Dehydrogenase, Gluco, Tranporter GLUT 1-4, GLUT 1-5,
Glutamate
2 5 Dehydrogenase, Glutamic Acid decarboxyla (GAD), Glutathione,
Glyceraldehyde-3-
phosphate dehydrogenase GAPDH, Glycerol-3-phosphate dehydrogenase, Glycerol
kinase,
glycine transporter (GLYT1, GLYT2), Glycogen Phosphoralase Isoenzyme BB
(GPBB),
Glycophorin A (CD235a), GM-CSF, C receptor alpha, Golgi Complex, Gonadotropin-
Releasing Hormone Receptor (GnRHR), GP130, Granzyme, GRB2, GRB1, Green
3 0 Fluorescent Protein (GFP), Growth Hormone, Growth Hormone Receptor, Growth
Hormone
Releasing factor, GRP78, Hantavirus, HCG, HDL (high density lipoprotein), Heat
Shock
-74-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Protein HSP-27, HeK 293 Host Cell Proteins, Helodermin, helospectin,
Hemeoxygenase,
Hemoglobin, Heparin, Hepatitis A, Hepatitis B Core Antigen, Hepatitis B virus
surface
antigen, Hepatitis C virus, Hepatistis E virus, Hepatitis G Virus, Hepatocyte
Growth Factor,
Heregulin (Neu differentiation factor/Neuregulin), Herpes Simplex Virus,
Hexokinase,
Histamine, His Tag, 6-His vector tags, HIV-1 p24, p55/17, gp4l, gp120, tat,
nef, rev, HIV
reverse transcriptase, HLA Class I, HLA Class II, HLA-DM, HLA DQwl, HLA DRw
52,
Peroxidase, HPV 16 Late I Protein, human free kappa light chains, human lambda
light
chains, Human IgA, human I heavy chain, human IgAl, human IgD, human IgE,
human IgG
heavy chain, human IgGl, human IgG3, human IgG4, human IgM, human IgM heavy
chain,
human J chain, human kappa lig, chains, human lambda light chains, Human Serum
Amyloid
P, Human Serum Amyloid P, Interleukin 1 beta converting enzyme, ICH-1 (caspase
2),
Indian Hedgehog Protein (IHH), Influenza virus, Inhibin, Insulin, insulin like
growth factor II,
insulin growth factor binding protein 1, 2, 3, 4 or 5, insulin like growth
factor, insulin like
growth factor I receptor, insulin receptor, insuliuproinsulin, Interferon
alpha, interferon alpha
receptor, Interferon beta, Interferon gamma, interferon gamma receptor alpha
and beta,
Interleukin 1 alpha, Interleukin Receptor alpha type II, Interleukin 1-beta,
Interleukin 10,
interleukin 10 receptor, Interleukin 1 l, Tnterleukin 12, interleukin 12
receptor, Interleukin 13,
Interleukin 15, Interleukin 16, Interleukin 17, Interleukin 18, Interleukin 2,
Interleukin 2
receptor alpha, Interleukin receptor alpha chain (GD25), Interleukin 2
receptor beta,
2 0 Interleukin 2 receptor beta chain(CD 122), Interleukin 2 receptor gamma,
Interleukin 3,
Interleukin 3/interleukin 5/GM-CSF Receptor common chain, Interleukin 4,
Interleukin 5,
Interleukin 6, Interleukin 6 receptor alpha chain, Interleukin 7, Interleukin
7 receptor alpha,
Interleukin 8, Interleukin 8 receptor, Interleukin 9, invertase, Involucrin,
IP-10, Keratins,
KGF, Ki67, KOR-SA3544, Kt3 epitope tag, lactate dehydrogenase, Lactoferrin,
2 5 lactoperoxidase, Lamins, Laminin, La (SS-B), LCMV (Lymphocytic
Choriomeningitis
Virus), Legionella pneumophilia serotype, Legionella pneumophila LPS, Leptin
and Leptin
Receptor, Lewis A Antigen, LH (leutenizing Hormone), LHRH (leutenizing Hormone
Releasing), L, (leukemia Inhibitory Factor), 5-Lipoxygenase, LPS Francesella
tularensis,
luciferase, Cancer Marker (MOC-1, MOC-21, MOC-32, Moc-52), Lymphocytes,
30 lymphotactin, Lysozyme, M13, F1 Filamentous Phages, Macrophages/monocytes,
Macrophage Scaveng, Receptor, Matrix metalloproteases, M-CSF, Major Basic
Protein,
-75-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
malate dehyrogenase, Maltose Binding Protein, Mannose Receptor (macrophage),
Mannose-
6-phosphate receptor, MAP kinase antibodies (ERK, ERK, ERK2, ERK3), MASHl
(Mammalian achaete schute homolog 1 and 2), MCL-1, Mcm3, M, (MCAF), MCP-2, MCP-
3, Melanocortin Receptors (1 through 5), Met (c-met), Mineralcortocoid
Receptor
(MR/MCR), Melanoma Associated Antigen, MGMT (methylguanine-DNA-
methyltransferase), MHC Antibodies (incl. HLA DATA PACK), Milk F, Globule
Membrane,
Milk Mucin Core Antigen, MIP-1 alpha, MIP-1 beta, Mitochondrial markers,
Mitosin, MMP-
l, MM, MMP3, MMP7, MMPB, MMP-9 and MMP13 (matrix metalloproteases), MMP-
14(MT1-MM, MMP15 (MT2-MMP), MMP16(MT3-MMP) and MMP19, Morphine, motili,
Mucin related antibodies (Muc-1, muc-2, muc-3, muc-Sac), Mucin-6 glycoprotein,
Mucin-
like Glycoprotein, Mycobacterium tuberculosis, Myelin, Myelin Basic Protein,
Myeloperoxidase, MyoD, Myoglobin, Myosin, Na+ Ca+ Exchanger Protein,
Na+/K+/ATPase, Na+/K+/ATPa, NCAM (CD56), pan N-Cam, (neural cell adhesion
marker),
Nerve Growth Factor, Neu-Oncogene (c-erb B2), Neurofibrillary Tangle,
Neurofilament 70 +
200kD, Neurofilament 145Kd, neurofilament 160kd, Neurofilament 68Kd,
Neurofilament
200kd, Neurofilament 200kd, neurokin, A/substance K, neuromedin U-8 (NMU-8),
Neuromodulin, neuronal pentraxin, Neuro- Specific Enolase, Neuropeptide Y
(NPY),
Neurophysin I (oxytocin precursor), Neurophysin, (vasopressin precursor),
Neuropsin,
Neurotensin, NFKB, Nicotinic Acetylcholine Receptor, (Beta2 and Alpha 4), NMDA
2 0 receptors, N-MYC, Norepinephrine Transporter (NET), N, (Nitric Oxide
Syntase) eNos,
iNos, NT-3, NT, (neurotroph, 4), Nucleolar Helicase, Nucleolar Protein N038,
Nuclear
Protein xNopp180, Nucleoplasm, Protein AND-l, Nucleolus Organizing Region
(NOR),
Nucleolin, occludin, Oncostatin M, ORC, Ornithine Decarboxylase, Ovalbumin,
Ovarian
Carcinoma, Oxytocin, P15, P16, P2, P27, P53 Oncoprotein, p62 Protein, p97
Atpase,
membrane associated and cytosolic 42kDa inositol (1,3,4,5) tetrakisphosphate
receptor, PP44
Podocyte Protein (Synaptopodin), PAH (Polyaxomatic Hydrocarbons), PACAP
(pituitary
adenylate cyclase activating peptide), Pancreas Polpeptide (PP),
Pancreastatin, Pancreatic
Islet Cell, papain, Papillomavirus (HPV), Parainfluenza type 2 viruses,
Parathion, Parlcin,
PARP (Poly-A, Riobose Polymerase) PARP-1 aazd PARP-2, Patched-1, Patched-2,
Paxillin,
3 0 polychlorinated biphenyls, Pemphigus vulgaris (desmoglein 3), Penicillin,
penicillinase, pep-
caxboxylase, pepsin, Peptide YY, Perform and polyclonals, Perilipin,
Peripherin, Perlecan,
-76-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Petrole, Hydrocarbons (total), PPAR (peroxisome proliferation activated
receptors), P-
Glycoprotein (mufti-drug resistance), PGP9.5, Phenanthrene, Phencyclidine
(PCP),
Phenylethanolamine, methyltransferase (PNMT), Phospholamban, Phospholipase A2,
Phosphoserine, Phosphothreonine, Phosphotyrosine, Phosphothreonine-proline,
phosphothreonine-lysi, phophotyrosi, Phosphotyrosine Kinase, Pichia pastoris,
Placental
Alkaline Phosphatase, Plakoglobin, Plakophilin l, Plakophilin 2, Plakophilin
3, Plasminogen,
Platelet Derived Growth Factor AA and BB and AB, Plectin, PM, ATPase (plasma
membrane
Ca pump), Pneumocystis carinii, Pneumolysin, Polychlorobiphenyl (PCB), PP 17/
TIP47,
PPAR (peroxisome proliferation activated receptors), Prednisone, Prednisolone,
Pregnancy
associated Plasma Protein A (PAPP-A), Pregnenolone, Prepro NPY 68-97,
Presenilin-l,
Presenilin-2, Prion protein, Progesterone, Progestero, Receptor, Prohibitin,
Proinsulin,
Prolactin, Proliferation Ce, Nuclear Antigen, Proline Transporter, Prostatic
Acid Phosphatase
(PAP), Prostatic Specif, Antigen (PSA), Proteasome 265, Protein 4.1 M ascites,
Protein G,
Protein Kinase C, Pseudomonas mallei, PTH, Pulmonary Surfactant Associated
Proteins,
Puromycin, Pyruva, kinase, Rabies Virus, RAC-1 and Rac-2, RAGE (receptor for
AGE),
RANTES, RDX, RecA, Receptor for advanced glycation end products (RAGE), Red
Blood
cells, Regulatory subunit, RELM alpha and Beta (resistin like molecules),
Renin, Rennin,
Replication Protein A (RPA p32 and p70), Resistin, Respiratory syncytial virus
(RSV),
Retinoblastoma (Rb), phospho-specific RB (ser780), Ribonuclease A, RNA
Polymera, Arna3,
2 0 RNP (70KdaU1), A Protein, B Protein, RO (R052, Ro60), Rotavirus group
specific antigen,
Rubella virus structural glycoprotein E1, Ryanodine Receptor, S-100 Protein,
saccharomyces
cerevisiae, Salmonella O-antigens, Salmonel, typhimurium, Sarcosine Oxidase,
SDF-1 Alpha
and SDF-1 Beta, secretin, Selenoprotein P, Serotonin, Serotonin Receptor,
Serotonin
Transporter, Sex Hormone Binding Globulin (SHBG), SFRPS (secreted frizzled-
related
2 5 protein 5), SF21 and SF9, SIV gp120, SIV p28, Smooth muscle actin,
Somatostatin,
Staphylococcus aureus, Staphylococcus aureus enterotoxin, STATl, Stat2, Stat,
Stat4, StatS
Stat6, Stem Cell Factor (SCF) and SCFR/C-kit, Streptavidin, Streptococcus B,
Stromal Cell
Derived Factor-1 (SDF-1 alpha and beta), Substance P, Sufentanil AB,
Superoxide
Dismutase, Surfactant Associated Proteins (A,B,C,D), Symplekin, Synapsin I,
Synapsin IIa,
3 0 Synaptophysin, Synaptopodin (Podocyte Protein), Syndecan l, Synphilin-1,
Synuclein
(alpha), SV40 Large T antigen and small T antigen, Talin, TARC, TAU, Taurine
transporter,
_77_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Tenascin, Testosterone, TGF-alpha, TGF-beta, TGF beta receptor (Endoglin),
THC, Thomsen
Friedenreich Antigen (TF), THY-1 25kd Brain (CDw90), Thymocytes, Thrombin and
Thrombin Receptor, Thyroglobulin (24TG/SE6 and 24Tg/SF9), Thyroid Binding
Globulin,
Thyroid Hormone Receptors, Thyroid Peroxidase, Thyroid Stimulating Hormone
(TSH),
Tyrosine Hydroxylase, Thyrotropin Releasing Hormone (TRH), Thyroxine (T4), TIe-
1 and
TIe-2, TIMP-1, TIMP-2, TIMP-3 (Tissue Inhibitors, metalloproteinase), Titin,
TNF receptor
associated factors 1 and 2, TNF Receptor, TNF receptor II, TNF-Alpha, TNF-
Alpha, TNF-
beta, Toxoplasma gondii p30 antigen, TPO (thrombopoietin), TRAF,
Traf2,Traf3,TRAF4,TRAFS, TRAF6, Transferrin, Transferrin Receptor,
Transforming
Growth Factor A, Transformi, Growth Factor Beta, Transportin, Trepone,
pallidium,
Triiodothyronine (T3), Trinitrotoluene (TNT), TRK A, TRIG B, TRK C, Tropon,
(cardiac),
Troponin I, Troponin T, trypsin, trypsin inhibitor, trypsinogen, TSH, TUB
Gene, Tubulin
alpha and beta, Tubulin beta specific, Tumor Marker related Antibodies, Tumor
Necrosis
Factor Alpha, Tyrosinase, Tweak, (caspase-4), Ubiquitin, Ubiquitin-L1,
Uncoupling Proteins
(UGPl, UCP2, UCP3, UCP 4 and UCPS), Urease, Uricase, Urocortin, Uroplakin,
Vasopressin, Vasopressin Receptor, VEGF, Vesicular acetycholine transport,
(VACht ),
Vesicular monoamine transporter (VMAT2), Villin, Vimentin, Vinculin, VIP
(Vasoactive
Intestinal Peptide), Vitamin B12, Vitamin B12, Vitamin D metabolites, Vitamin
D3
Receptor, Von Willebrand Factor, VSV-G Epitope Tag, Wihn's tumor Protein X,
Oxida,
2 0 Yeast, hexokinase, SOD, cytochrome oxidase, carboxypeptidase, and Yersinia
eterocolotica.
Alternatively, many of the substances noted above (e.g. folate, PGP,
cytochrome P
450, and EGF) may in and of themselves be useful as targeting substances and
may be
incorporated into the particles of the present invention. In addition, other
chemical
compounds such as PEG may also be used for targeting and may be incorporated.
2 5 It is important to point out that in addition to targeting compounds per
se, active
compounds, functional excipients such as absorption enhancers, and other
bioactive materials
as gleaned from the lists of materials given herein can be incorporated in any
of these
localization sites.
In addition to the targeting of particles to specific sites for release of
drug, as
3 0 mentioned above particles incorporating certain radiopaque or optically
dense materials
could themselves be used for imaging, and when coupled to targeting compounds
as
_78_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
described herein could target specific sites in the body and allow their
visualization. As an
example, somatostatin receptors are known to be localized at certain tumor
sites, so that the
attachment of a target to coated particles as per the instant invention that
would bind
selectively to somatostatin receptors could target a tumor and allow
visualization via, e.g., x-
ray, MR imaging, or radioimaging. To extend this idea, a similarly targeted
particle could
then carry a radioactive material that would emit radiation intended to induce
necrosis of the
tumor.
Polymerized liquid crystals as interior phases.
U.S. patent 5,244,799 (the contents of which are hereby incorporated by
reference in entirety)
reports the polymerization of nanostructured cubic and hexagonal phase liquid
crystals, with
retention of their nanostructure. The retention of structure was demonstrated
by small-angle
x-ray scattering (SAXS) and transmission electron microscopy (TEM).
The possibility of polymerizing the cubic phase in the interior of a particle
of the
instant invention opens up a number of possibilities, particularly as relate
to increasing the
stability of the interior phase and modulating its interaction with the body,
and cell
membranes in particular. For an example of the latter, whereas an
unpolymerized cubic
phase might be expected to molecularly disperse when coming into contact with
a
biomembrane, polymerization of the same interior matrix might create a
particle interior that
2 0 would retain its integrity throughout its interaction with the same
biomembrane, and this
could have dramatic consequences as to the fate of the particle and to a drug
inside the
particle. Furthermore, the retention of a bilayer-bound drug (hydrophobic
small molecule,
membrane protein, etc.) might be increased tremendously by polymerization,
yielding a slow-
release particle. And the presence of a more permanent, precisely-defined pore
structure,
2 5 with precisely tunable poresize, might make possible improved controlled
release of a drug,
and/or sequestration of the drug from degradative or other enzymes by size-
exclusion from
the pores of the polymerized matrix.
The following examples illustrate the present invention but are not to be
construed as
limiting the invention.
-79-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
EXAMPLES
In the following examples, Examples 14, 15, 16, and 34 demonstrate systems
with
coatings made of physically robust mineral materials, such as cupric
ferrocyanide and calcium
phosphate, that can provide for stability of the intact particles under
stronger shear conditions,
such as during pumping of a dispersion of the coated particles, for example,
for recycling or
transport. These minerals are also of low aqueous solubility, making them of
potential interest
in applications requiring release of the particle coating by strong shear,
while at the same time
protecting against release due to simple dilution with water. An example of
such an
application would be where a rodent deterrent such as capsaicin, or rodent
toxin, would be
encapsulated in the coated particles of the present invention, the particles
impregnated into
electrical wires, corrugated boxes, and other products requiring protection
against pawing by
rodents, and the pawing action of a rodent would induce release of the active
deterrent or
toxin. The low water solubility would prevent the deterrent from premature
release due to
damp conditions.
A robust organic material that provides a coating that is also of low aqueous
solubility
is ethylhydrocupreine, as in examples 17 and 33, and this compound has the
additional
characteristic that it has an extremely bitter taste that could provide an
additional deterrent
effect in a rodent-deterent application.
Examples 1, 2, 3, 6, 7, 8, 9, 10, 17, 18, 19, 20, 23 and 33 provide examples
of
2 0 coatings that are of low water solubility at neutral pH, but that increase
substantially in
solubility as the pH becomes either acidic or basic, depending on the
compound. This can
make the coated particles of importance in, for example, drug delivery, where
a coating that
releases preferentially in a particular pH range is desired, such as for
intestinal release. Or
such a coating could release, allowing the release of an antibacterial
compound, at sites of
2 5 bacterial activity, where pH is typically acidic. Or the release of the
coating at a particular pH
could allow the release of a pH stabilizing compound or a buffer system, for
example in
microparticles designed to control the pH of water in swimming pools.
Example 4 gives an example of particles with a coating, silver iodide, that
could
provide very useful properties as a cloud-seeding agent, since the silver
iodide coating is
3 0 well-known for cloud-seeding effectiveness, and the surface area and
surface morphology
-80-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
afforded by the particle shape and size could amplify the effect of the silver
iodide. This
could be of commercial importance due to the expense of silver compounds, in
which case
the inexpensive liquid crystal interior could serve the role as a filler that
would provide the
same or greater seeding potential at a fraction of the cost of simple silver
iodide. A similar
increase in effectiveness due to amplification of surface area might prove of
interest in the use
of the particles as local anesthetics for mucous membranes, and the proper
balance of lipids
and active anesthetic hydrophobes (such as lidocaine) in the particle interior
could be used to
enhance the effect.
Example 5 demonstrates that compounds such as sulfides and oxides can be used
as
coatings in the coated particles of the present invention, even when they
require gaseous
reactants for formation. Such compounds are well-known for being not only high-
stiffness
materials, but also chemically extremely resistant, which could make such
coated particles of
interest in applications where the particles encounter harsh chemical and
physical conditions,
such as would be expected in use of the particles as polymer additives, or in
processing
involving high shear, such as impregnation of dye-containing particles in
nonwoven
materials, etc.
Examples 12 and 13 demonstrate the use of high water-solubility compounds as
coatings, that can be of importance in applications requiring quick and
convenient release of
the coating by simple dilution with water. For instance, a spray system that
would merge two
2 0 streams, one containing the dispersion and the other water, could provide
an aerosol in which
the particle coating, useful for preventing agglomeration prior to spraying,
would be dissolved
after spraying when the particles were already aerosolized -- in flight, so to
speak. Since this
dissolution could expose, for example, a nanostructured cubic phase interior
that was very
tacky, the particles could be used to adhere to, e.g., crops, or the bronchial
lining, etc. The
2 5 capsaicin loaded into the interior in Examples 12 and 13 would make this
product of potential
importance in providing rodent resistance after deposition of the tacky
aerosolized particles
onto crops, for example, since rodents are generally strongly repelled by the
taste of capsaicin
even at very low concentrations.
Examples 39 and 40 demonstrate the use of amorphous materials as exterior
coatings
3 0 for the particles of the present invention. Example 39 utilizes the
amorphous polymer
(PLGA), and Example 40 utilizes a small molecule (the sugar trehalose).
_81_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Examples 41 and 42 demonstrate variations in the processes used to create
particles,
and in particular they involve processes in which milling or particle size
reduction are applied
after formation of the coating material, in some cases in addition to
sonication or
microfluidization that is applied prior to coating material precipitation.
Examples 1 (part E), 27, 28 and 43 demonstrate the incorporation of active
targets,
including receptors, lectins, and antibodies, in particles according to the
instant invention, and
the retention of their binding capabilities.
All percentages in the following examples are weight percentages unless
otherwise
noted. The amounts of the components used in the following examples can be
varied as
desired, provided that the relative amounts remain as in the example: thus,
these amounts can
be scaled proportionately to the desired amount, recognizing of course that
scaling up to large
amounts will require larger equipment to process.
In the following examples, unless otherwise noted, the exterior coating of
each coated
particle comprises a nonlamellar material, and each interior core comprises a
matrix
consisting- essentially of at least one nanostructured liquid phase, at least
one nanostructured
liquid crystalline phase or a combination of at least one nanostructured
liquid phase and at
least one nanostructured liquid crystalline phase.
EXAMPLE 1
This Example shows that a wide range of active compounds, including compounds
of
2 0 importance in pharmaceutics and biotechnology, can be incorporated into
nonlamellar
material-coated particles of the present invention
An amount of 0.266 grains of sodium hydroxide was dissolved in 20 ml of
glycerol
using heating and stirring to aid in dissolution. An equimolar amount, namely
1.01 grams, of
methyl paraben was then dissolved, again with heating. From this solution,
0.616 grams were
2 5 taken out and mixed with 0.436 grams of lecithin and 0.173 grams of oleyl
alcohol in a test
tube. The active ingredient (or agent) identified below was incorporated at
this point and the
solution thoroughly mixed to form a nanostructured liquid crystalline phase
material with an
active ingredient disposed within it. An "upper solution", which was obtained
by dissolving
0.062 grams of Pluronic F-68 (a polvpropyleneoxide-polvethyleneoxide block
copolymer
3 0 surfactant commercially available from BASF), and 0.0132 grams of acetic
acid together and
_8~_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
adding to the test tube as a layer of solution above the previous solution
that included the
active agent. Immediately the test tube containing the liquid crystalline
mixture and the upper
solution was shaken vigorously and sonicated for three hours in a small, table-
top
ultrasonicator (Model FS6, manufactured by Fisher Scientific). The resulting
dispersion
showed a high loading of particles coated with methyl paraben with size on the
order of one
micron upon examination with an optical microscope.
Example 1 A 2.0 wt% of salicylic acid (based on the weight of the internal
core of
liquid crystalline phase material) was incorporated as an active agent.
Example 1B 2.0 wt% Vinblastine sulfate (based on the weight of the internal
core of
liquid crystalline phase material) was incorporated as an active agent.
Example 1 C 2.4 wt% Thymidine (based on the weight of the internal core of
liquid
crystalline phase material) was incorporated as an active agent.
Example 1 D 1.6 wt% Thyrotropic hormone (based on the weight of the internal
core
of liquid crystalline phase material) was incorporated as an active agent.
l 5 Example 1 E 2.9 wt % Anti 3', 5'cyclic AMP antibody (based on the weight
of the
internal core of liquid crystalline phase material) was incorporated as an
active agent.
Example 1 F 2.0 wt% L-Thyroxine (based on the weight of the internal core of
liquid
crystalline phase material) was incorporated as an active.
Particles such as these with a coating which increases substantially in
solubility as the
2 0 pH increases, could be useful in drug delivery, where the increase in pH
moving along the
gastrointestinal tract from the stomach to the intestines could result in
effective delivery to the
lower gastrointestinal tract, giving rise to a more uniform delivery rate over
time.
EXAMPLE 2
This example demonstrates the long-term stability of a dispersion of particles
of the
2 5 present invention.
The amino acid D,L-leucine, in the amount of 0.132 grams, was dissolved in
2.514
grams of 1 M hydrochloric acid, resulting in the formation of leucine
hydrochloride in
solution. The solution was dried on a hot plate under flow of air, but was not
allowed to dry
to complete dryness: drying was stopped when the weight reached 0.1666 gram,
which
3 0 corresponds to one molar equivalent addition of HCl to the leucine. An
amount of 0.130
-~3-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
grams of tlus compound were added to 0.879 grams of a nanostructured reverse
bicontinuous
cubic phase material prepared by mixing sunflower oil monoglycerides and
water,
centrifuging, and removing the excess water. An upper solution was prepared by
mixing 1.0
grams of 1 M sodium hydroxide with 3 grams of water. All water used was triply-
distilled.
The upper solution was overlaid on the cubic phase, the test tube sealed and
sonicated,
resulting in the formation of a milky-white dispersion of microparticles
coated with leucine.
A similar dispersion was prepared with the use of Pluronic F-68 as stabilizer.
An
amount of 0.152 grams of leucine hydrochloride was added to 0.852 grams of
nanostructured
reverse bicontinuous cubic phase material as above, and an upper phase
consisting of 0.08
grams of F-68, 1.0 gram of 1 M sodium hydroxide, and 3.0 grams of water was
overlaid on
the nanostrucrured reverse bicontinuous cubic phase material and sonicated.
Again, a milky-
white dispersion of leucine-coated microparticles was formed, where this time
the F-68
amphiphilic block copolymer surfactant coated the outer (leucine-based)
surface of the
particles.
As a control experiment to show the necessity of the leucine for the formation
of
crystalcoated panicles, 1.107 grams of Dimodan LS (hereinafter "sunflower
monog lycerides)
were mixed with 1.000 gram of water to form a nanostructured reverse
bicontinuous cubic
phase material. An upper solution was prepared by adding 0.08 grams of
Pluronic F-68 to
4.00 grams of water. As per the same procedure used to make the dispersions
above using
2 0 leucine, the upper solution was overlaid on the nanostructured reverse
bicontinuous cubic
phase material and the test tube sealed and sonicated. In this case,
essentially no
microparticles were formed: the nanostructured reverse bicontinuous cubic
phase material
remained as large, macroscopic chunks even after several hours of sonication
under the same
conditions as the leucine experiment.
2 5 This dispersion of the coated particles of the present invention was
examined
regularly for a period of twelve months and did not show signs of irreversible
flocculation.
With even slight agitation, it showed no signs of irreversible flocculation
over time scales of
weeks. In the absence of agitation, it did show signs of flocculation, but
upon mild shaking
for 5 seconds or more, any flocculation reversed. A droplet of the dispersion
was examined in
30 an Edge Scientific 8400 3-D) microscope at 1,000 magnification (100x
objective, oil
immersion, transmitted light) and shown to have a very high loading of
submicron particles.
-84-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Particles such as these, with relatively weak organic coatings, can be used,
for
example, in acne creams, where an active material such as triclosan could be
incorporated and
the shear associated with applying the material to the skin would release the
coating.
EXAMPLE 3
In this example paclitaxel was incorporated at the level of 0.5% of the
internal core.
The particle coating was leucine, which in other examples herein has been
shown to provide
longterm stability.
A paclitaxel-containing nanostructured reverse bicontinuous cubic phase
material was
produced by mixing 4 mg of paclitaxel, dissolved in 2 ml t-butanol, in a
nanostructured
reverse bicontinuous cubic phase material containing 0.280 gm lecithin, 0.091
gm of oleyl
alcohol, and 0.390 am glycerol: after evaporation of the butanol under argon,
a nanostructured
reverse bicontinuous cubic phase material formed that was viscous and
optically isotropic.
The sample was centrifuged for one hour, during which time no precipitate
appeared. Optical
isotropy was verified in a polarizing optical microscope. A leucine
hydrochloride solution in
glycerol was produced by mixing 0.241 grams of leucine, 2.573 grams of 1 M
HCI, and 0.970
grams of glycerol, after which the water and excess HCl were evaporated under
air flow on a
50°C hot plate, drying for three hours. Next, 0.882 grams of this
leucine-HCl in glycerol
solution were added to the nanostructured reverse bicontinuous cubic phase
material. The
2 0 upper solution was then prepared by adding 0.102 grams of Pluronic F-68 to
4.42 grams of an
aqueous buffer at pH 5Ø After overlaying the upper solution onto the
nanostructured reverse
bicontinuous cubic phase material, the nanostructured reverse bicontinuous
cubic phase
material was dispersed into microparticles by sonicating for 2 hours.
Particles such as these could be used for the controlled release of the
antineoplastic
2 5 agent paclitaxel.
EXAMPLE 4
In this example, the coating was silver iodide, which has the potential to
make the
particles useful in photographic processes. Silver iodide is somewhat unusual
in that, even
though it is a simple salt (with monovalent ions only), it has a very low
solubility in water.
3 0 A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing 0.509
-85-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
grams of Dimodan LS (commercially available as from Grinstedt AB and referred
to herein as
"sunflower monoglycerides"), 0.563 grams of triply-distilled water, and 0.060
grams of
sodium iodide. An upper solution was prepared by adding 0.220 grams of silver
nitrate, 0.094
grams of Pluronic F-68, and 0.008 grams of cetylpyridinium chloride to 3.01
grams of water.
A dispersion of microparticles was then produced by overlaying the upper
solution onto the
nanostructured reverse bicontinuous cubic phase material and sonicating for
one hour. The
particle coating was silver iodide, which has a low solubility in water.
EXAMPLE 5
In this example cadmium sulfide was used as the coating. It is a nonlamellar
crystalline compound that exhibits large changes in physical properties when
doped with
small amounts of other ions. This example also demonstrates that a gas, such
as hydrogen
sulfide gas, can be used in the present invention to induce crystallization
and particle
formation.
A nanostructured reverse bicontinuous cubic phase material was prepared by
thoroughly mixing 0.641 grams of Dimodan LS with 0.412 grams of water, and to
this was
added 0.058 grams of cadmium sulfate hydrate. After this, 0.039 grams of
calcium sulfide
was overlaid on the mixture, and the test tube was purged with argon gas and
capped. An
upper solution was prepaxed by adding 0.088 grams of Pluronic F-68 and 1.53
grams of
glycerol to 1.51 grams of 1M HCl and then sparging the solution with argon.
The upper
2 0 solution was taken up in a syringe, and added to the first test tube. Upon
addition, the smell of
hydrogen sulfide gas could be detected in the test tube, as well as the
formation of a yellowish
precipitate: this indicated the action of hydrogen sulfide gas in producing
cadmium sulfide
(CdS) from the cadmium sulfate. The system was sonicated, resulting in a
dispersion of
microparticles which had a cadmium sulfide coating.
2 5 EXAMPLE 6
This example demonstrates that the interior is substantially protected from
contact
with conditions outside the paxticle by the crystalline coating, which here is
leucine. Any
contact with zinc dust chances methylene blue to colorless in less than one
second; here,
addition of zinc did not cause a loss of color for some 24 hours. Although
there was an
-86-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
eventual loss of color, that loss is believed to be due simply to the effect
of the zinc on the
leucine coating.
A solution of leucine hydrochloride in water was made by mixing 0.122 grams of
leucine with 1.179 grams of 1M HCl and evaporating until approximately 1 gram
of solution
remained. To this was added 0.922 grams of sunflower monoglycerides, and 10
drops of a
strongly colored aqueous solution of methylene blue. An upper solution was
produced by
adding 0.497 grams of 1M NaOH and 0.037 grams of Pluronic F-68 to 3.00 grams
of pH 5
buffer. The upper solution was overlaid, the system sonicated, and a
dispersion of
microparticles formed. An aliquot of the dispersion was filtered to remove any
undispersed
liquid crystal, and 0.1 grams of 100 mesh zinc dust added. (When zinc dust is
shaken with a
solution of methylene blue, the reducing effect of the zinc removes the blue
color, normally in
a matter of a second, or almost instantaneously.) However, in the case of the
microencapsulated methylene blue produced by this process, it took on the
order of 24 hours
for the color to disappear, finally resulting in a white dispersion. Thus,
despite interactions
between the zinc and the leucine that can disrupt the coatings of these
particles, the coatings
provided substantial protection of the methylene blue against the effect of
the zinc, increasing
the time required for zinc reduction of the dye some 4-5 orders of magnitude.
If particles such as these are employed in a product in which two active
ingredients
must be sequestered from contact with each other (such as the oxidation-
sensitive
2 0 antibacterial compound triclosan and the strongly oxidizing cleansing
agent benzoyl
peroxide), this experiment demonstrates the feasibility of using leucine-
coated particles in
preventing contact between an encapsulated compound and the environment
outside the
particle.
EXAMPLE 7
2 5 In this example a leucine coating protects the methylene blue dye in the
particle
interior from contact with ferrous chloride, as easily seen by the absence of
the expected color
change when ferrous chloride is added to the dispersion. This indicated that
the coating was
substantially impermeable even to ions.
A solution of leucine hydrochloride in glycerol was made by mixing 0.242 grams
of
3 0 leucine, 2.60 grams of 1M HCI, and 1.04 grams of glycerol, and then drying
on a 50°C hot
_87_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
plate under flow of air for 1.5 hours. A nanostructured reverse bicontinuous
cubic phase
material was prepared by mixing this leucine-HCl solution, 0.291 agams of
lecithin (Epikuron
200, from Lucas-Meyer), 0.116 grams of oleyl alcohol, and 0.873 grams of
glycerol; this was
colored by the addition of a pinch of methylene blue. An upper solution was
prepared by
adding 0.042 grams of Pluronic F-68 surfactant to 4.36 grams of pH 5 buffer,
overlaid on the
nanostructured reverse bicontinuous cubic phase material, and the system
sonicated to
produce a dispersion of microparticles. To an aliquot of this dispersion was
added 0.19 grams
of ferrous chloride, a reducing agent. The absence of a color change indicated
that the
methylene blue was protected against contact with the ferrous compound by
encapsulation in
the leucine-coated particles, since the addition of ferrous chloride to
methylene blue solutions
normally changes the color to blue-green (turquoise).
Similarly to Example 6, this experiment shows that encapsulated compounds such
as
methylene blue which are sensitive to, in this case, reducing agents, can be
protected against
reducing conditions outside the particle until release of the coating. This
could be useful in,
for example, electrochemical applications where the effect of application of
electrical current
would be gated by the chemical release of the coating.
EXAMPLE 8
This example, when considered along with Example lA and Example 10,
demonstrates that particles of the present invention coated with methyl
paraben can be
2 0 produced in two entirely different ways: either by a thermal process, such
as a heating-cooling
method, or by a chemical reaction, such as an acid-base method.
To a nanostructured reverse bicontinuous cubic phase material, produced by
mixing
0.426 grams of sunflower monoglycerides (Dimodan LS) with 0.206 grams of
acidic water at
pH 3, were added 0.051 grams of methyl paraben and a trace of methylene blue
dye. The
2 5 mixture was heated to 110 °C, shaken and put on a vibro-mixer, and
plunged into 23°C water
for 5 minutes. Two milliliters of a 2% Pluronic F-68 solution, acidified to pH
3 with HCl
were overlaid, the test tube scaled with a twist cap, and the tube shaken and
then sonicated for
30 minutes. This produced a dispersion of microparticles coated with
methylparaben.
Since this experiment alone with Example 10 demonstrate that particles coated
with
3 0 the same compound,n this case methyl paraben, can be produced either by a
thermal method
_88_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
or by a chemical precipitation method, this provides an extra degree of
versatility which can
be important in optimizing production efficiency and minimizing costs, for
example in large-
scale pharmaceutical production of microencapsulated drugs.
EXAMPLE 9
The nanostructured reverse bicontinuous cubic phase material in this example
is based
on nonionic surfactants, which are generally approved for drug formulation,
and which yield
liquid crystalline phase materials with properties tunable by small
temperature changes. For
example, in an acne cream this could be used to achieve detergent (cleansing)
properties at
the temperature of application, but insolubility at the temperature of
formulation.
l 0 Furthermore, since it is based on a tuned mixture of two surfactants, and
since the phase and
properties thereof depend sensitively on the ratio of the two surfactants,
this provides a
convenient and powerful means to control the properties of the internal core.
In addition, this
example resulted in a transparent dispersion. This is noteworthy because even
a small
fraction of particles with a size larger than about 0.5 microns gives rise to
an opaque
dispersion.
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
0.276 grams of "OE2" (an ethoxylated alcohol surfactant commercially available
as
"Ameroxol OE-2", supplied by Amerchol, a division of CPC International, Inc.)
with 0.238
grams of "OES" (an ethoxylated alcohol surfactant commercially available as
"Ameroxol OE-
2 0 2", supplied by Amerchol, a division of CPC International, Inc.), and
adding 0.250 grams of
water (includes excess water). To this was added 0.054 grams of methyl paraben
and a trace
of methylene blue dye. The mixture was heated to 110° C, shaken and put
on a vibro-mixer,
and plunged into 23 C water for 5 minutes. Two milliliters of a 2% Pluronic F-
68 solution,
acidified to pH 3 with HCI, were overlaid, the test tube sealed with a twist
cap, and the tube
2 5 shaken and then sonicated for 30 minutes. This produced a dispersion of
microparticles
coated with methylparaben. Interestingly, the sub-micron size of the particles
resulted in a
transparent dispersion.
EXAMPLE 10
This example shows that methyl paraben-coated particles can be created by a
heating-
_8g_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
cooling process, in addition to the acid-base method of the previous example.
This example
also demonstrates that a mixture of two phases can be dispersed.
Lecithin (Epikuron 200, 0.418 grams) was mixed with 0.234 grams of oleyl
alcohol
and 0.461 grams of acidic water at pH 3, resulting in a mixture of
nanostructured reverse
bicontinuous cubic phase material and nanostructured reversed hexagonal phase
material.
Out of this was taken 0.50 grams, to which were added 0.049 grams of methyl
paraben, and
mixed well. This was heated to 120°C, stirred while hot, then reheated
to 120°C. The test
tube was removed from the oven, and the test tube plunged into cold water for
5 minutes.
After this the twist-cap was taken off, two milliliters of a 2% Pluronic F-68
solution, acidified
to pH 3 with HCI, were overlaid, and the sample stirred, shaken, and finally
sonicated. This
resulted in a milky-white dispersion of microparticles coated with methyl
paraben.
Examination in an optical microscope showed microparticles with sizes in the
range of 2-10
microns. Excess methyl paraben crystalline material was also seen.
This example demonstrates that a mixture of two co-existing nanostructured
phases
can provide the interior of the microparticles. This could be important in,
for example,
controlled-release drug delivery, where a mixture of two phases, each loaded
with drug, could
be used to achieve a desired pharmacokinetics: for example, with a mixture of
a reversed
hexagonal phase and a cubic phase, the release from these two phases follows
different
kinetics, due to the different geometry of the porespaces, and the resulting
kinetics would be a
2 0 combination of these two profiles.
EXAMPLE 11
This example shows that water-free particle interiors can be produced, such as
for
protection of water-sensitive compounds.
The same procedure used in the preparation of Example 10 was used, but the
water
2 5 was replaced by glycerol (which was present in excess) in the preparation
of the
nanostructured bicontinuous reversed cubic phase liquid crystalline material.
The amounts
were: lecithin 0.418 grams, oleyl alcohol 0.152 grams, glycerol 0.458 grams,
and methyl
paxaben 0.052 grams. The result was a milky-wlute dispersion of micropaxticles
coated with
methyl paraben.
3 0 The protection of water-sensitive active compounds is important in, for
example, oral
-90-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
health care products incorporating actives that are hydrolytically unstable.
EXAMPLE 12
In this example capsaicin was incorporated in particles coated with potassium
nitrate,
and where the nanostructured reverse bicontinuous cubic phase material is
based on
extremely inexpensive surfactants. The coating is easily removed by simply
adding water --
such as in a crop-spraying gun which merges a stream of the dispersion with a
stream of
water, as it aerosolizes the liquid into droplets. Note that the potassium
nitrate would serve a
dual purpose as a fertilizer.
The nonionic surfactants "OE2" (0.597 grams)'and "OES" (0.402 grams) were
mixed
with 0.624 grams of water which had been saturated with potassium nitrate. To
this mixture
the active compound capsaicin (in pure crystalline form, obtained from Snyder
Seed
Corporation) was added in the amount of 0.045 grams. Next, 0.552 grams of this
mixture
were removed, 0.135 grams of potassium nitrate were added, and the complete
mixture
heated to 80°C for 5 minutes. An upper solution was prepared by taking
a 2% aqueous
solution of Pluronic F-68 and saturating it with potassium nitrate. The melted
mixture was
shaken to mix it, then put back in the 80°C oven for 2 minutes. The
test tube was the plunged
in 20°C water for 5 minutes, at which point the upper solution was
overlaid, and the entire
mix stirred with a spatula, capped, shaken, and sonicated. The result was a
dispersion of
microparticles coated with potassium nitrate, and containing the active
ingredient capsaicin in
2 0 the interior.
When the dispersion was diluted with an equal volume of water, the coating was
dissolved (in accordance with the high solubility of potassium nitrate in
water at room
temperature), and this was manifested as a rapid coagulation and fusion of the
particles into
large clumps. The interior of each particle was a tacky liquid crystal, so
that in the absence of
2 5 a coating, flocculation and fusion occur.
The example that we discuss here is that of a spray that would be used on
decorative
plants and/or agricultural crops, and would deter animals from eating the
leaves. We have
succeeded in encapsulating the compound capsaicin, which is a non-toxic
compound (found
in red peppers and paprika) that causes a burning sensation in the mouth at
concentrations in
3 0 the range of a few parts per million. Capsaicin has a record of commercial
use as a deterrent
-91-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
to rodents and other animals.
Pure capsaicin was encapsulated in the cubic phase interior of particles
having a
coating of crystalline potassium nitrate -- saltpeter. The exterior solution
outside the particles
was a saturated aqueous solution of potassium nitrate, which prevents
dissolution of the
coating until it is diluted; a dilution of the dispersion with water by
approximately 1:1
resulted in nearly complete dissolution of the particle coatings. (This
dissolution was captured
on videotape and, on viewing the tape, it was clear that there was dissolution
of the coating
and subsequent fusing of the particle interiors.)
Upon dilution and subsequent dissolution of the coating, the interior of the
particle
was exposed, this being a cubic phase with the following crucial properties:
A) it was insoluble in water;
B) it was extremely tacky, adhesive; and
C) it has very high viscosity.
Together these three properties imply that the de-coated cubic phase particles
should adhere
to plant leaves, and property A means that it will not dissolve even when
rained on.
The same three properties were also crucial to the success of animal tests of
the bulk
cubic phase, used as a controlled-release paste, in the delivery of
photodynamic therapy
(PDT) pharmaceutical agents for the treatment of oral cancer.
The concentration of capsaicin achieved in the cubic phase particles was two
orders of
2 0 magnitude higher than in pharmaceutical preparations used in the treatment
of arthritis.
Higher loadings, perhaps as high as 20%, may be possible.
From the standpoint of commercialization, the components in the dispersion are
extremely inexpensive, and all are approved for use in foods, for topical
application, and the
like. In addition, potassium nitrate is a well-known fertilizer.
2 5 EXAMPLE 13
This example used capsaicin / potassium nitrate as in the previous example,
but here
the nanostructured reverse bicontinuous cubic phase material is based on
lecithin, which is an
essential compound in plant and animal life, and can be obtained cheaply. This
nanostructured reverse bicontinuous cubic phase material is also stable over a
wide
3 0 temperature range, at least to 40°C as might be encountered under
normal weather conditions.
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
Soy lecithin (Epikuron 200), in the amount of 1.150 grams, was mixed with
0.300
grams of oleyl alcohol, 1.236 grams of glycerol, and 0.407 grams of potassium
nitrate. The
active capsaicin was added to this in the amount of 0.150 grams, and the
mixture thoroughly
mixed. Next, 0.50 grams of potassium nitrate were added, and the complete
mixture heated
to 120°C for 5 minutes. An upper solution was prepared by taking a 2%
aqueous solution of
Pluronic F-68 and saturating it with potassium nitrate. The melted mixture was
stirred, then
put back in the 120°C oven for 3 minutes. The test tube was the plunged
in cold water for 5
minutes, at which point the upper solution was overlaid, and the entire mix
stirred with a
spatula, capped, shaken, and sonicated, then alternated between shaking and
sonicating for 30
cycles. The result was a dispersion of microparticles coated with potassium
nitrate, and
containing the active ingredient capsaicin in the interior at a level of
approximately 5%. Also
present were crystals of excess potassium nitrate.
The applications are similar to those of Example 12, except that the use of
lecithin in
the interior could provide for better integration of the particle interior
with the plant cell
membranes, possibly yielding better delivery.
EXAMPLE 14
In this example cupric ferrocyanide-coated particles were shown to be
resistant to
shear.
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
2 0 0.296 grams of sunflower monoglycerides (Dimodan LS) with 0.263 grams of a
10% aqueous
solution of potassium ferrocyanide. An upper solution was prepared by adding
0.021 grams
of cupric sulfate and 0.063 grams of Pluronic F-68 to 4.44 grams of water. The
upper
solution was overlaid onto the nanostructured reverse bicontinuous cubic phase
material, the
test tube sealed with a twist cap, and the system sonicated for 45 minutes.
The result was a
2 5 high concentration of microparticles, coated with cupric ferrocyanide, and
with diameters on
the order of 3 microns. This process produces microparticles without requiring
temperature
excursions, except those associated with sonicating, and these can be
circumvented by using
another form of emulsification. Furthermore, no excursions in pH were
required.
When a droplet was placed between microscope slide and cover slip for
microscopic
3 0 examination, it was found that the cupric ferrocyanide-coated particles
were fairly resistant to
-93-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
shear; when the cover slip was massaged over the dispersion, light pressure
with the fingers
did not induce any noticeable loss of shape or fusion of the particles. This
was in contrast
with, for example, particles coated with magnesium carbonate hydroxide, where
light
pressure induced a high degree of shape loss and fusing of particles. These
observations were
in accordance with the high stiffness of cupric ferrocyanide.
Particles with coatings resistant to sheax could be important in applications
requiring
pumping of the particles, where traditional polymer-coated particles are known
to suffer
lifetime limitations due to degradation of the coating with shear.
EXAMPLE 15
In this example capsaicin was incorporated at a fairly high loading, namely 9
wt %,
into the interiors of crystal-coated particles of the present invention. A
nanostructured
reversed bicontinuous cubic phase was produced by mixing 0.329 grams of
lecithin, 0.109
grams oleyl alcohol, 0.611 grams glycerol, and 0.105 grams of capsaicin
(obtained in
crystalline form as a gift from Snyder Seed Corp., Buffalo, NIA. To this cubic
phase were
added 0.046 grams of cupric sulfate. An upper solution was prepared by mixing
0.563 grams
of 10% potassium ferrocyanide aqueous solution with 2.54 grams of water. The
upper
solution was overlaid onto the cubic phase-cupric sulfate mixture, and the
tube sonicated for
two hours. The reaction that forms cupric ferrocyanide was easily evidenced by
the deep
reddish-brown color of the compound. At the end of this time, the cubic phase
was dispersed
2 0 into cupric ferrocyanide-coated particles. The coating was made of cupric
ferrocyanide, which
is a strong material and has some selective permeability to sulfate ions.
Since this coating
material is a robust crystal, as seen from Example 14, and capsaicin is
extremely unpleasant
to the taste of rodents, these particles could be useful as rodent deterrents
in preventing
damage to corrugated boxes, agricultural plants, etc., particularly where the
particles must be
2 5 resistant to mild shear (as during production of the particle-laced boxes,
or deposition of the
particles onto plants), prior to the gnawing action of rodents which would
open the
microparticles and expose the capsaicin to the animal's tastebuds.
EXAMPLE 16
In this example microparticles with a cupric ferrocyanide coating were
produced using
-94-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
the same procedure as in Example 14, but in this case an antibody was
incorporated as the
active agent. In particular, anti 3', 5' cyclic adenosine monophosphate (AMP)
antibody was
incorporated as an active agent at a loading of 1 wt% of the interior. A cubic
phase was
prepared by mixing 0.501 grams of sunflower monoglycerides with 0.523 grams of
water.
Potassium ferrocyanide, in the amount of 0.048 grams, was added to the cubic
phase, together
with approximately 0.010 grams of the antibody. Excess aqueous solution was
removed after
centrifuging. An upper solution was prepared by adding 0.032 grams of cupric
nitrate and
0.06 grams of Platonic F-68 to 3.0 grams of water. After overlaying the upper
solution and
sonicating, a milky-white dispersion of microparticles, coated with cupric
ferrocyanide, was
obtained. Such particles could be useful in a biotechnology setting such as a
bioreactor, in
which the stiff cupric ferrocyanide coating would be useful in limiting
release during mild
shear conditions encountered (for example, in a pressurized inlet), prior to
the desired release
of coating and availability of the bioreactive antibody.
EXAMPLE 17
l5 In this example ethylhydrocupreine forms an extremely hard shell. In this
example an
acid-base process was used.
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
0.648 grams of sunflower monoglycerides (Dimodan LS) with 0.704 grams of
water. To this
were added 0.084 grams of ethylhydrocupreine hydrochloride, and a trace of
methylene blue.
2 0 An upper solution was prepared by adding 1.01 grams of 0.1 M sodium
hydroxide and 0.052
grams of Platonic F-68 to 3.0 grams of water. After overlaying the upper
solution onto the
liquid crystal, the system was sonicated, resulting in a dispersion of
microparticles coated
with ethylhydrocupreine (free base). Most of the particles were less than a
micron in size,
when examined with optical microscopy.
2 5 Particles which maintain integrity with dessication could be useful in,
for example,
slow-release of agricultural actives (herbicides, pheromones, pesticides,
etc.), where dry
weather conditions could cause premature release of less resistant particles.
EXAMPLE 18
In this example leucine-coated particles were created by a heating-cooling
protocol.
-95-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
1.51 grams of sunflower monoglycerides (Dimodan LS) with 0.723 grams of water.
To 0.52
grams of the nanostructured reverse bicontinuous cubic phase material taken
from this
mixture were added 0.048 grams of DL-leucine. The mixture was stirred well and
heated to
80° C, then cooled to room temperature by plunging in water.
Immediately a 2% solution of
Platonic F-68 in water was overlaid, the mixture shaken, and then sonicated.
This resulted in
a milky dispersion of microparticles coated with leucine.
The ability to make the same coating (in this case leucine) by either a
thermal method
or an acid-base method provides important flexibility in production, since,
for example,
certain actives (proteins, for example) are very easily denatured with
temperature but can be
quite resistant to pH, whereas other compounds can be resistant to temperature
but can
hydrolyze at acidic or basic pH.
EXAMPLE 19
This example shows that interior components can be protected from contact with
oxygen, even when oxygen was bubbled into the exterior medium (here water).
A nanostructured reverse bicontinuous cubic phase material (with excess water)
was
prepared by mixing 2.542 grams of sunflower monoglycerides with 2.667 grams of
water.
From this, 0.60 grams of nanostructured reverse bicontinuous cubic phase
material were
removed. Next, 0.037 grams of DL-leucine and 0.497 grams of 1M HCl were mixed
and
2 0 dried, after which 0.102 grams of water were added, to yield a solution of
leucine
hydrochloride, which was added to the 0.60 grams of nanostructured reverse
bicontinuous
cubic phase material, along with a trace of methyl red dye. The nanostructured
reverse
bicontinuous cubic phase material was a strong yellow color, but when spread
out as a film it
turned crimson-red in about 3 minutes, due to oxidation. An upper solution was
prepared by
mixing 0.511 grams of 1M sodium hydroxide, 0.013 grams of Platonic F-68, and
2.435 of
water. A dispersion of leucine-coated, methyl red-containing microparticles
was prepared by
overlaying the upper solution onto the liquid crystal and sonicating. It was
first checked that
a solution of methyl red in water, with or without F-68 added, quickly changes
from yellow to
crimson-red when air was bubbled through. Then, when air was bubbled through
the
3 0 dispersion of methyl red-containing microparticles, it was found that the
color did not change
-96-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
from yellow, thus demonstrating that the encapsulation of the methyl red
inside the
microparticles protected the methyl red against oxidation.
Particles such as these which are able to protect the active compound from
contact
with oxygen could be useful in protecting oxygen-sensitive compounds, such as
iron dietary
supplements for example, during long storage.
EXAMPLE 20
In this example the water substitute glycerol was used both in the interior
nanostructured reverse bicontinuous cubic phase material, and as the exterior
(continuous)
coating, thus substantially excluding water from the dispersion.
A dispersion of microparticles was prepared using glycerol instead of water,
by
mixing soy lecithin and oleyl alcohol in the ratio 2.4:1, then adding excess
glycerol and
mixing and centrifuging. An amount of 0.70 grams of this nanostructured
reverse
bicontinuous cubic phase material was mixed with 0.081 grams of methyl
paraben. An upper
solution was prepared by adding cetylpyridinium bromide to glycerol at the
level of 2%. The
nanostructured reverse bicontinuous cubic phase material-methyl paraben
mixture was sealed
and heated to 120°C, mixed well, reheated to 120°C, and then
plunged into cold water, at
which point the upper solution was overlaid and the test tube re-sealed (with
a twist-cap) and
sonicated. This resulted in microparticles, coated with methyl paraben, in a
glycerol
continuous phase. Such a glycerol-based dispersion is of interest in the
microencapsulation
2 0 of water-sensitive actives.
Using microparticle dispersions such as these, hydrolytically unstable
actives, which
are encountered in a wide range of applications, can be protected against
contact with water
even after release of the coating.
EXAMPLE 21
2 5 Similar to Example 6 above, where zinc is used to challenge encapsulated
methylene
blue, but here the coating is potassium nitrate. In addition, the same
dispersion is also
subjected to challenge by potassium dichromate.
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
0.667 grams of soy lecithin, 0.343 grams of oleyl alcohol, 0.738 grams of
glycerol, and a trace
-97-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
of methylene blue. To 0.469 grams of the equilibrated phase was added 0.225
grams of
potassium nitrate. An upper solution was prepared by adding 2% Pluronic F-68
to a saturated
aqueous solution of potassium nitrate. This was overlaid onto the liquid
crystal, and the
system sonicated until the liquid crystal was dispersed into microparticles,
coated with
potassium nitrate. The color of the dispersion was light blue. Two tests were
then used to
show that the methylene blue was protected by encapsulation in the
microparticles. To
approximately 1 ml of this dispersion was added approximately 0.1 grams of
finely powdered
zinc; when powdered zinc contacts methylene blue in solution, it causes a loss
of color. After
shaking, the mixture was centrifuged very briefly, with about 10 seconds total
time loading
into the centrifuge, centrifuging, and removing from the centrifuge; this was
done to avoid
interference from the zinc in determining the color of the methylene blue-
containing particles.
It was found that there was very little, if any, decrease in blue color from
the treatment with
zinc, showing that the microparticle coating protected the methylene blue from
contact with
the zinc. Then, potassium dichromate was added to another aliquot of the
original light-blue
dispersion. This changed the color to a greenish color, with no hint of the
purplish-brown
that results if methylene blue in solution were contacted with potassium
dichromate.
Coated particles of this Example feature an extremely cost-effective coating
material,
potassium nitrate, and yet protect active compounds against chemical
degradation from
outside conditions, making them of potential importance in, for example,
agricultural slow-
2 0 release.
EXAMPLE 22
This provides an example of microparticles with a permselective coating of a
inclusion compound. This particular inclusion compound, a so-called Werner
complex, has
the property that the porosity remains when the guest molecule is removed.
Clathrate and
2 5 inclusion compound coatings are of interest as coatings of selective
porosity, where
selectivity for release or absorption can be based on molecular size, shape,
and/or polarity.
A nanostructured reverse bicontinuous cubic phase material was first prepared
by
mixing 0.525 grams of sunflower monoglycerides and 0.400 grams of water. To
this were
added 0.039 grams of manganese chloride (MnCl2) and 0.032 grams of sodium
thiocyanate.
3 0 An upper solution was prepared by adding 0.147 grams of 4-picoline (4-
methylpyridine) to
_98_
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
3.0 ml of a 2% aqueous solution of Pluronic F-68. The upper solution was
overlaid on the
liquid crystal mixture, and the test tube sealed and sonicated. The
nanostructured reverse
bicontinuous cubic phase material was thus dispersed into microparticles
coated with the
manganese form of the Werner complex, namely Mn(NCS)2(4-MePy)4.
The coating in this example may find use in the removal of heavy metals from
industrial streams. In this case the coating can be a porous crystal -- known
as a clathrate --
which permits atomic ions to pass across the coating and into the cubic phase
interior, which
is an extremely high-capacity absorbent for ions due to the high surface
charge density (using
an anionic surfactant, or more selective chelating groups such as bipyridinium
groups, etc.).
Most likely permanent pores would be the best. The selectivity afforded by the
clathrate
coating circumvents the reduction in sorbent power that is inevitable with
traditional sorbents
(such as activated carbon and macroreticular polymers), due to larger
compounds that
compete with the target heavy metal ions for the available adsorption sites.
Regeneration of
the sorbent could be by ion-exchange, while keeping the particles and coatings
intact (this
latter step would, incidently, be an example of release).
EXAMPLE 23
In this example coated particles with an outer coating comprising methyl
paraben and
having a special dye disposed in the nanostructured reverse bicontinuous cubic
phase material
were challenged with a cyanide compound, which would cause a color change in
the event of
2 0 contact with the dye. Since the cyanide ion is extremely small, the
success of this test shows
that the coating is impervious even to very small ions.
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
0.424 grams of sunflower monoglycerides and 0.272 grams of water. To this were
added
0.061 grams of methyl paraben and a trace of the dye 1,2-pyridylazo-2-
naphthol. An upper
2 5 solution of 1 % cetylpyridinium bromide was prepared. The liquid crystal
was heated in a
120°C oven for five minutes, stirred vigorously, reheated, then plunged
into cold water, and
which time the upper solution was overlaid, the test tube sealed, and put in a
sonicator. The
result was a dispersion of methyl paraben-coated microparticles, with average
size on the
order of 1 micron. Cuprous cyanide was then used to demonstrate that the dye
was protected
3 0 from contact with the exterior phase. When cuprous cyanide was added to a
solution of 1,2-
-99-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
pyridylazo-2-naphthol (whether in the presence of F-68; or not), the color
changes from
orange to strong purple. However, when cuprous cyanide was added to an aliquot
of the
dispersion of dye-containing particles, there was no color change, showing
that the dye was
protected from contact with the cuprous cyanide by the methyl paraben coating.
One can
calculate that the diffusion time of a cuprous ion into the center of a 1
micron particle is on
the order of a few seconds or less, which would not have prevented the color
change had the
coating not sealed off the particle.
The protection of active compounds from contact with ions from the outside
environment could be useful in, for example, drug delivery, in particular in
delivery of a
polyelectrolyte which could be complexed and inactivated by contact with
multivalent ions.
EXAMPLE 24
In this example the cyanide ion test of the previous example was repeated for
potassium nitrate-coated particles.
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
0.434 grams of sunflower monoglycerides and 0.215 grams of water. To this were
added
0.158 grams of potassium nitrate and a trace of the dye 1,2-pyridylazo-2-
naphthol. An upper
solution of 1% cetylpyridinium bromide in saturated aqueous potassium nitrate
was prepared.
The liquid crystal was heated in a 120°C oven for five minutes, stirred
vigorously, reheated,
then plunged into cold water, at which time the upper solution was overlaid,
the test tube
2 0 sealed, and put in a sonicator. The result was a dispersion of potassium
nitrate-coated
microparticles. When cuprous cyanide was added to an aliquot of the dispersion
of dye-
containing particles, there was only a slight change of color, showing that
the dye was
substantially protected from contact with the cuprous cyanide by the potassium
nitrate
coating.
2 5 The utility of these particles is similar to those in Example 23, but the
cost-effective
coating potassium nitrate was used in this Example.
EXAMPLE 25
A nanostructured reverse bicontinuous cubic phase material was prepared by
mixing
3 0 0.913 grams of soy lecithin (Epikuron 200), 0.430 grams of oleyl alcohol,
and 0.90 grams of
-100-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
glycerol (excess glycerol). After mixing thoroughly and centrifuging, 0.50
grams of the
nanostructured reverse bicontinuous cubic phase material were removed and
0.050 grams of
dibasic sodium phosphate added. An upper solution was prepared by adding 0.10
grams of
calcium chloride to 3 ml of an aqueous solution containing 2% Pluronic F-68
and 1%
cetylpyridinium bromide. After overlaying the upper solution on the liquid
crystal - sodium
phosphate mixture, the test tube was sealed and sonicated. The result was a
dispersion of
microparticles coated with a calcium phosphate. Calcium phosphate coatings
were of
inherent interest in biological contexts since calcium phosphates were a major
component of
bone, teeth, and other structural components.
EXAMPLE 26
This example shows that the magnesium carbonate-coated particles in the
example
retain their integrity upon dessication, that is, when the exterior water
phase was dried off.
Thus, dry powders can be produced while retaining the interior as a water-rich
liquid
crystalline phase material.
"Tong-sorbitol compound" preparation. Initially, a"tong-sorbitol compound" was
prepared as follows:
An amount of 110 grams of tong oil (obtained as Chinese Tung Oil from Alnor
Oil) was
combined in a reaction flask with 11.50 grams of sorbitol. The flask was
purged with argon,
sealed and heated to 170° C, and stirred magnetically. Sodium carbonate
(3.6 grams) were
2 0 added and the mixture stirred at 170° C for 1 hour. At this point,
3.4 grams of 3-chloro-1,2-
propanediol were added, and the mixture was cooled to room temperature.
Seventy-five
milliliters of the oily phase from this reaction were mixed with 300 ml of
acetone, and a white
precipitate removed after centrifugation. Next, 18 grams of water and 100 ml
of acetone were
added, the mixture centrifuged, and an oil residue on the bottom removed. Then
44 grams of
2 5 water were added, and the bottom phase again collected and discarded.
Finally, 20 grams of
water were added and this time the oily residue on the bottom collected and
dried under argon
flow. This yielded approximately 50 ml of a tong fatty acid ester of sorbitol,
which was
referred to hereinafter as "tong-sorbitol product".
Example 26A. A nanostructured reverse bicontinuous cubic phase material was
3 0 prepared by mixing 0.110 grams of the "tong-sorbitol product", 0.315 grams
of soy lecithin,
-101-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
and 0.248 grams of water, mixing thoroughly, and centrifuging. To this was
added 0.085
grams of potassium carbonate. An upper solution was then prepared by adding
0.118 grams
of Pluronic F-68 and 0.147 grams of magnesium sulfate to 5.34 grams of water.
The upper
solution was overlaid onto the liquid crystal, and the test tube sealed,
shaken, sonicated for 2
hours, and finally shaken well again. The result was a milky-white dispersion
of
microparticles coated with magnesium carbonate hydroxide. This was diluted, by
adding two
parts water to one part dispersion, in order to dissolve excess inorganic
crystalline material.
A small drop of the dispersion was spread gently onto the surface of a
microscope slide, and
allowed to dry. After ten minutes of drying, the water exterior to the
particles was almost
completely evaporated. Microscopic examination showed that the particles
nevertheless
retained their shape, and did not become amorphous blobs, as was observed if
uncoated
particles were dried in a similar manner (as the dried liquid crystalline
mixture turns to a
liquid).
Example 26B. The dispersion produced in Example 26A was heated to
40° C.
According to phase behavior determinations, at this temperature the interior
phase was a
nanostructured liquid L2 phase material. The dispersion remained milky-white,
and under the
microscope showed the retention of microparticles as well. Since this L2 phase
contains oil,
water, and surfactant (namely the lecithin), it was also a nanostructured
microemulsion.
EXAMPLE 27
2 0 In this example receptor proteins are disposed within the matrix of a
nanostructured
reverse bicontinuous cubic phase material in the internal core of magnesium
carbonate-coated
particles, then the coated particles were in turn embedded in a hydrogel. The
coating on the
particles can be used to protect the receptor protein during shipping and
storage, and then
easily removed by washing just before use. This example and Example 28 presage
the use of
2 5 coated particles of the present invention for, e.g., affinity
chromatography, using hydrogel
beads with coated particles of the present invention embedded in them.
An amount of 0.470 grams of soy lecithin (Epikuron 200) was mixed with 0.183
grams of the "tong-sorbitol product" (described above), and 0.359 grams of
water. To this
was added 0.112 grams of potassium carbonate. This was centrifuged for several
hours and
3 0 the excess aqueous phase removed. A preparation of torpedo nicotinic
acetylcholine receptor
-102-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
was prepared according to the protocol described by L. Pradier and M. G.
McNamee in
Structure and Function of Membranes (ed. P. Yeagle, 1992, pp. 1047-1106). In
this
preparation, 50 micrograms of receptor protein was contained in 50 microliters
of lipid, most
of which was dioleoylphosphatidylcholine (DOPC). (The remainder was other
membrane
lipid components, such as other phospholipids, cholesterol, etc.) This amount
of preparati~n
was added to the nanostructured reverse bicontinuous cubic phase material-
potassium
carbonate mixture, and the entire mixture stirred gently but long enough to
ensure good
mixing, as checked by the absence of birefringence. An upper solution was
prepared by
adding 0.328 grams of magnesium sulfate, 0.324 grams of Pluronic F-68, and
0.0722 grams
of cetylpyridinium bromide to 20.02 grams of water. Five grams of the upper
solution were
overlaid onto the test tube contaiung the receptor-loaded nanostructured
reverse bicontinuous
cubic phase material, and the test tube sealed, shaken, and sonicated for 2
hours. This
resulted in a dispersion of magnesium carbonate hydroxide-coated, receptor-
containing
microparticles, a substantial fraction of which were in the size range of 0.5
to 1 micron.
The microparticles were then immobilized in a polyacrylamide hydrogel.
Acrylamide
(0.296 grams), methylene-bis-acrylamide (0.024 grams, as crosslinker),
ammonium persulfate
(0.005 grams, as initiator), and tetramethylethylene diamine (TMED, 0.019
grams, as co-
initiator) were added to the dispersion, resulting in polymerization of the
acrylamide into a
crosslinlced hydrogel in less than 30 minutes. A thin slice of the hydrogel
was examined
2 0 under a microscope, and a high concentration of microparticles was seen,
just as with the
original dispersion.
The hydrogel was furthermore fragmented into bits with size approximately 30
microns. This was accomplished by pressing the hydrogel through a wire mesh
with a 40
micron mesh size.
2 5 EXAMPLE 28
In this example receptor proteins were disposed in the internal core of coated
particles of the present invention where the coating was potassium nitrate,
and the coated
particles in turn immobilized in hydrogel beads. The receptor-laden beads were
successfully
tested for binding activity in radioassays performed at UC Davis.
3 0 An amount of 0.470 grams of soy lecithin (Epikuron 200) was mixed with
0.185
-103-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
grams of the "tang-sorbitol product" (described above), and 0.368 grams of
water. To this
was added 0.198 grams of potassium nitrate, and the contents thoroughly mixed.
A
preparation of torpedo nicotinic acetylcholine receptor was prepared as
described in the
previous Example. In this preparation, per every 50 micrograms of receptor
protein was
contained in 50 microliters of lipid, most of which was dioleoylphosphatidyl-
choline
(DOPC). Fifty-five milligrams of preparation was added to the nanostructured
reverse
bicontinuous cubic phase material-potassium carbonate mixture, and the entire
mixture stirred
gently but long enough to ensure good mixing. An upper solution was prepared
by adding
0.128 grams of Platonic F-68 and 0.015 grams of cetylpyridinium bromide to
6.05 grams of
saturated aqueous potassium nitrate solution. The nanostructured reverse
bicontinuous cubic
phase material-potassium nitrate preparation was heated to 40° C to
dissolve potassium
nitrate, then plunged into 10° C water for 10 minutes. The upper
solution was overlaid onto
the test tube containing the receptor-loaded nanostructured reverse
bicontinuous cubic phase
material, and the test tube sealed, shaken, and sonicated for 2 hours. This
resulted in a
dispersion of potassium nitrate-coated, receptor-containing microparticles, a
substantial
fraction of which were in the size range of 0.3 to 1 micron.
The microparticles were then immobilized in a polyacrylamide hydrogel.
Acrylamide
(0.365 grams), methylene-bis-acrylamide (0.049 grams, as crosslinker),
ammonium persulfate
(0.072 grams of a 2% solution, as initiator), and tetramethylethylene diaxnine
(TMED, 0.011
2 0 grams, as co-initiator) were added to the dispersion, resulting in
polymerization of the
acrylamide into a crosslinked hydrogel in a matter of hours. A thin slice of
the hydrogel was
examined under a microscope, and a high concentration of microparticles was
seen (except
near the very bottom of the hydrogel), just as with the original dispersion.
The hydrogel was furthermore fragmented into bits with size approximately 30
2 5 microns. This was accomplished by pressing the hydrogel through a wire
mesh with a 40
micron mesh size. At a 40 micron bit size, one can estimate that the diffusion
time for a
small molecule into the center of a bit is on the order of a second or less,
which does not have
a significant impact on the receptor tests reported next.
Using'ZSI-labeled bungaxotoxin as the ligand, an assay of receptor binding was
3 0 performed using the nanostructured reverse bicontinuous cubic phase
material microparticle-
immobilized acetylcholine receptor system just described. The standard assay
for binding has
-104-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
been described in publications from Dr. Mark McNamee's group. The results
showed that the
nanostructured reverse bicontinuous cubic phase material microparticle-
immobilized
acetylcholine receptor system exhibited binding of the bungarotoxin at
approximately 70% of
the level measured with the standard receptor preparation, demonstrating the
retention of
protein binding properties throughout not only the immobilization procedure,
but also the
period (more than two months) that elapsed between the date on which the
sample was
prepared and the date it was tested.
In addition to demonstrating the production of particles of the instant
invention
incorporating receptor proteins capable of targeting specific sites in the
body, the foregoing
Examples 26-28 actually show the application of the particles in biochemical
assays, with a
high degree of improvement in stability over the commonly used liposomes,
which are
inconvenient due to their inherent instabilities. Such assays are important in
clinical
diagnoses, as well as in pharmaceutical drug screening.
EXAMPLE 29
As in Example 22 above, clathrate-coated particles were produced in this
example. In
this example the nanostructured reverse bicontinuous cubic phase material
interior can be
polymerized, by the effect of oxygen which can pass through the coating (the
coating
nevertheless prevents passage of water).
Lecithin extracted from Drill shrimp was obtained as Drill shrimp
2 0 phosphatidylcholine from Avanti Polar Lipids of Birmingham, Alabama. An
amount of
0.220 grams of this lecithin was mixed with 0.110 grams of "tung-sorbitol
product", 0.220
grams of water, 0.005 grams of a cobalt dryer (from the art materials supply
company
Grumbacher) containing cobalt naphthenate, and 0.30 grams of potassium
thiocyanate. This
formed a green-colored nanostructured reverse bicontinuous cubic phase
material. An upper
2 5 solution was prepared by adding 0.309 grams of manganese chloride, 0.105
grams of 4-
picoline (4-methyl pyridine), 0.113 grams of Pluronic F-68, and 0.021 grams of
cetylpyridinium bromide to 5.10 grams of water. The upper solution was
overlaid on the
nanostructured reverse bicontinuous cubic phase material, the test tube
sealed, shaken, and
sonicated, with ice water filling the sonication bath water. As the green-
color nanostructured
3 0 reverse bicontinuous cubic phase material was dispersed into
microparticles, the reaction
-105-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
caused a color change to brown. After two hours, substantially all of the
nanostructured
reverse bicontinuous cubic phase material had been dispersed into particles,
which were
mostly submicron in size. The coating was a Werner compound, which according
to the
literature has chamlels that allow the absorption of (or passage of) molecular
oxygen. The
high degree of unsaturation in the Krill lecithin, as well as that in the tung-
sorbitol product,
together with the catalytic action of the cobalt dryer, makes it possible to
polymerize this
microencapsulated nanostructured reverse bicontinuous cubic phase material by
contact with
atmospheric oxygen.
The clathrates described in this example were discussed above (Example 22).
EXAMPLE 30
In this example a nanostructured reversed hexagonal phase material was
dispersed.
A nanostructured reversed hexagonal phase material was prepared by mixing
0.369
grams of soy lecithin (Epikuron 200), 0.110 grams of sorbitan trioleate, and
0.370 grams of
glycerol. To this nanostructured reversed hexagonal phase material was added
0.054 grams
of magnesium sulfate. An upper solution was prepared by adding 0.10 grams of
potassium
carbonate, 0.10 grams of Pluronic F-68, and 0.02 grams of cetylpyridinimn
bromide to 5
grams of water. The upper solution was overlaid on the nanostructured reversed
hexagonal
phase material, and the test tube sealed, shaken and sonicated for one hour,
resulting in a
dispersion of most of the nanostructured reversed hexagonal phase material
into
2 0 microparticles coated with magnesium carbonate hydroxide.
The dimensionality of the pores (cylindrical) in the reversed hexagonal phase
provides
a unique release kinetics profile which could be useful in, for example,
controlled drug
delivery.
EXAMPLE 31
2 5 In contrast with most of the above examples, the nanostructured reversed
hexagonal
phase material that was dispersed in this example was not in equilibrium with
excess water,
although it was insoluble in water.
Soy lecithin (0.412 grams), linseed oil (0.159 grams), and glycerol (0.458
grams) were
thoroughly mixed, producing a nanostructured reversed hexagonal phase material
at room
-106-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
temperature. To this nanostructured reversed hexagonal phase material was
added 0.059
grams of magnesium sulfate. An upper solution was prepared by adding 0.10
grams of
potassium carbonate, 0.10 grams of Pluronic F-68, and 0.02 grams of
cetylpyridinium
bromide to 5 grams of water. The upper solution was overlaid on the
nanostructured reversed
hexagonal phase material, and the test tube sealed, shaken and sonicated for
30 minutes,
resulting in a dispersion of most of the nanostructured reversed hexagonal
phase material into
microparticles coated with magnesium carbonate hydroxide.
The ability to disperse nanostructured phases which are not in equilibrium
with excess
water expands the range of chemistries which can be used in the present
invention. This
versatility is especially important in demanding applications, such as drug
delivery, where a
large number of product criteria must be simultaneously satisfied.
EXAMPLE 32
In this example, the nanostructured lamellas phase material was dispersed
using a
chemical reaction process.
A nanostructured lamellas phase material was prepared by mixing 0.832 grams of
soy
lecithin (Epikuron 200) and 0.666 grams of water. To approximately 0.80 grams
of this
nanostructured lamellas phase material was added 0.057 grams of magnesium
sulfate. An
upper solution was prepared by adding 0.10 grams of potassium carbonate, 0.10
grams of
Pluronic F-68, and 0.02 grams of cetylpyridinium bromide to 5 grams of water.
The upper
2 0 solution was overlaid on the nanostructured reversed hexagonal phase
material, and the test
tube sealed, shaken and sonicated for five minutes, resulting in a dispersion
of most of the
nanostructured lamellas phase material into microparticles coated with
magnesium carbonate
hydroxide.
The particles in this Example bear a structural relationship with polymer-
encapsulated
2 5 liposomes, but do not suffer from the harsh chemical conditions used to
produce polymer-
encapsulated liposomes; the ability to produce, in a single step, lamellas
phase-interior
particles coated with a wide range of crystalline coatings, and under mild
conditions, could
make the present invention of importance in controlled release drug delivery.
-107-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
EXAMPLE 33
Preparation of free bases. Both ethylhydrocupreine and neutral red were
purchased
in the protonated hydrochloride form. In each case this salt was dissolved in
water, to which
was added aqueous sodium hydroxide in 1:1 molar ratio. The mixture of the two
aqueous
solutions produced a precipitate that was washed with water (to remove NaCI
and any
unreacted NaOH), centrifuged and then dried above the melting point of the
free base.
Preparation of nanostructured reverse bicontinuous cubic phase dispersions.
Formulation of dispersions began with the following mixture:
0.417 gm glycerol monooleate (GMO)
0.191 gm glycerol
0.044 gm ethylhydrocupreine (or, as the case may be, neutral red, both in free
base
form).
Instead of the usual monoglyceride - water nanostructured reverse bicontinuous
cubic phase
material, the monoglyceride - glycerol nanostructured reverse bicontinuous
cubic phase
material was used in these examples.
An upper solution was made by dissolving Pluronic F-68 in water to a level of
2%.
After weighing the components into a test tube and mixing with a spatula, the
sealed
(twist-cap) test tube was put in a 140° C oven for at least 20 minutes,
and the
ethylhydrocupreine (or neutral red free base) was checked to have melted. The
test tube was
2 0 then plunged into water, which was below room temperature (about
10° C) in some cases and
room temperature water in others; no difference was found in the dispersions
in the two cases.
After the sample had been in the cooling water for about 5 minutes, the
viscosity was
checked to be very high, indicating a nanostructured reverse bicontinuous
cubic phase; in
some cases the sample was observed through crossed polars for optical isotropy
(the
2 5 crystalline coating domains are much smaller than the wavelength of light,
too small to affect
the optical properties). The Pluronic upper solution was poured into the test
tube until about
half full. The tube was then shaken, by hand and with the use of a mechanical
mixer. The
solution became increasingly opaque as the bulk nanostructured reverse
bicontinuous cubic
phase material disappeared and went into dispersion.
3 0 SEM characterization. Seaming electron microscope (SEM) preparation did
not
involve any fixation technique whatsoever. A drop of dispersion was simply
placed on a
-108-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
glass slide, the water evaporated, and a thin (2 nm) coating of carbon
sputtered on to avoid
charging effects. In the sputtering apparatus, before sputtering began the
sample was
deliberately held for about 5 minutes at a vacuum of 5 x 10-4 Toil. Tlus was
done to test the
robustness of the particle coating. The SEM used was a Hitachi S-X00 field-
emission SEM,
and was operated at 25 kV.
Figure 3 shows an SEM micrograph of an ethylhydrocupreine dispersion, and
particles
in the range of about 0.5-2 micron diameter are seen (the bottom half is a l
Ox magnification
of the area boxed in the top half, so that the magnification is 500 on top and
5,000 on the
bottom). Many of the particles, remarkably, distinctly show a polyhedral
shape.
The measured particle size distribution for this sample (see the next section)
showed
that particles on the order of 0.5 - 2 microns diameter dominate in this
dispersion, and this
agrees well with the particles seen in the micrograph. One can estimate that
the thickness of
the ethylhydrocupreine coating in a 0.5 micron particle was about 10 nm, and
this was clearly
thick enough that it was able to protect the liquid components in the interior
of the particles
from evaporation in the 0.5 mTorr vacuum.
In this dispersion, the nanostructured reverse bicontinuous cubic phase
material was
loaded with lithium sulphate as a marker before dispersing, and indeed the EDX
spectra of
particles in this dispersion showed a sulfur peak. Lithium cannot be detected
by the EDX
used, and other peaks in the spectrum were attributed to the glass substrate.
2 0 Figure 4 shows an SEM micrograph of a neutral red dispersion.
Substantially all of
the particles have sizes in the range of 0.3 - 1 micron.
Particle size distribution. A Malvern 3600E laser diffraction particle sizer
was used
to measure the distribution. For each dispersion checked, a few drops were
added to the
carrier fluid (water), resulting in a large dilution of the concentration so
as to avoid multiple
2 5 scattering. The particle size was computed as the diameter of a sphere of
the same volume,
which is a good measure considering the polyhedral shape of the particles.
(See below.) The
instrument is capable of measuring particles down to at least 0.5 micron, and
data on the
distribution include contributions at least down to 0.5 microns.
The particle size distribution of a dispersion prepared with a 13:1 ratio of
3 0 GMO:ethylhydrocupreine is shown in Figure 5. In general, as the ratio of
nanostructured
reverse bicontinuous cubic phase material to crystalline coating agent
increases so does the
-109-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
particle size. The data for this dispersion show that, on a volume average
basis, 10% of the
particles have particle size less than 0.6 microns, this being represented by
the equation
D(v,0.1)=0.6 micron. The narrowness of the distribution is indicated in two
ways. First, the
D(v,0.9) and D(v,0.1) are each a factor of 2 from the (volume-weighted)
average of
D(v,0.5)=1.2 micron. And second, the "span", which gives the width of the
distribution as:
span = [D(v,0.9) - D(v,0.1)]/D(v,0.5)
is computed to be 1.4. These results indicate a fairly low degree of
agglomeration.
A narrower distribution was indicated for a dispersion with GMO:neutral red =
10:1.
The span is given as 1.1, and the (differential) particle size distribution
was easily seen to be
quite sharp, dropping off quickly above 2 micron.
A small particle size was measured for a dispersion prepared with a lower
GMO:ethylhydrocupreine ratio, with a distribution averaging 0.8 microns, and a
span of 1.2.
Thus, particle size can be controlled by the ratio of nanostructured reverse
bicontinuous cubic
phase material to crystalline coating agent, with the particle size decreasing
with decreasing
ratio.
Small-angle X-ray scattering (SAXS). This was used to verify that the interior
of
the particles in an ethylhydrocupreine dispersion was a nanostructured reverse
bicontinuous
cubic phase material. The dispersion itself-not a concentrate of the particles-
was loaded
into a 1.5 rmn x-ray capillary, which was transported to the laboratory of Dr.
Stephen Hui at
2 0 Roswell Park Cancer Center Biophysics Department. The SAXS camera was
equipped with
a rotating anode, and measurements were performed at 100kV, 40 mV power (4
kW). Data
were collected using a linear position-sensitive detector connected through an
electronics
setup to a Nucleus multichannel analyzer. The MCA has the capacity for 8,192
channels, but
only 2,048 resolution was used to increase the counts per channel. Counting
times on the
2 5 order of an hour were used because the volume fraction of nanostructured
reverse
bicontinuous cubic phase material in the dispersion (which was about 85% of
the particle
volume) was on the order of 10%. The software package "PCA" was used for
analysis of the
data.
Figure 6 shows the measured SAXS intensity versus wave vector q plot. The wave
3 0 vector q is related to the diffraction angle 8 and the wavelength ~, of x-
rays by the formula:
q = 4~ (sin 8)/~,.
-110-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
A d-spacing is calculated from the q-value of a Bragg reflection by:
d = 2~t/q.
In Figure 6, vertical lines given the exact, calculated Bragg peak positions
for a lattice
with space group Pn3m and lattice parameter 7.47 nm. This space group is well-
established
for nanostructured reverse cubic phases in the monoolein-water system,
particularly for those
which are in equilibrium with excess water. (Indeed, in nanostructured reverse
cubic phases
which are in equilibrium with excess water, the space group Pn3m almost
exclusively
appears). Lattice parameters for the monoolein-water nanostructured reverse
cubic phase
with space group Pn3m are also close to 8 nm; a more exact comparison was
impossible
because of the substitution of glycerol for water in the present case. In any
case, the lattice
type and size deduced from this SAXS scan are in exact accord with literature
data for
monoglyceride nanostructured reverse cubic phases.
In the space group Pn3m, the Miller indices (hkl) for the allowed peak
positions, and
the value of h2+k2+12, are: (110), 2; (111), 3; (200), 4; (211), 6; (220), 8;
(221), 9; (222), 12;
and higher. Looking at the data and the expected peak positions, it is clear
that the peaks at
the (110) and (222) positions are strongly supported by the data. The (111)
peak appears as a
shoulder to the (110) peak on the right side of the scan, and as a small but
discernible peak on
the left side. The (200) peak is supported at least on the right side of the
scan; this peak is
always measured to be much less intense than the (110) and (111) peaks in
monoglyceride
2 0 Pn3m phases, and in Pn3m phases in general, and this has been found to be
in accord with
theoretical amplitude calculations [Strom, P. and Anderson, D.M. (1992)
Langmuir, 8:691].
The (211 ) peak is supported by data on the left side of the scan, and the
(221 ) by data on the
right side. The absence or low intensity of peaks between the (211) and (222)
is a
consequence of the low concentration (10%) of nanostructured reverse
bicontinuous cubic
2 5 phase in the dispersion, since the intensity of diffracted x-rays varies
as the square of the
volume concentration. Despite this, the definitive peaks at the (110) and
(222) positions, and
the perfect agreement of the deduced lattice and lattice parameter with
related systems in the
literature provide strong support for the conclusion that the SAXS data
demonstrate
nanostructured reverse bicontinuous cubic phase ordering in the particle
interiors.
3 0 These particles could be useful in, for example, controlled release of
antiseptics in
oral rinses, where the solubilities of the two coatings at slightly lowered pH
(on the order of
-111-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
5) was in the right range to make delivery preferential at sites of bacterial
activity.
EXAMPLE 34
High-performance liquid chromatography (HPLC) was used to characterize the
integrity under shear and pressure of two dispersions, one chosen to have a
rigid coating --
cupric ferrocyaude -- and the other a soft, easily disrupted coating, the
latter, to act
essentially as a control, to quantify any release under pressure of the more
rigid coating. In
other words, if the concentration of marker in the two dispersions were
approximately the
same, and the release of marker in the rigid system were a small fraction,
say. x% (where x is
substantially less than 100), of the release of marker in the soft system,
then one could
conclude that only x% of the particles in the rigid system broke up under the
pressure, and the
remaining (100 - x)% remained intact during the HPLC. Indeed, this percentage
100 - x is a
lower limit; the actual percentage of intact rigid particles would be
calculated to be higher if
it were found that some fraction of the soft particles in the control had
actually remained
intact, though this possibility is remote. In any case, the calculations were
assumed to be on a
worst case scenario, by assuming that all the control particles broke up.
Preparation of the dispersions.
Example 34A. A nanostructured reverse bicontinuous cubic phase material was
prepared by mixing 0.499 grams of soy lecithin, 0.163 grams of oleyl alcohol,
0.900 grams of
glycerol, and 0.1.24 grams of capsaicin. To 0.842 grams of the nanostructured
reverse
2 0 bicontinuous cubic phase material from this system was added 0.043 grams
of sodium
cholate. An upper solution was prepared by adding 1 drop of 1M HCl to 3.00
grams of pH 5
phosphate buffer. The upper solution was overlaid onto the liquid crystalline
material, and the
test tube sealed and sonicated, resulting in a milky-white dispersion of
microparticles.
Example 34B. A nanostructured reverse bicontinuous cubic phase material was
2 5 prepared by mixing 0.329 grams of soy lecithin, 0.108 grams of oleyl
alcohol, 0.611 grams of
glycerol, and 0.105 grams of capsaicin. To this were added 0.046 grams of
cupric sulfate. An
upper solution was prepared by adding 0.563 grams of 10% potassium
ferrocyanide solution
to 2.54 grams of water. The upper solution was overlaid onto the liquid
crystal and the test
tube sealed and sonicated, resulting in a milky-white dispersion of
microparticles coated with
3 0 cupric ferrocyanide.
-112-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
The concentration of marker, namely capsaicin, was comparable in the two
samples.
The final concentration in the cupric ferrocyanide dispersion was 2.44%,
compared to 3.19%
for Example 34B -- a 30% difference, which will be accounted for in the
calculations below.
Purified capsaicin was then run in HPLC and found to have an elution time of
22
minutes (data not shown). Under these identical conditions, the two
dispersions prepared
above were run. The data for the particles of Example 34B is shown in Figure
7, and for the
cupric ferrocyanide particles in Figure 8. Tables l and 2 give the integrated
peaks
corresponding to Figures 7 and 8, respectively, as output from the HPLC
computer; sampling
rate was 5 Hz.
Clearly there is a strong peak in Figure 7 at 22 minutes elution time
(numbered peak
13 by the computer), and Table 1 gives the integrated intensity of this peak
as 3,939,401. A
much smaller peak is seen at 22 minutes in Figure 8 (numbered 10 by the
computer), and
Table 2 gives the intensity as 304.29.
If these integrated peak values are normalized according to the concentration
of
capsaicin in the two samples, namely 3,939,401 /0.0319 for the Example 34B
case and
304,929/0.0244 for the cupric ferrocyanide case, the ratio of the normalized
peak intensity for
the cupric ferrocyanide case to the Example 34B case is 0.101 -- that is, at
most 10.1 % of the
cupric ferrocyanide particles released the capsaicin marker under the HPLC
conditions.
These particles have a coating which is a mineral of low aqueous solubility,
making
2 0 them of potential utility in applications requiring release of the
particle coating by strong
shear, while at the same time protecting against release due to simple
dilution with water. An
example of such an application would be where a rodent deterrent such as
capsaicin, or rodent
toxin, would be encapsulated, the particles impregnated into electrical wires,
corrugated
boxes, and other products requiring protection against gnawing by rodents, and
the gnawing
2 5 action of a rodent would induce release of the active deterrent or toxin.
The low water
solubility would prevent the deterrent from premature release due to damp
conditions.
TABLE 1: Integrated peak intensities corresponding to Figure 7 for HPLC
analysis of
Example 34B particles containing capsaicin. Peak #13 is the main capsaicin
peak.
Peak Area
3 0 1 2914
-113-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
2 8096
3 2848
4 29466
11304
5 6 2254
7 12871
8 4955
9 124833
113828
10 11 19334
12 7302
13 3939401
14 39153
255278
15 16 755868
17 52623
18 19395
19 4899
10519
2 21 5101
0
22 1481
23 344230
24 9971
194442
2 26 89831
5
27 80603
28 105163
29 186224
194020
3 31 36805
0
32 2115
-114-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
33 23296
34 4327
35 5166
36 90236
37 62606
38 44523
39 110347
40 4391
41 1275597
1 0 42 1353000
43 238187
TABLE 2: Integrated peak intensities corresponding to Figure 8 for HPLC
analysis of cupric
ferrocyanide-coated
particles
containing
capsaicin.
Peak
#10 is
the main
capsaicin
peak.
Peak Asea
1 1681172
2 3011240
3 106006
4 2760
5 59059
2 0 6 38727
7 163539
8 44134
9 6757
10 304929
11 10466
12 141800
13 332742
14 14442
15 6996
16 15008
-115-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
17 11940
19 91446
20 250214
21 251902
22 203000
23 44658
24 110901
25 24296
26 19633
27 25527
28 15593
29 75442
30 40245
31 421437
EXAMPLE 35
A nanostructured cubic phase liquid crystal was prepared by mixing 0.77 grams
of soy
lecithin (Epikuron 200, from Lucas-Meyer), 0.285 grams of oleyl alcohol, and
0.84 grams of
glycerol, to which was added 0.11 grams of auric chloride. No heating was used
in the
equilibration of this mixture, only mechanical stirring with a spatula. An
amount 0.595 grams
2 0 of this mixture was removed and smeared along the bottom half of the inner
surface of a test
tube. An upper solution was prepared by dissolving 0.14 grams of ferrous
chloride and 0.04
grams of Pluronic F-68 in 1.74 grams of distilled water. After overlaying the
upper solution
the test tube containing the cubic phase was sonicated, resulting in a
dispersion of
microparticles coated with a gold coating. A control sample, in which the
upper solution
2 5 contained the F-68 but no ferrous chloride, was sonicated side by side
with the first sample
and did not result in a dispersion of microparticles. The reaction between
ferrous chloride and
auric chloride results in the precipitation of elemental, nonlamellar
crystalline gold, which in
the case of the first sample resulted in the creation of microparticles
covered with gold, with
cubic phase interior. A glycerol- water mixture with a density approximately
1.2 gram/cc was
3 0 then prepared by mixing 0.628 of glycerol with 0.205 grams of water, and
approximately 0.1
grams of the dispersion was added to this, and the new dispersion centrifuged.
A substantial
-116-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
fraction of the microparticles could be centrifuged to the bottom of this test
tube after
centrifuging for 3 hours, demonstrating that the density of these particles
was significantly
higher than 1.2; this was due to the presence of the gold coating, since the
density of the cubic
phase was less than 1.2 -- indeed, a portion of the cubic phase which was not
dispersed during
the time of sonication could be centrifuged out of the original dispersions as
a lower-density
band, showing that this liquid was even less dense than the original
dispersion.
Because gold is well-known for exhibiting chemical inertness- as well as good
mechanical properties when in the form of very thin films, and since it is
also approved by the
FDA for many routes of administration, gold-coated particles could be useful
in safe,
environmentally-friendly products demanding chemically and physically stable
coatings.
Furthermore, such particles could be effective in the treatment of arthritis,
by providing
greatly increased surface area of gold over other colloidal forms.
EXAMPLE 36
A nanostructured liquid phase containing the antineoplastic drug Paclitaxel
was
prepared by solubilizing 0.045 grams of Paclitaxel, 0.57 grams of eugeno1,15
grams of soy
lecithin (Epikuron 200), 0.33 grams of glycerol, and 0.06 grams of cupric
nitrate with 0.61
grams of methanol, and then evaporating off the methanol in an evaporating
dish, with
stirring during evaporation. An glycerol-rich upper solution was prepared by
dissolving 0.09
grams of potassium iodide, 0.05 grams of Pluronic F-68, 0.44 grams of water
and 1.96 grams
2 0 of glycerol. After overlaying the upper phase, the system was sonicated,
resulting in the
dispersing of Paclitaxel-containing, nanostructured liquid phase into
microparticles coated
with crystalline iodine. Since these ingredients were chosen for their general
acceptance as
safe, inactive (except for the Paclitaxel itself) excipients in pharmaceutical
preparations, this
formulation or a variation thereof could be of importance in the delivery of
Paclitaxel for the
2 5 treatment of cancer. The loading of Paclitaxel in the particle interior
was quite high, namely
on the order of 3 wt%, which in this case was so high that precipitation of
some of the
Paclitaxel within the interior of each particle may occur since the
solubilization of Paclitaxel
in this cubic phase at this high loading was metastable. However, studies
indicate that the
precipitation is very slow, taking hours or even days, at such loadings, so
that substantially all
3 0 of the Paclitaxel remains in solution during the course of the production
of particles;
-117-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
thereafter, the confinement of the Paclitaxel within the coated particles
prevents the formation
of large crystals (larger than a micron). If the concentration of Paclitaxel
in this system were
lowered, to 0.7% or less of the interior, then the solubilization of
Paclitaxel becomes a truly
stable solubilization (thermodynamic equilibrium), so that precipitation is
prevented
altogether, and microparticles of the present invention coated with
nonlamellar crystalline
iodine can be produced as described in this Example. Thus this system provides
several
scenarios for use in Paclitaxel delivery for cancer treatment.
EXAMPLE 37
A Paclitaxel-containing cubic phase liquid crystal was prepared by mixing
0.345
grams of soy lecithin (Epikuron 200), 0.357 grams of anisole, 0.26 grams of
water and 0.02
grams of Paclitaxel (from LIST Laboratories): equilibration was speeded by
plunging a test
tube of the mixture, after vigorous stirring, into boiling water for one
minute then cooling to
room temperature. To provide a coating material, 0.07 grams of propyl gallate
was stirred in
and the test tube again heated in boiling water. It had previously been
checked that propyl
gallate does not dissolve appreciably in this cubic phase at room temperature,
but that the
solubility increases substantially at 100°C. An upper solution
consisted of 2.25 grams of a 2%
Pluronic F-68 solution. The cubic phase-propyl gallate mixture was heated to
100°C, cooled
to about 80 °C, stirred with a spatula at the elevated temperature, and
reheated to 100°C.
After cooling the mixture for about 30 seconds, the upper solution was then
overlaid on this
2 0 mixture and the test tube placed in a sonication bath for one hour. A
dispersion of
microparticles with Paclitaxelcontaining interior and coated with propyl
gallate was obtained.
The dispersion had a high concentration of extremely fine microparticles
(estimated particle
diameter less than 0.4 micron), which were observable in the optical
microscope at 1000x by
virtue of their Brownian motion. The overall particle size distribution was
fairly broad, with
2 5 some particles as large as 1-2 microns. Only a very small amount of
precipitated Paclitaxel, in
the form of needles, was observed, so that nearly all of it must be in the
interiors of the
microparticles. The concentration of Paclitaxel in this example was high
enough that the
solubilization was metastable, which has implication as discussed in the
previous example.
Since the concentration of the antineoplastic drug Paclitaxel in the interiors
of these particles
3 0 was about 2%, and the components of the formulation are on the FDA list of
approved
-118-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
inactive excipients for oral delivery, (and nearly all of them for injection
as well), this
formulation could be very important as a drug-delivery, formulation for the
treatment of
cancers.
EXAMPLE 38
The amphiphilic polyethyleneoxide-polypropyleneoxide block copolymer Pluronic
F-
68 (also called Poloxamer 188), in the amount of 1.655 grams, was mixed with
0.705 grams
of eugenol and 2.06 grams of water. Upon centrifugation, two phases resulted,
the bottom
phase being a nanostructured liquid phase, and the top a nanostructured cubic
phase. An
amount of 0.68 grams of the liquid crystalline phase was removed, and to it
were added 0.05
grams of sodium iodide. A drop of eugenol was added to 2.48 grams of the lower
phase to
ensure low viscosity, and this nanostructured liquid phase, with 0.14 grams of
silver nitrate
added, served as the "upper solution" in dispersing the liquid crystalline
phase. Thus, the
liquid phase was overlaid on the liquid crystalline phase containing the
iodide, and the
mixture sonicated for 1.5 hours. The result was a dispersion of sliver iodide-
coated particles
in an external medium of the nanostructured liquid phase.
This Example illustrates the use of nanostructured liquid crystalline phases
based on
block copolymers as interior matrices for particles of the present invention.
In this case, water
was used as a preferential solvent for the polyethyleneoxide blocks of the
bloclc copolymer,
and eugenol as preferential solvent for the polypropyleneoxide blocks of the
block copolymer
2 0 (which are insoluble in water).
This Example also illustrates the use of a general approach discussed above,
namely
the use of a nanostructured phase as the mixture that serves as the "upper
solution", providing
moiety B which reacts with moiety A in the interior phase to cause
precipitation of a
crystalline coating material. In this case, B is the silver nitrate, which
induces precipitation of
2 5 silver iodide on contact with the interior matrix A (the cubic phase)
which contains sodium
iodide. As discussed above, it is generally desirable to choose this upper
solution so that it is
in equilibrium with the interior matrix, or, as in this case, very nearly so
(the only deviation
from true equilibrium being due to the addition of a single drop, about 0.01
grams or less than
0.5% of eugenol to the upper solution). As in this approach, is generally
useful to choose the
3 0 interior matrix so that it is a viscous material, much more so than the
upper solution which
-ll9-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
should be of relatively low viscosity.
EXAMPLE 39
A poly(lactic-glycolic acid) polymer (PLGA), with a 59:41 lactide:glycolide
ratio and
an inherent viscosity of 0.51 dl/gm, was obtained from Purac Biochem (The
Netherlands).
This copolymer is known to be amorphous, and this was evidenced by lack of
birefringence.
An amount 0.307 grams of this polymer was dissolved in 3.002 gm of ethyl
acetate. A cubic
phase was prepared by mixing 0.042 grams of the prothrombogenic compound
menadione,
0.272 grams of oil of ginger, 0.224 grams of water, and 0.540 grams of the
ethoxylated
hydrogenated castor oil surfactant Arlatone G (obtained from Uniquema). This
was heated to
50°C in order to dissolve the menadione. An amount 0.302 grams of this
cubic phase was
added to a second 16 ml glass tube, overlaid with 9.707 ml of water, and
dispersed into the
water by shaking. The~PLGA solution was added to the cubic phase dispersion,
the mixture
shaken immediately, and sonicated for 10 minutes. Following this, the contents
were
transferred into a round bottom flask, placed on a rotovap apparatus, and
evaporated to a final
volume of approximately 9.7 ml.
This resulted in PLGA-coated particles of two types. First, in the water
phase,
comprising approximately 2% by volume, were microparticles of cubic phase
coated with
PLGA. A significant fraction of these microparticles were large enough to see
structural
detail in a phase-contrast optical microscope. An optical micrograph is shown
in Figure 9.
2 0 The shell is visible in the larger particles. The irregular thickness of
this shell layer is
evidence that this layer is not an optical artifact. This is also evident when
adjusting the focus
on the microscope: if this were an artifact, its thickness would change as the
focus changed,
and this does not occur.
The second type of particle that came out of the process was a large,
millimeter-sized
2 5 particle that clearly behaved as a solid-coated particle. In one
experiment, a reddish-orange
dye, methyl red, which is of low solubility in both water and ethyl acetate,
was dissolved in
the cubic phase prior to dispersing. In addition to a reddish-orange tinge to
the microparticles
seen in the microscope, the millimeter-sized particles were strongly red-
orange,
demonstrating that the cubic phase is encapsulated inside the PLGA. Millimeter-
sized
3 0 particles of this type could be suspended on the tip of a needle, for
example, without flowing,
-120-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
in contrast with uncoated cubic phase which could not be suspended in this
fashion.
One of these large particles was placed in linalool, which is a solvent for
the cubic
phase but not for the PLGA. The particle did not dissolve in this solvent even
after one week,
whereas the cubic phase without PLGA coating dissolved in less than 5 minutes.
Figure 10
shows a side-by-side comparison of the PLGA-coated (on the left) and uncoated
(on the right)
cubic phases soaking in linalool, demonstrating cleaxly the insolubility of
the coated cubic
phase-the original color photograph shows that there is essentially no color
to the linalool
for the PLGA-coated sample, whereas the linalool containing the uncoated cubic
phase is
strongly red-orange. This experiment proves that the cubic phase is truly
encapsulated by the
PLGA.
EXAMPLE 40
A cubic phase containing solubilized methyl red was first prepared by mixing
2.118
grams of Arlatone G, 0.904 grams of water, 1.064 grams of oil of ginger, and
0.012 grams of
methyl red, and stirring thoroughly. A trehalose solution was prepared by
dissolving 2.00
grams of trehalose in 10.005 grams of water. Then 1.002 grams of the cubic
phase were
dispersed in the trehalose solution by a combination of shaking and mild
sonication. This
dispersion was then freeze-dried in a lyophilizes. Trehalose solutions are
known to yield
amorphous solid on freeze-drying. The resulting material flowed freely, and
gave no hint of
the greasy, sticky feel and behavior that characterizes the uncoated cubic
phase. There was
2 0 no second phase present, as the material was homogeneous to the eye, and
had a strong,
uniform, red-orange color. A large particle of the material was speared with
the point of a
push-pin and photographed, as shown in Figure 11; an uncoated cubic phase
would not have
been possible to spear and suspend indefinitely in this fashion.
In the phase-contrast optical microscope, thin portions of this material were
readily
2 5 seen to contain a fine-scale structure, which is consistent with the
presence of cubic phase
microparticles (submicron to 5 microns in size) within the trehalose solid
matrix. The
material was brittle and could therefore be crushed into small particles with
ease. Upon
mixing the material into water at, say, a 1:10 ratio, a dispersion was
immediately obtained
which was indistinguishable in the optical microscope from dispersions of this
cubic phase in
3 0 water.
-121-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
EXAMPLE 41
This Example demonstrates a method of production of coated microparticles in
which
a precursor to the coating material, which is surface-active when dissolved in
water, is used to
disperse a cubic phase into particles; then after reacting to convert this
precursor to a solid
coating, energy input is again applied to reduce the particle size to
submicron. As discussed
above, one advantage of this method is that it localizes the coating precursor
at the particle
surface, so that the cubic phase readily becomes encapsulated upon conversion
of this
precursor to the coating. The active compound in this Example was triclosan.
A cubic phase was prepared by mixing 0.886 grams of linalool, 0.960 grams of
Pluronic P123 (BASF), 0.104 grams of triclosan, 0.189 grams of 2-ethylhexanoic
acid, and
0.879 grams of distilled water, and then stirring thoroughly. This cubic phase
was then
smeared onto the sides of a test tube, 3.33 grams of a sodium N-
acetyltryptophan (Na-NAT)
solution (6 wt% based on the NAT) overlain, and the mixture shaken and
sonicated briefly to
disperse the cubic phase; the Na-NAT thus acts as a dispersant or surfactant
in this step. A
30% zinc acetate solution, in the amount 0.37 grams, was then added and mixed
with the
dispersion, followed by 0.52 grams of 2N NaOH. Five minutes were allowed for
the reaction
to begin, after which the material was further sonicated. A surfactant
solution (0.10 grams)
containing Cremophor EL (9%) and Pluronic F-68 (12.5%) was then added, and the
mixture
sonicated for 15 minutes. The solid-coated nature of the resulting
microparticles was evident
2 0 in phase contrast optical microscopy, where shearing the dispersion
between glass and
coverslip clearly showed that the microparticles behaved as solid-coated
particles rather than
as the readily-deformable cubic phase particles that result without
application of the coating.
EXAMPLE 42
This Example reports a process in which coating material is melted, and a
cubic phase
2 5 dispersed therein, following which the temperature is lowered to solidify
the coating, after
which energy input is applied to create particles. Such a process can be
applied to crystalline
materials as well as to amorphous or semi-crystalline coating materials, where
in the case of
an amorphous material the cooling may result in an amorphous material (and is
thus not a true
"freezing", but rather a vitrification).
3 0 The nutriceutical compound Coenzyme Q 10 was incorporated into a cubic
phase
-122-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
based on the ethoxylated, hydrogenated castor oil surfactant Arlatone G (from
Uniquema).
Coenzyme Q10 (10 mg) was solubilized in a mixture of 0.302 grams of essential
oil of
ginger, 0.201 grams of water, and 0.606 grams of Arlatone G. This cubic phase
was placed in
a test tube and 2.994 grams of hydrogenated cottonseed oil added, and the
entire contents
were heated to 90°C to melt the oil. The sample was immediately
sonicated in a hot water
bath with vigorous shaking every 30 seconds, for 3 minutes. The test tube was
then placed in
an ice bath to solidify the oil with particles dispersed throughout the
trigylceride. The
resulting solid was then milled by the application of mechanical energy to an
average particle
size of several hundred microns; further reduction in size can readily be
accomplished by
milling methods well known in the ant.
EXAMPLE 43
This Example shows that a lectin incorporated into a cubic phase microparticle-
the
microparticle that would result after the dissolution of the zinc N-
acetyltryptophan coating of
a particle of the type produced in Example 41-retains its ability to bind
oligosaccharides.
A cubic phase was first prepared by mixing 0.752 grams of Pluronic P123 (an
insoluble surfactant), 0.705 grams of linalool, and 0.703 grams of water. An
amount of 1.005
grams of tlus cubic phase was put in a glass flask together with 0.054 grams
of the
rhamnolipid surfactant JBR-99 (Jeneil Biosurfactant, Inc.) and 35 ml of pH 4.5
acetate buffer
containing 4 mM MnClz and 4 mM CaCl2. The flask was then sonicated to disperse
the cubic
2 0 phase. Following this, the dispersion was microfluidized in a model 1 l OS
Microfluidizer
(Microfluidics, Inc.) to a particle size that was fme enough where the
absorbance measured on
an Ultrospec 3000 UV-Vis spectrometer, at a wavelength of 620 nm, was about
0.2
absorbance units.
The following reagents were then added to 2 ml of the cubic phase dispersion:
2 5 Anti Concanavalin A, Vector AS-2004, Lot 0321, 1 mg/ml stock solution
prepared; working
solution prepared by diluting 1:10 to 0.1 mg/ml: 51 microliters added.
Concanavalin A,
Sigma C-5275, Lot 60I~8934 prepared as 1 mg/ml stock solution; working
solution prepared
by diluting 1:10 to 0.1 mg/ml: 16 microliters added. Biotinylated mannotriose
, V-labs,
NGB1336, prepare a 1 mg/ml stock solution, working solution prepared by
diluting 1:100 to
3 0 0.01 mg/ml: 20 microliters added. HRP/Avidin; 0.28 mg/ml stock solution:
90 microliters
-123-
CA 02488643 2004-12-06
WO 03/106168 PCT/US02/18654
added.
Fifteen minutes were allowed for diffusion and equilibration after the
addition of the
antibody and Con A solutions. Another fifteen minutes were allowed after the
addition of the
biotinylated mannatriose and HRP/avidin. To 10 drops of a Dextran Blue
solution, at 3.9
mg/ml water, were added 6 drops of fast red TR salt, 2.4 mg/ml, 1 drop of
3°/a H~O2, and 800
ul 50 mM sodium acetate pH 4.5 containing 4 mM MnCl2 and 4 mM CaCh. This
solution
has been found to show disappearance of absorbance at 620 nm upon addition of
HRP, or the
entire antibody-Con A-biotinylated mannatriose-avidin/HRP. At the end of all
these
additions, the total volume in the cuvette was 3.0 ml.
After the addition of the Dextran Blue-based Detection System, absorbance
readings
at 620 nm were monitored continuously. After the readings stabilized at 0.40
absorbance
units, 500 microliters of Displacement Solution were added. This solution was
composed of
saturated alpha methylmannoside in 50 mM sodium acetate pH 4.5 containing 4 mM
MnCh
and 4 mM CaCl2.
Upon addition of this alpha methylinannoside-the analyte-the absorbance
dropped
from 0.40 to 0.26 absorbance units. This decrease, 35%, is far greater than
the 14% that one
would expect based on the dilution from 3.0 to 3.5 ml volume, and was
reproducible, as seen
in several repetitions. The majority of the decrease in absorbance was due to
the enzymatic
action of displaced HRP on the Dextran Blue.
2 0 It is apparent that many modifications and variations of the invention may
be made
without departing from the spirit and scope of the present invention. It is
understood that the
invention is not confined to the particular construction and arrangement
herein described, but
embraces such modified forms of it as come within the appended claims. The
specific
embodiments described are given by way of example only and the invention is
limited only
2 5 by the terms of the appended claims.
-124-
