Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR THE PRODUCTION OF PROTEIN PARTICLES
USEFUL FOR DELIVERY OF PHARMACOLOGICAL AGENTS
FIELD OF THE INVENTION
The present invention relates to methods for the
production of protein-based colloidal particulate vehicles,
and compositions produced thereby. In a particular aspect,
the present invention provides a novel procedure for the
preparation of a colloidal system of proteins such as
albumin and other molecules in the form of microparticles
and nanoparticles, in the presence of divalent cations such
as calcium.
BACKGROUND OF THE INVENTION
Intravenous drug delivery permits rapid and
direct equilibration with the blood stream, which then
carries the medication to the rest of the body. It is
desirable, however, to avoid the peak serum levels which
are achieved within a short time after intravascular
injection. A solution to this problem is to administer
drugs carried within stable carriers, which would allow
gradual release of the drugs inside the intravascular
compartment following a bolus intravenous injection of
therapeutic nanoparticles.
Injectable controlled-release nanoparticles can
provide pre-programmed duration of action ranging from days
to weeks to months from a single injection. Such
nanoparticles can also offer several profound advantages
over conventionally admininstered medicaments, such as, for
example, automatic assured patient compliance with the dose
regimen, as well as drug targeting to specific tissues or
organs (see, for example, Tice and Gilley, in Journal of
Controlled Release 2:343-352 (1985)). Use of submicron
size microspheres (nanospheres) minimizes the incidence of
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pulmonary embolism often encountered with particles greater
than 7 microns or particles which aggregate upon in vivo
administration (see, for example, Gupta, et al., in
International Journal of Pharmaceutics 43:167-177 (1988)).
Particulate microspheres facilitate the delivery
of drugs to specific organs of the body. For example, a
major aim in cancer chemotherapy is to improve the
efficiency of cytostatic treatment. Killing of sensitive
tumor cells is facilitated by exposing the cancerous
lesions to high concentrations of anticancer drug, but
conventional methods of administration prohibit the use of
very high doses of the drugs due to their toxic secondary
effects. However, intravenous delivery of drug-containing
microspheres can prolong the serum half-life and
bioavailability of the drug (see, for example, Leucuta, et
al., in International Journal of Pharmaceutics 41:213-217
(1988)).
Since albumin microspheres have been used
extensively in the diagnosis of reticuloendothelial
abnormalities and in the measurement of blood flow, it has
been desirable to develop albumin microspheres for active
as well as passive drug targeting (see, for example, Gupta,
et al., supra) . An important prerequisite for clinical use
of drug-containing microspheres is a stable product with
reproducible characteristics. Microspheres were originally
manufactured in suspension form for use in patients within
a few hours, but techniques such as freeze-drying have been
employed to increase the shelf-life of the product. For
example, albumin microspheres have been prepared by
formation of water-in-oil emlusions, where the droplets
contain a protein and a drug, and the droplets are further
crosslinked by covalent bonds by using a crosslinking agent
such as glutaraldehyde. This method is capable of yielding
suspensions of drug-loaded albumin microspheres in aqueous
buffers for intravascular administration (see, for example,
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Willmott and Harrison, in International Journal of
Pharmaceutics 43:161-166 (1988)).
Heat-denaturation of albumin to form
drug-containing microspheres is often preferred to the use
of crosslinking agents such as glutaraldehyde, because
heat-denaturation avoids potentially undesirable chemical
crosslinking reactions involving the therapeutic drug
itself. For example, cotton seed oil has been added to a
solution of bovine serum albumin, and then ultrasonicated
to obtain a water-in-oil emulsion. This emulsion is then
added dropwise to a larger volume of rapidly stirred
cottonseed oil which has been preheated to temperatures
between 105 C and 150 C. Such high temperatures are needed
to achieve denaturation and precipitation of the serum
albumin from the microdroplets of the emulsion, thereby
forming stabilized microspheres which can be washed and
dried to obtain a free-flowing powder of drug-containing
microspheres (see, for example, Gupta, et al., supra).
US Patent No. 5,041,292, issued to J. Feijen,
also discloses a process for preparation of microspheres,
by forming a water-in-oil emulsion of olive oil and an
aqueous solution of bovine serum albumin and a
polysaccharide such as Heparin, and then heating the
emulsion to 100-170 C, or adding a crosslinking agent such
as glutaraldehyde. The microspheres are loaded later with
a suitable pharmacologically active agent.
US Patent No. 5,133,908, assigned to S.
Stainmesse, describes a method to form nanoparticles by
dissolving the protein, e.g., serum albumin, at low
temperature, and at a pH far above or below the isoelectric
point, and adding this solution into a larger volume of
water at very high temperature (e.g., boiling).
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EP Application No. 349,428, by Devissaguet et
al., describes a method for producing colloidal suspensions
of spherical protein particles employing the following
steps:
a. dissolving the protein and optionally an
active agent in aqueous medium below the coagulation
temperature of the protein, optionally with added
surfactant,
b. heating an aqueous medium, optionally
containing biologically active substance and surfactant(s),
to a temperature above the coagulation temperature of the
protein, and
c. adding the second solution to the first
solution, under moderate agitation at a pH far from the
isoelectric point of the protein.
Unfortunately, some drugs are destabilized or
inactivated by the conditions typically used to obtain
heat-denatured microspheres. It is therefore highly
desirable to develop a general method for manufacturing
drug-containing protein microspheres by heat denaturation
which can be carried out at temperatures low enough to
prevent drug inactivation, but which is still capable of
producing an effective and stable carrier product with well
controlled uniformity of particle sizes.
The formation of nanomatrixes of proteins has
recently been reported (see US Patent No. 5,308,620, by
R.C.K. Chen, issued in 1994) . The method is based on
mixing at least two types of proteins (hemoglobin and
albumin), and addition of a solution containing organic
solvent (e.g., an alcohol), without heat treatment. The
resulting system is a turbid suspension of monodispersed
nanomatrixes which are typically larger than 1 micron but
less than 4 microns in diameter.
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US Patent No. 5,049,322, issued to Devissaguet et
al., discloses a method of producing a colloidal system
containing 150-450 nm particles by dissolving a single
protein in a solvent and adding ethanol or ethanol-
5 containing surfactant. These particles have a core
material which is encapsulated inside a wall material made
of the protein.
In spite of these recent developments, there is
still a need in the art for a general method for
manufacturing drug-containing protein microspheres without
the need for organic solvents and/or surfactants.
Especially desirable would be methods for manufacturing
drug-containing protein microspheres by heat denaturation
at temperatures low enough to prevent drug inactivation,
but which is still capable of producing an effective and
stable carrier product with well controlled uniformity of
particle sizes.
OBJECTS OF THE INVENTION
It is an object of the present invention to
provide a method for the formation of particles by
heat-denaturation of proteins (e.g., human serum albumin),
at a pH above the iso-electric point without the need for
organic solvents and/or surfactants.
This and other objects of the invention will
become apparent to those of skill in the art upon review of
the specification and appended claims.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, we have
discovered that stable protein particles can be produced by
heat-denaturation of proteins (e.g., human serum albumin),
at a pH above the iso-electric point in the presence of an
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appropriate concentration of polyvalent cations (e.g.,
calcium ions) at temperatures below the heat denaturation
temperature of the protein. Heating of the protein
solution in the presence of polyvalent (e.g., calcium)
cations allows co-precipitation of other negatively charged
molecules such as hemoglobin, antibodies (at pH above the
isoelectric point) and DNA or pharmacologically active
molecules (including uncharged molecules) which cannot
withstand high temperatures.
The addition of multivalent ions (such as
calcium) leads to precipitation of protein particles by
heat treatment at temperatures which are significantly
lower than the denaturation temperature of the protein. In
accordance with the present invention, it has been
discovered that this ability of polyvalent cations to
promote the precipitation of protein particles occurs only
within a very narrow range of electrolyte concentrations.
Thus, the present invention relates to performing
a heat induced precipitation, which, in the presence of
divalent ions (e.g., calcium) at a specific concentration
range, forms submicron particles of albumin or particles of
albumin and other molecules such as DNA. The heat
denaturation takes place at temperatures significantly
below that of the protein in the absence of the divalent
cations. In addition, the protein particles form without
the need for formation of a water-in-oil emulsion, or
addition of any organic solvent, cross-linking agent or
surfactants, at the defined cation concentration range.
Thus, the present invention provides a method for
the formation of submicron particles (nanoparticles) by
heat-denaturation of proteins (e.g., human serum albumin)
in the presence of multivalent ions (e.g., calcium). The
use of an appropriate concentration of cations (e.g.,
calcium ions) induces the precipitation of the protein at
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a temperature which is significantly lower than the regular
heat denaturation temperature of the protein. The process
also allows co-precipitation of other molecules (including
those which cannot withstand high temperatures), which are
then incorporated within the nanoparticles. This procedure
facilitates the production of nanoparticles containing
various proteins, pharmacologically active agents or DNA
for various purposes such as drug delivery, diagnostics,
gene therapy, and the like. The present invention
ultimately yields a colloidal system in the form of
particles composed of proteins and other active molecules,
having a diameter of several micrometers down to about 50
nm, depending on the specific cation and protein used, the
concentrations of each, pH and temperature.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there
are provided methods for the preparation of protein
particles having a diameter less than about 30 microns.
Invention methods comprise:
dissolving at least one protein, and
optionally other non-proteinaceous materials, in
an aqueous solution containing an amount of at
least one multivalent cation sufficient to
promote the formation of insoluble particles at
a temperature below that of the heat denaturation
temperature of said protein in purified form,
wherein said aqueous solution is
substantially free of organic media, and
heating the solution to a temperature below
that of the heat denaturation temperature of an
aqueous solution of said protein in purified form
for a period of time sufficient to form insoluble
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particles containing the protein and optional
non-proteinaceous materials.
Invention methods can readily be carried out in
the substantial absence of organic solvent, i.e., organic
solvent is not required to facilitate the invention method.
Avoidance of organic solvent substantially reduces the
possibility for denaturation of sensitive pharmacologically
active agents as a result of the microparticle-forming
process.
Proteins contemplated for use in the practice of
the present invention include structural proteins, enzymes,
antibodies, peptides, and the like. Specific proteins
contemplated for use herein include albumins, collagen,
gelatin, immunoglobulins, insulin, hemoglobin, transferrin,
caesins, pepsin, trypsin, chymotrypsin, lysozyme, a-2-
macroglobulin, fibronectin, vitronectin, fibrinogen,
laminin, lipase, interleukin-1, interleukin-2, tissue
necrosis factor, colony-stimulating factor, epidermal
growth factor, transforming growth factors, fibroblast
growth factor, insulin-like growth factors, hirudin, tissue
plasminogen activator, urokinase, streptokinase,
erythropoietin, Factor VIIi, Factor IX, insulin,
somatostatin, proinsulin, macrophage-inhibiting factor,
macrophage-activating factor, muramyl dipeptide,
interferons, glucocerebrosidase, calcitonin, oxytocin,
growth hormone, a-1 antitrypsin, superoxide dismutase
(SOD), catalase, adenosine deaminase, lactalbumin,
ovalalbumin, amylase, and the like.
Optional non-proteinaceous materials contemplated
for use herein include nucleic acids, oligonucleotides,
polynucleotides, DNA, RNA, polysaccharides, ribozymes,
pharmacologically active compounds capable of inclusion
within the protein particles, and the like.
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Nucleic acids contemplated for use in the
practice of the present invention include sense or anti-
sense nucleic acids encoding (or complementary to nucleic
acids encoding) any protein suitable for delivery by
inhalation.
DNA introduced as part of invention particles can
be used for a variety of purposes, e.g., for gene delivery,
for cell transfection, and the like.
Polysaccharides contemplated for use in the
practice of the present invention include starches,
celluloses, dextrans, alginates, chitosans, pectins,
hyaluronic acid, and the like.
Pharmacologically active compounds capable of
inclusion within the protein particles contemplated for
incorporation into invention particles include antisense
nucleic acids, antiviral compounds, anticancer agents,
immunosuppressive agents, and the like. Exemplary
pharmacologically active agents contemplated for use herein
include antiviral agents (e.g., interferon gamma,
zidovudine, amantadine hydrochloride, ribavirin, acyclovir,
and the like), anticancer agents, immunosuppressants (e.g.,
glucocorticoids, buspirone, castanospermine, ebselen,
edelfosine, enlimomab, galaptin, methoxatone, mizoribine),
and the like.
Multivalent cations contemplated for use in the
practice of the present invention include calcium, zinc,
magnesium, barium, copper, iron, manganese, nickel,
aluminium, gadolinium, technecium, strontium, cobalt, and
the like. Isotopes of these elements are also contemplated
for potential use in such applications as diagnostics and
radionuclide therapy.
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In accordance with the present invention,
multivalent cations are introduced in an amount sufficient
to promote the formation of insoluble particles at a
temperature below that of the heat denaturation temperature
5 of said protein in purified form. Typically, the
concentration of multivalent cations falls in the range of
about 0.1 mM up to about 10 mM. This concentration range
depends on the ion type used, the pH of the protein-
containing solution, the presence of solvent(s), and the
10 like.
In accordance with the present invention,
suitable protein(s), optional non-proteinaceous materials,
and multivalent cation(s) are combined in a suitable
aqueous solution. As readily recognized by those of skill
in the art, suitable solutions for such purpose include
distilled water, deionized water, buffered aqueous media,
solutions of water and water-miscible solvent(s), and the
like.
In accordance with the present invention, once
the combination of protein(s), optional non-proteinaceous
materials, and multivalent cation(s) have been combined in
a suitable aqueous solution, the combination is subjected
to heat to a temperature below that of the heat
denaturation temperature of an aqueous solution of said
protein in purified form for a period of time sufficient to
form insoluble particles containing the protein and
optional non-proteinaceous materials. Such heating is
typically carried out at a temperature less than about
100 C. It is presently preferred that such heating be
carried out at a temperature less than about 80 C.
A detailed understanding of the thermal
properties of proteins (e.g., albumin) has been essential
in the development of the invention process. In
particular, conventional preparation of human serum albumin
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for clinical purposes involves treatment at 60 C for about
hours to inactivate the hepatitis virus. In the absence
of multivalent ions, little denaturation or aggregation
occurs at 60 C and the native albumin structure is largely
5 retained, even without a stabilizer. However, when the
temperature is kept above 80 C, about 40% of the albumin
becomes irreversibly denatured and a considerable degree of
aggregation is observed. The formation of intermolecular
disulfide bridges by free sulfhydryl groups plays an
10 essential role in the aggregation process (see, for
example, Wetzel, et al., European Journal of Biochemistry
104:469-478 (1980)).
Suitable pHs contemplated for use in the
invention process are in the range 1.5 - 11. Electrolyte
concentration in the aqueous medium containing the protein,
peptide, nucleic acids, pharmacological agents is in the
range 0.01 mM - 1.0 M.
A typical process is based on preparation of the
protein solution at pH above the isoelectric point (e.g.,
1% serum albumin at pH 6.4), together with multivalent
cation (e.g., calcium) at a defined, very low
concentration, and optionally a non-proteinaceous material
(i.e., an active molecule such as DNA), followed by heating
the solution at a temperature below the usual heat
denaturation temperature (for example, serum albumin should
be heated to about 60 C). The duration of heating, the
temperature, and the concentration of the multivalent ions
will determine the average size of the particles, and their
concentration in solution.
The method of the present invention does not
require formation of a water-in-oil emulsion and heating to
very high temperatures, nor addition of crosslinking
agents, as commonly described in other patents and
publications.
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In addition to the formation of solid particles,
hollow particles comprising a shell of proteinaceous
material may be formed by dispersion of gas into the
aqueous medium during the process of formation of the
particles. The invention process results in a lower
formation temperature for the hollow particles such as
described in US Patent No. 4,957,656 (Cerny et al., 1990).
In accordance with another embodiment of the
present invention, there are provided products produced by
the above-described methods. Such products are useful for
a variety of purposes, e.g., controlled release of a
variety of pharmacologically active agents, for protected
delivery of sensitive reagents (e.g., DNA, peptides,
enzymes, and the like), and the like.
In accordance with yet another embodiment of the
present invention, there are provided methods for the
controlled release delivery of pharmacologically active
agents to a patient in need thereof, said method comprising
administering to said patient an effective amount of said
pharmacologically active agent incorporated into a product
as described herein.
The invention will now be described in greater
detail by reference to the following non-limiting examples.
Example 1
Formation of microparticles and nanoparticles
from albumin in the presence of divalent cations
Human serum albumin was dissolved in deionized,
sterile water, to yield a clear 1% w/w stock albumin
solution, having a pH 6.3, which is above the isoelectric
point of the protein. The stock solution was used for the
following experiments:
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A) Three ml of this solution was immersed into a
water bath at 65 C, for 5 minutes. The solution remained
clear, and no particles were observed by light microscope.
B) Two ml of the stock solution were mixed with
0.08 ml of 100 mM calcium chloride solution, and 0.92 ml
sterile water (final calcium chloride concentration
2.66 mM). The solution was immersed into a water bath, at
65 C, for 5 minutes. The solution became cloudy about two
minutes after immersion. When viewed by light microscopy
(magnification x 1000), particles, having a size 1 m and
below were observed. The average size of these particles
(conducted by a Malvern light scattering instrument),
proved that these are albumin submicron particles, having
a Z-average diameter of 460 nm. The particles were
negatively charged, and have a Zeta-potential of -10 mv
(measured by Malvern Zetasizer) . This colloidal system was
stable for at least two weeks at 4 C, without any
precipitation. A frozen sample was found to retain its
initial size, after thawing. The stability of the
particles was also tested by centrifugation, washing and
redispersion in sterile water: it was found that the
particles were resuspended to give a colloidal system which
contains particles having a diameter 1 m and below.
C) A similar experiment was conducted, in which
the final calcium chloride concentration is 13.3 mM, or
6.6 mM. In both cases, larger particles, in the
micron-size range (up to 50 m) were observed. These
particles were aggregates of smaller particles, as observed
by light microscopy.
These experiments indicate that at a given
temperature and incubation time, colloidal particles of
albumin can be formed if calcium chloride is present at
very low concentration, and that submicron particles can be
formed only within a very narrow range of calcium chloride
concentrations.
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Example 2
Effect of calcium concentration
A series of solutions was prepared in which the
concentration of human serum albumin remained constant,
0.66 % w/v, and the concentrations of calcium chloride were
in the range of 0 - 20 mM. Each solution was heated in a
water bath at 60 C for 2 minutes, and than was observed by
an optical microscope. It was found that the solution
without calcium chloride added, and the solutions which
have calcium chloride at concentrations of 5.33 - 20 mM,
were clear, and no particles were detected. However, only
at a specific, narrow range of calcium chloride
concentrations (i.e., 0.67 - 2.7 mM), the solutions were
turbid, indicating the presence of a colloidal system.
Microscopic evaluation revealed that particles having a
size below 2.5 um were present in these turbid systems.
A similar experiment was performed at 65 C, with
similar results. Thus, the albumin particles were formed
only at a specific, narrow range of calcium chloride
concentrations.
However, it was found that at the higher
temperature, the calcium concentration range in which the
albumin particles are formed is slightly extended, up to
6.7 mM. Above this calcium chloride concentration, the
solutions remained clear.
This experiment indicates that the formation of
protein particles is not a result of a simple salting - out
precipitation process, and that, surprisingly, at high
calcium concentration, colloidal particles are not formed,
as would be expected from a salting-out process.
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Example 3
Formation of DNA-Albumin colloidal particles
The purpose of this example is to demonstrate the
formation of microparticles and nanoparticles which contain
5 both a protein and DNA.
DNA PREPARATION
The RSV-SGal reporter plasmid was introduced into
Escherichia coli JM109 cells by calcium chloride
transformation (see, for example, Sambrook et al. in
10 Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1.
1989, Plainview, NY: Cold Spring Harbor Laboratory Press.
The cells were grown from a single colony in 1 liter LB
Broth (GibcoBRL #12780-029) containing 50 (g/ml ampicillin
to an optical density of 1.32 at 600 nm. The plasmid was
15 purified using the QIAGEN Plasmid Mega Kit (QIAGEN #12181)
according to the manufacturer's instructions. The final
yield was 2 ml RSV-f3Gal plasmid at a concentration of 1.24
g/l in TE (10 mM Tris-HC1, 1 mM EDTA pH 8.0). 806 l of
the sample was diluted to 1 ml with TE to give a final
concentration of 1 g/l, and this sample was divided into 50
l aliquots for nanoparticle formation studies.
FORMATION OF HSA-DNA PARTICLES
a. 1 ml HSA 1.25% w/w solution was mixed with 0.41 ml
deionized water, 0.04 ml of 100 mM calcium chloride
solution (to yield a final calcium chloride concentration
of 2.66 mM), 0.41 ml sterile deionized water and 0.05 ml
DNA solution (1 mg/ml). The solution was immersed in a
water bath (60 C) and after about one minute, the solution
becomes turbid. The solution is removed from the water
bath after about 2 minutes. Microscopic observation
reveals the presence of particles having a size of 1pm and
below. Light scattering measurments proved the presence of
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one population of nanoparticles, having a Z-average
diameter of 420 nm.
b. larger HSA-DNA particles, having a size about 10
um, were prepared by the same procedure but the temperature
was 65 C, while the heating period was only one minute.
ANALYSIS OF HSA - DNA NANOPARTICLES
In the present study, two classes of HSA-DNA
particles were made in the micron and submicron size
ranges. The presence of DNA in both classes of colloidal
particles was determined by two independent methods:
Following formation, the particles were separated
from the supernatant by centrifugation and the two
fractions collected and analyzed in parallel. Equivalent
aliquots of both the pellet and the supernatant fractions
were digested for two hours at 60 C with Proteinase K
(GibcoBRL #255530-049; 100 g/ml in 20 mM Tris-HC1, 20 mM
EDTA, 0.5% sodium dodecylsulfate pH 7.5), extracted twice
with phenol:chloroform:isoamyl alcohol (25:24:1 v/v),
extracted once with chloroform, then precipitated with 2
vol. ethanol in the presence of 0.3 M sodium acetate, pH
5.2. The samples were then electrophoresed on a 0.7%
agarose gel and stained with ethidium bromide to ascertain
the presence and integrity of the DNA.
It was found that the vast majority (estimated
greater than 90%) of the DNA was in the particle fraction
of both classes of samples, i.e., microparticles (having a
mean diameter of 10 m) and nanoparticles (having a mean
diameter of 420 nm).
In contrast, very little DNA was associated with
the pellet of the control samples in which no particles
were formed. Thus, for example, in the "no heating"
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control sample, the same components as used for the
preparation of invention particulate samples were combined,
but not heated and therefore did not contain any particles.
In the "adsorbed DNA" control sample, the DNA was added
after the formation of particles with HSA alone, i.e., the
HSA particles were formed prior to the addition of DNA.
Furthermore, the position of the DNA bands indicates that
the DNA in the particles is intact, and may be used to
obtain specific biological activity.
Interestingly, the DNA could not be extracted
from the particles using the standard
phenol:chloroform:isoamyl alcohol (25:24:1 v/v) procedure
alone, even after addition of both 10 mM EDTA and 1% sodium
dodecylsulfate. Since most proteins are extracted from DNA
by these procedures (see, for example, Sambrook et al.,
su ra), this is indicative of very tight associations
within the HSA-DNA complexes.
The second method employed to verify the presence
of DNA was based on specific DNA staining. The particles
were stained with 1 g/ml Hoechst 33258 (a DNA specific
dye) in 50 mM sodium phosphate pH 7.4/2M NaCl [Labarca),
and then visualized with an Olympus IX70 fluorescent
microscope.
It was found that the particles containing DNA
fluoresced bright blue, while particles of HSA alone were
not fluorescent.
These experiments demonstrate that the DNA was
indeed substantially entrapped within the micro and
nanoparticles, and not simply adsorbed on the surface of
the particles, making it possible to use such colloidal
particles for gene delivery.
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Example 4
Formation of Protein Particles with multivalent ions
other than calcium
The formation of protein particles during heating
in the presence of calcium ions at a specific concentration
can also be achieved by using other multivalent ions (e.g.,
barium, copper, magnesium, and the like) which are capable
of reducing the heat denaturation temperature of the
protein. The type and required concentration of the
specific multivalent ion used should be evaluated according
to the type of protein (and other optional molecules)
present in the system.
Example 5
Formation of albumin - IgG colloidal particles
The purpose of this example is to demonstrate the
possibility to form particles which contain more than one
protein. Therefore, a fluorescently lablled monoclonal IgG
(rabbit) was used in order to allow simple and quick
evaluation of the presence of the antibody by fluorescence
microscope. One ml human serum albumin, at pH 7.1
(phosphate buffer, 0.25mM), was mixed with 0.1 ml FTIC
lablled IgG (Sigma F-4151), 0.04 ml calcium chloride 100 mM
solution, and 0.36 ml deionized water. The solution was
heated in a water bath, at 60 C, for 5 minutes. The turbid
dispersion which was obtained contained aggregates of
particles in the size range of 50-100 m, while each
aggregate was composed of many small (about 2 m)
particles. The experiment was conducted at prolonged
heating, in order to obtain large particles which can be
easily viewed by an optical microscope.
It was found that all particles were fluorescent,
giving bright green light due to the FITC labelling.
Taking into consideration that the concentration of HSA is
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much higher than the concentration of the IgG, (about two
orders of magnitude in the final solution), it can be
concluded that the particles contain both albumin and
antibodies.
Example 6
Targeting of Protein particles
The above prepared albumin-antibody particles can
be used to deliver a drug, diagnostic agent or
radiopharmaceutical and target it to a specific site in the
body by incorporating into the particles, a recognition
molecule or molecules. A non-limiting list of such
molecules include proteins, peptides, antibodies, sugars,
polysaccharides and combinations thereof. For example, an
antibody against a human mammary carcinoma (MX-1) can be
incorporated into these particles which are then
subsequently introduced into the blood stream of a cancer
patient suffering from the disease in order to carry a
payload of therapeutic drug or radioactive isotope to treat
the disease, or a diagnostic marker to locate the extent of
disease. These targeting agents can be incorporated into
the particles during the process of formation or after the
protein particles are formed.
Example 7
Gene therapy with protein-DNA particles
The above prepared protein-DNA particles can be
used for gene therapy by delivering the intact DNA into
cells, under such conditions that effective transfection
can be achieved. To increase the efficacy of particle
uptake, addition of agents such as glycocholic acid,
polylysine, gelatin, phospholipids, calcium ions, glycerol,
and the like, into a suspension of the protein-DNA
particles or using techniques such as electroporation and
the like can improve the uptake of particles by various
CA 02263765 1999-02-18
WO 98/07410 PCTIUS97/14661
cells. In addition, these particles can be also linked to
molecules capable of targeting the particles to specific
organs and tissues, cells or even to specific sites inside
the cell.
5 Example 8
Other uses of protein particles
The present invention may be used in order to
achieve small particles of proteins with specific
biological activity. For example, hemoglobin particles can
10 be formed by a similar process as described above for
albumin. Hemoglobin particles can be used, for example, as
a blood substitute, as a therapeutic to treat anemias, and
the like. In addition, the present invention can be used
in order to form protein shells around air bubbles, thus
15 enabling their use as an echocontrast agent, for medical
diagnostics.
While the invention has been described in detail
with reference to certain preferred embodiments thereof, it
will be understood that modifications and variations are
20 within the spirit and scope of that which is described and
claimed.
