Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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B&P File No. 3244-014~/JRR
Title: NOVEL NON-PHOSPHOLIPID LIPOSOMES MODIFIED WITH A PH
AND TEMPERATURE SENSITIVE COPOLYMER
FIELD OF THE INVENTION
This invention relates to the field of liposomes, more
particularly it relates to pH and temperature sensitive liposomes for use as
a delivery system.
BACKGROUND OF THE INVENTION
Liposome-based materials are versatile delivery systems
(Gregoriadis, G., 1992; New, R.R.C., 1990; and Rolland, A., 1993).
Practically any water-soluble material can be encapsulated into the internal
aqueous pool of a liposome and the surrounding membrane can be loaded
with lipophilic components.
The outer surface of liposomes can be endowed with specific
properties by choosing appropriate combinations of membrane-forming
lipids or by further modification with specific receptors or mediators.
Phospholipid liposomes modified by a copolymer sensitive to pH and
temperature changes have been studied and while being versatile systems,
they can be adjusted to respond effectively to only a limited number of
specific triggers. For most therapeutic applications the lipids of choice have
been phospholipids, since they are the constituents of natural membranes
(Iga, K., 1992; Zoy, Y., 1993; Maruyama, K., 1993). To date a number of
pH-responsive phospholipid liposomes have been prepared using specific
lipid combinations (Torchilin, V.P. et al., 1993; Wilschut, P.A., 1991). Also
employed with varying success are pH-sensitive phospholipid liposomes
containing in their bilayer fusogenic molecules (White, J. et al. 1996), such
as viral fusion proteins or fusion peptides (Lear, J.D. et al. 1987).
Recently it has been observed that a large number of other
lipids form liposomes, known as non-phospholipid liposomes, or NPL's
(Philipot, J.R., 1995). These vesicles are obtained from mixtures of three
families of lipids: (a) a main amphiphilic component, such as
n-octadecyldiethylene oxide, (b) a modulator, usually cholesterol, and (c)
an ionogen, such as dioctadecyldimethylammonium bromide (DDAB) in
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the case of cationic NPL's or dioctadecylphosphate (DP) in the case of
anionic NPL's. While from a morphological viewpoint the two classes of
liposomes bear significant similarity, from a chemical standpoint, the
hydrophilic surfaces of the vesicles are quite different. The surface of
NPL's is characterized by a high density of hydroxyl and ether functional
groups able to form hydrogen bonds with suitable partners. Generally,
NPLs have not been studied extensively and not much if at all in
biological systems. In biological systems uncoated NPLs are either too
fusegenic or not fusegenic at all.
The design of responsive liposomes has been stimulated by
the need to develop efficient delivery vehicles in medicine, building on
the inherent advantages of liposomes which have been shown both to
enhance the efficiency and reduce the toxicity of several drugs (Rolland,
A., 1993; Lopes-Berestein, G. et al. 1989; Roerdink, F.I-i. et al. 1989;
Gregoriadis, G. 1988; Machy, P. et al., 1987; Ostro, M.J. 1987; Oku, N., 1994;
Lasic, D.D., 1997). Several factors contribute to successful drug delivery to
cells by liposomes. One of the key events that has to occur is the release of
encapsulated material into a cell's cytoplasm once the liposomes are
internalized by a process known as endocytosis. In this context, a
particularly attractive feature of surface-modified liposomes as delivery
systems is their responsiveness to a change in external pH, enabling them
to fuse with an endosomal membrane and release an encapsulated drug
into the cytoplasm (Straubinger, R.M. et al., (1983; Straubinger, R.M. et al.,
1990; Straubinger, R.M., 1993.
Intermolecular hydrogen bonds are of crucial importance in
inducing aggregation in nature and can also be employed with great
effectiveness to design nanostructures via spontaneous association of
molecules (Whitesides, G.M. et al., 1991; Lehn, J.-M. 1990). This strategy
has been used to generate liquid crystal structures (Paleos, C.M. et al.
1995),
macromolecular assemblies, such as supramolecular columns, to improve
the compatibility of polymer blends (Zhou, C. et al. 1998), and modify the
thermal and mechanical properties of polymers (Wang, Q. et al. 1997).
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Polyelectrolyte/liposome complexes are sensitive to pH
changes. In certain lipid bilayers, polyelectrolytes can promote phase
changes, create local defects in the membrane, or cause aggregation or
fusion either by themselves or in the presence of added salt (Thomas, J.L.
et al., 1992). Alternatively, as demonstrated in the work of Thomas and
Tirrell, a polyelectrolyte can promote pH-dependent lateral diffusion of
charged lipids in the bilayer and induce reversible permeability of the
membrane. Particularly effective in promoting such effects are derivatives
of poly(2-ethylacrylic acid) anchored into the bilayer through phospholipid
residues (Thomas, J.L. et al., 1992).
Aqueous solutions of poly(N-isopropylacrylamide)
(PNIPAM) exhibit well characterized phase changes. When the
temperature of a PNIPAM solution exceeds 32°C, the polymer chain
collapses from an extended coil into a globule, a transition revealed on the
macroscopic scale by a sudden increase in turbidity (known as the cloud
point or the lower critical solution temperature (LCST), the LCST for a
solution of PNIPAM is 32°C) (Schild, H.G., 1992). Polymers bearing
carboxylic acid functional groups, such as polyacrylic acid, are pH-sensitive,
adopting a coiled conformation in solutions of low pH, where the
carboxylic acid groups are protonated, and an extended conformation in
solutions of high pH, where the negatively charged carboxylates undergo
strong electrostatic repulsion. Graft and random copolymers of
N-isopropylacrylamide (NIPAM) and acrylic acid (AA) have been shown
to be responsive to both pH and temperature (Chen, G. et al., 1995; Ghen G.
et al., 1995; Kim, J.C., et al., (199. Derivatives of PNIPAM carrying a small
number of hydrophobic groups, such as octadecyl groups, maintain their
sensitivity to changes of temperature. Such copolymers, known as
hydrophobically-modified PNIPAM (HM-PNIPAM) have been used to
modify the surface of liposomes and to impart to them some level of
sensitivity to changes in temperature (Ringsdorf, H. et al., 1993). However,
the utility of such complexes is rather restricted due to the narrow range of
possible critical temperatures and the limited ability of the polymer to
influence the liposome morphology. Further, as mentioned, the
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attachment of responsive polymers to lipids in the bilayer has been
investigated as another means of sensitizing phospholipid liposomes to
external stimuli (Thomas, J.L. 1992; Kono, K. et al., 1994; Hayashi, H. et
al.,
1996). However, existing polymer-phospholipid liposome complexes are
rather limited in their flexibility and effective cytoplasmic delivery is
seldom achieved.
SUMMARY OF THE INVENTION
The present inventors have prepared a new type of pH and
temperature sensitive copolymer that possesses not only
temperature-responsive groups (NIPAM) and pH-responsive groups, but
also hydrophobic groups. According to a preferred embodiment this
hydrophobic group is an octadecyl chain linked to a secondary amide
nitrogen. According to one embodiment there is provided a copolymer of
a formula:
~(CH=--CH)x--(CH= H)y--(CH~H)z--
~~O ~=O C
NH NH NH
I~ I
HsC~CH~ R R
C 04H
According to a preferred embodiment, R=C18H3~ ; R' is
selected from the group consisting of: a hydrophobic amino acid; an
hydroxylated amino acid, a charged amino acid; and an aromatic amino
acid; and x = 20 to 80, y = 20 to 80, z = 1 to 5 (where x + y + z = 100).
Preferably R' - CH2; CH2-CH2; CH(CH3); CH(iPr);CH(CH2~);
CH[CH2)CH(CH3)2]; CH(iBu); CH(secBu); CH2CH2SCH3; CH2(C3N2H4+);
(CH2)4NH2; CH20H; or CH2 CH2 CH2 CH2NH3; and x = 20 to 80, y = 20 to
80, z = 1 to 5 (where x + y + z = 100). More preferably R' = CH2 (GLYCINE)
and x = 20 to 80, y = 20 to 80, z = 1 to 5 (where x + y + z = 100) which is
referred to herein as PNIPAM-Gly.
The present inventors have also found that the copolymer
of the present invention, PNIPAM-Gly, when used to modify
non-phospholipid liposome complexes results in an unexpectedly efficient
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delivery system. The present inventors have demonstrated controlled
release and fusion of NPL's with a single coating of a copolymer of the
present invention and several vesicle compositions. Accordingly, the
present invention provides an NPL, coated with a copolymer of a formula:
~(CH=--CH )x--.~C H~ --C H )y--(C H~ H )z-
=o ~=o
NH NH NH
~ R~ R
HsC~GH~
C 04H
According to a preferred embodiment, R=C18H 3~ ; R' is
selected from the group consisting of: a hydrophobic amino acid; an
hydroxylated amino acid, a charged amino acid; and an aromatic amino
acid; and x = 20 to 80, y = 20 to 80, z = 1 to 5 (where x + y + z = 100). More
preferably R' = CH2 (GLYCINE) and x = 20 to 80), y = 20 to 80, z = 1 to 5
(where x + y + z = 100).
According to another preferred embodiment, the NPL is
comprised of n-octadecyl diethylene oxide, cholesterol and
dimethyldioctadeclammonium bromide, more preferably, the n-octadecyl
diethylene oxide, cholesterol and dimethyldioctadeclammonium bromide
are in a ratio of 7.5: 2: 0.5, respectively.
The present invention also provides for coating
phospholipid liposomes with any one of the copolymers of the present
invention. According to a preferred embodiment the phospholipid
liposomes comprise dimyristoyl phosphatydylcholine, cholesterol, and
dimethyldioctadecylammonium bromide in a ratio of 7.5:2:0.5,
respectively.
The present invention may be used advantageously in the
delivery of pharmaceutical compositions to target locations, including
delivery of drugs to the interior of a cell. In addition, delivery may also be
targetted to specific vesicles or organelles within a cell type. Accordingly
the prsent invention provides various pharmaceutical compositions.
Application of these complexes is not restricted only to drug
delivery: They can be employed in any area that requires controlled
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release of sensitive encapsulated materials, such as vaccine technologies,
topical encapsulations including heat activitated sun-screen, and
transfection of cells in a variety of settings including gene therapy.
Accordingly, the present invention provides a variety of methods of
delivering a selected composition of matter to an organism.
Other features and advantages of the present invention will
become apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific
examples while indicating preferred embodiments of the invention are
given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the
drawings in which:
Figure 1A illustrates a synthetic scheme for the preparation
of PNIPAM-Gly-Py.
Figure 1B illustrates a synthetic scheme for the preparation
of PNIPAM-Gly.
Figure 2 illustrates changes in the cloud point of aqueous
PNIPAM-Gly-Py (full diamond) and PNIPAM-Gly (full circle) solutions as
a function of pH.
Figure 3 illustrates a fluorescence spectrum of PNIPAM-Gly-
Py in water.
Figure 4 illustrates the structure of the polymer of PNIPAM-
Gly-Py- of the present invention.
Figure 5 illustrates a fluorescence spectra of PNIPAM-Gly-
Py-- in aqueous solution without (top) and with cationic liposomes
(bottom).
Figure 6 illustrates changes in the ratio, IE / IM of excimer to
monomer emission intensities as a function of lipid concentration in
mixtures of PNIPAM-Gly-Py-- with liposomes of various types.
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Figure 7 illustrates changes in the ratio, IE/IM of excimer to
monomer emission intensities as a function of the amount of charged
surfactant in mixtures of PNIPAM-Gly-Py-- with liposomes of different
types.
Figure 8 illustrates changes in the ratio IE/IM of excimer to
monomer emission intensities in PNIPAM-Gly-Py-- mixtures with
liposomes as a function of lipid concentration in the absence of added salt.
Figure 9 illustrates electropherograms of (a) PNIPAM-Gly-
Py-- solution, (b) -(EO)2C18H3~/cholesterol/DDAB (7.5:2:0.5) liposomes, (c) -
mixture of PNIPAM-Gly-Py-- with (EO)2C18H3~/cholesterol/DDAB
(7.5:2:0.5) liposomes.
Figure 10 shows a binding curve for mixture of PNIPAM-
Gly-Py- with (EO)2C18H3~/cholesterol/DDAB (7:2.5:0.5) liposomes.
Figure 11 shows changes of the cloud point of PNIPAM-Gly-
Py-/liposome mixtures as a function of lipid concentration.
Figure 12 shows changes of the cloud point of PNIPAM-Gly-
Py/liposome mixtures as a function of the amount of cationic surfactant.
Figure 13 illustrates an idealized representation of the
interactions between a cationic NPL and PNIPAM-Gly-Py--.
Figure 14 illustrates the structure of PNIPAM-Gly of the
present invention.
Figure 15A illustrates leakage of NPL coated with PNIPAM-
Gly in response to solution acidification showing the extent of leakage
after 2000 sec as a function of final solution pH.
Figure 15B illustrates leakage of NPL coated with PNIPAM-
Gly in response to solution acidification and illustrates time trace of
leakage initiated by acidification of the solution from pH 7.4 to 5.
Figure 16 shows fusion of NPL coated with PNIPAM-Gly
with target phospholipid liposomes triggered by solution acidification.
Figure 17 shows images of cells treated with liposomes
where part (a) in the figure illustrates those coated with PNIPAM-Gly, and
part (b) illustrates those which are uncoated.
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Figure 18 shows the generic structure of a copolymer of the
present invention where Structure 1 shows hydrophobic groups grafted
along the polymer chain, and structure 2 shows the hydrophobic groups
attached at one end of the chain.
DETAILED DESCRIPTION OF THE INVENTION
As already mentioned, the present invention comprises a
new type of pH and temperature sensitive polymer that possesses not only
temperature-responsive groups and pH-responsive groups, but also
hydrophobic groups which, in a preferred embodiment serve as anchors in
a lipid bilayer. According to a preferred embodiment the hydrophobic
group consists of an octadecyl chain linked to a secondary amide nitrogen.
According to a preferred embodiment the copolymer has the following
formula:
~(CH=r-CH)x-'(CH~ H)y--(CH~H)z"-
~=O
NH NH
~ R
H C"CH3
C OOH
PNIPAM-Glv an~paration of CoyolJ~xners
The copolymer of the present invention, PNIPAM-Gly,
shown in Figure 1(with marker Py) (Spafford, M. et al., 1998) consists of a
PNIPAM chain that carries, at random, approximately 15 mole percent of
glycine residues as well as a small number of hydrophobic groups. This
unique copolymer combines the phase transition characteristics of
PNIPAM (Scarpa, J.S. et al., 1967; Schild, H.G. 1992), with the
responsiveness to changes in pH, via protonation/deprotonation of the
glycine carboxyl groups. In neutral water the copolymer forms polymeric
micelles, with an average diameter of 20 nm, as a result of inter- and
intrapolymeric aggregation of the hydrophobic substituents (Spafford, M.
et al., 1998). The hydrophobic core of such micelles is composed of
octadecyl groups and, if desired for experimental spectroscopic analysis,
pyrene (Py) labels (shown in Figure 1). It is surrounded by a corona
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consisting of hydrated poly-(N-isopropylacrylamide) moieties and partially
neutralized glycine residues.
Other pH and temperature sensitive copolymers which
have been used for modification of phospholipid liposomes are based on
the copolymerization of NIPAM (N isopropylacrylamide) with acrylamide
in aqueous solutions. This produces a copolymer modified with
carboxylate groups located very close to the polymer backbone.
Furthermore, the copolymer produced is restricted in the number and
type of modified polymers that can be synthesized. In contrast, chemical
synthesis of the the copolymer of the present invention permits many
variations on the basic structural theme of PNIPAM-Gly. By substituting
Gly with any other natural or non-natural amino acid you can make
PNIPAM-NAA where NAA is any natural or non-natural amino acid.
The polymers included within the scope of the present
invention are described as follows. The generic polymer name is
hydrophobically-modified copolymers of N-Isopropylaczyl:arnide and N-
acryloylacid (HM-PNIPAM-co-NAA), with the formula:
----(CH=.---CH)x--(CHI --CH)y---(CH~H)z--
=O
NH NH ~H
I~ I
HsC"CH3 R R
C OOH
(first formula)
or
.---NCH=--~H)xr/CH=--CH)y ~.-X-NHR
p C=O
NH NH
~ R'
H3C"CH3 I
COOH (second formula)
where 11=CnH2n+1 (n- 12, 14, 16, 18); R' is selected from the group
consisting of: a hydrophobic amino acid; an hydroxylated amino acid, a
30 charged amino acid; and an aromatic amino acid; and [ x = 20 to 80, y = 20
to 80, z = 1 to 5 (where x + y + z = 100) first formula] and [ x = 20 to 80, y
= 20
to 80 (where x + y = 100) second formula]. Preferably, R' = CH2; CH2-CH2;
CH(CH3); CH(iPr);CH(CH2~); CH[CH2)CH(CH3)2]; CH(iBu); CH(secBu);
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CHZCH2SCH3; CH2(C3N2H4+); (CHZ)4NH2; CH20H; or CH2 CH2 CH2
CH2NH3;andx=20to80,y=20to80,z=1 to5(wherex+y+z=100).
More preferably, R' = CH2 (GLYCINE), and x = 20 to 80, y = 20 to 80, z = 1 to
(where x + y + z = 100). In respect of the structure where hydrophobic
5 groups are attached at one end of the chain, X= an attachment group such
as an amine.
The copolymers of the present invention consist of three
components: 1) NIPAM which provides the temperature sensitivity; 2)
NAA which provides pH sensitivity (and H-bonding capability); and 3)
HM which acts as a hydrophobic anchor in the liposome bilayer.
The composition of the copolymer can be varied: a) by
changing the molar ratio of the three co-monomers, x, y, and z;or x and y
for second formula); b) by using various N-acyloylacid derivatives
(obtained from natural or unnatural amino acids); and c) by using
anchoring groups of various hydrophobicity. According to a preferred
embodiment, R=C18H3~ ; R' = CH2 (GLYCINE) and x = 20 to 80, y = 20 to 80,
z = 1 to 5 (where x + y + z = 100 first formula) x = 20 to 80, y = 20 to 80,
where x + y = 100, for second formula) which is referred to herein as
PNIPAM-Gly. All copolymers are prepared in a two-step procedure
involving a free radical copolymerization in an organic solvent of
NIPAM, HM, and N-AA alkyl ester, where the alkyl group is methyl, ethyl
and the like, and subsequent hydrolysis of the NAA alkyl ester group.
This permits a random distribution of the three comonomers. As set out
above, the present invention contemplates that the HM-PNIPAM-co-
NAA polymers can be prepared with two different architectures,
depending on the point of attachment of the hydrophobic group: a) grafted
along the polymer backbone (Figure 18, structure 1) or attached at one end
of the polymer chain (Figure 18, structure 2). Optionally, for experimental
tracking and spectroscopic analysis, the polymers can be labelled with a
fluorescent dye (for tracking), or a pyrene group (for spectroscopic analysis)
linked to a secondary amide nitrogen (see Figure 1A for the preparation of
PNIPAM-Gly which incorporates pyrene (Py)).
Non-phospholipid and phospholipid liposome complexes
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The copolymer of the present invention has direct
application to phospholipid and non-phospholipid liposomes. According
to a preferred embodiment, non-phospholipid liposomes are coated with a
copolymer of the invention. Non-phospholipid liposomes form
complexes with responsive polymers very efficiently. Compared to
phospholipid liposomes, coated non-phospholipid liposomes: 1) have
high encapsulation capacity; 2) outstanding chemical stability; and 3) they
exhibit higher release efficiency under identical conditions. Non-
phospholipid liposomes may be prepared from a variety of constituents.
The type of non-phospholipid liposome and combinations of constituents
to prepare them are well known to those skiled in the art. Preferably, non-
phospholipid liposomes of the present invention are prepared with a
weight ratio of n-octadecyl diethylene oxide, cholesterol and
dimethyldioctadeclammonium bromide of 7.5: 2: 0.5, respectively.
Phospholipid liposomes can also be prepared from a variety of
constituents. The type of phospholipid liposome and combinations of
constituents to create them are well known to those skiled in the art.
Preferably liposomes of the present invention are prepared wherein the
liposome is composed of dimyristoyl phosphatidylcholine, cholesterol,
dimethyldioctadecylammonium bromide in a weight ratio of 7.0:2:0.5,
respectively. While methods of preparation of both phospholipid and
non-phospholipid liposomes are well known in the prior art, details of
preparation of liposomes are set out in the examples detailed below.
Applications
By providing a highly versatile temperature and pH
responsive non-phospholipid liposome, the present invention greatly
expands the range of lipid compositions of liposomes that can be used in
application. Currently, mostly only phospholipid liposomes and
phospholipid liposomes coated with polyethylene glycol (or other similar
molecules) are used. Accordingly, the present invention provides a
method of treating an animal in need of a therapeutic agent comprising
administering a drug delivery composition comprising liposomes selected
from the group consisting of phospholipid liposomes and non-
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phospholipid liposomes. These liposomes are, according to the present
invention coated with a coploymer having the formula:
---(C Htr-C H
)x--(C H~ --C
H )y---(C H~
H)z-
=p ~-p
NH NH NH
~ R
C~CH
~
Hs C OOH
or ----(CHZ--~H)xr(CH=--CH)y --X-NHR
C=O
I NH NH
~ R'
HsC"CH3 t
COOH X~ a~chment group
where in either structure R=CnH2n+1 (n- 12, 14, 16, 18); R' is selected from
the group consisting of: a hydrophobic amino acid; an hydroxylated amino
acid, a charged amino acid; and an aromatic amino acid; x = 20 to 80; y = 20
to 80; and z = 1 to 5; where x + y + z = 100 (first formula), and x = 20 to
80; y
= 20 to 80; where x + y = 100 (second formula) wherein the liposome
contains the therapeutic agent.
Preferably this method is practised where R' = CH2; CH2-
CH2; CH(CH3); CH(iPr);CH(CH2~); CH[CH2)CH(CH3)2]; CH(iBu); CH(secBu);
CH2CH2SCH3; CHZ(C3NZH4+); (CH2)4NH2; CH20H; or CH2 CHZ CH2
CH2NH3. More preferably, the method is practised where R' is CH2and
R=C18H3~. Where the method is practised with a non- phospholipid
liposome the liposome is preferably composed of n-octadecyl diethylene
oxide, cholesterol and dimethyldioctadeclammonium bromide. Here, the
preferred weight ratio of n-octadecyl diethylene oxide, cholesterol and
dimethyldioctadeclammonium bromide is 7.5: 2: 0.5, respectively. W h a r a
the method is practised with phospholipid liposomes they are preferably
composed 20 of dimyristoyl phosphatidylcholine, cholesterol, and
dimethyldioctadecylammonium bromide. The preferred ratio here is
7.0:2:0.5, respectively.
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Set out herein are a number of examples of applications of
liposomes of the present invention. These are in no way intended to be
limiting of the scope of applications for this invention. Rather, they are
merely illustrative.
Vaccine technologies
Liposomes have already been demonstrated to have
adjuvant properties. The stabilizing effect of the coat copolymer of the
present invention would increase the variety of lipid compositions that
could be used. In addition, liposomes that become unstable when the pH
drops from 7.5 to 5 would be useful to stimulate cell mediated immunity
as they would release the antigen for cell mediated presentation by MHC
class II by leakage after endocytosis and for presentation by MHC class I by
fusion with the endosomal membrane and release of the antigen into the
cytoplasm. Stabilized yet regulated release liposomes might be more
useful for the generation of mucosal immunity or for the delivery of DNA
(or RNA) into cells in DNA (or RNA) vaccines.
Drug Delivery
Liposomes of the present invention may be conveniently
"loaded" with a target substance according to techniques well known to
those skilled in the art, see for example I. Lasic, "Liposomes in Gene
Delivery" CRC Press, Boca Raton, 1997 Chapter 6 and the references
contained therein. For drug delivery applications NIPAM co-polymers can
be used to both stabilize liposomes and regulate release of liposome
contents (contents could include drugs, nucleic acids, proteins etc.).
Release can be either pH or temperature sensitive. Release can also be
both pH and temperature sensitive. Thus vesicle contents can be
selectively released by endocytosis (due to the drop in pH from 7.5 to 5 that
occurs in endocytic vesicles after internalization) or directed to a specific
tissue or tumour by selective heating of the area (by microwaves or
ultrasound, or light or any other form of radiation having sufficient
energy to bring about modification).
According to one embodiment the present invention
contemplates a method for treating tumors in a subject comprising
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administering to the subject an effective amount of a drug delivery
composition comprising liposomes containing at least one
chemotherapeutic agent wherein the liposomes deliver the agent to the
target cells.
The drug delivery compositions may be delivered to a target
site through a variety of known routes of administration. For example, a
drug delivery composition comprising liposomes containing a given
pharmaceutical compound for treating an animal, for example a human,
in need of the pharmaceutical compound may be administered by
intravenous injection. Alternately in treating a tumor a drug delivery
composition comprising liposomes containing a given pharmaceutical
compound may be administered by intratumor injection. Other
administration routes may include oral delivery; rectal delivery and
topical adminstration such as creams for dermal or transdermal
application, ophthalmic application, containing drugs such as antibiotics,
antifungal agents, anticancer drugs, antiglucoma agents, anti-
inflammatory, and analgesic agents. Accordingly, the present invention
provides a variety of drug delivery compositions comprising liposomes
which may be either non-phospholipid or phospholipid liposomes,
wherein the liposome used is coated with a coploymer having the
formula:
~(CH=.- H)x""(CH=--CH)y--(CH H)z~-
=4 =p
NH NH NH
~ R
H C"CH3 I
COOH
or
Hz-~CH)y ---~X--NHR
~~O
NH
R'
OOH
X~ attachment ~~oup
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where in either case R=CnH2"+i (n- 12, 14, 16, 18); R' is selected from the
group consisting of: a hydrophobic amino acid; an hydroxylated amino
acid, a charged amino acid; and an aromatic amino acid; x = 20 to 80; y = 20
to 80; and z = 1 to 5; where x + y + z = 100 (first formula); and x = 20 to
80; y
= 20 to 80; where x + y = 100 (second formula) and the liposome contains
at least one therapeutic agent. Preferably the drug delivery composition
contains liposomes coated with a copolymer where R' = CH2; CH2-CH2;
CH(CH3); CH(iPr);CH(CH2~); CH[CH2)CH(CH3)2]; CH(iBu); CH(secBu);
CH2CH2SCH3; CHZ(C3N2H4+); (CH2)4NH2; CH20H; or CH2 CH2 CH2
CH2NH3. and more preferably R' is CH2 and R=C18H3~. Where the drug
delivery composition contains non-phospholipid liposomes they should,
preferably be composed of n-octadecyl diethylene oxide, cholesterol and
dimethyldioctadeclammonium bromide. The preferred ratio of
combination of these lipids is 7.5: 2: 0.5, respectively. Where the drug
delivery composition contains phospholipid liposomes they are
preferably composed of dimyristoyl phosphatidylcholine, cholesterol,
dimethyldioctadecylammonium bromide, more preferably the lipids are
in a ratio of 7.0:2:0.5, respectively.
The drug delivery compositions of the invention can be
intended for administration to humans or animals. Dosages of a
therapeutic agent incorporated in the drug delivery composition will
depend on individual needs, on the desired effect and on the chosen route
of administration. As used herein, the term animal means all members
of the animal kingdom, including mammals, and preferrably, humans.
while the preferred targets for applications of the present invention is
animals, in no way is the invention limited to animals. included within
the scope of the present invention is any organism, whether unicellular or
mulitcellular, to which delivery of a selected composition is required.
Other uses
A liposome of the present invention may also be used in
targetted topical applications including heat activated sunscreen, heat or
pH activated deodorants, and the design of responsive cosmetics.
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Further, liposomes of the present invention may be used in
the transfection of cells with nucleic acids (DNA or RNA), with, for
example, application in gene therapy as ideal vehicles for the introduction
of nucleic acids into cells or tissues in animals in a controlled fashion (for
example by local heating of a tissue in which the gene is to be introduced).
The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
A. Materials and Instrumentation For Examples 1 to 5
Materials
Water was deionized with a Barnstead NANOpureTM water
purification system. Dioxane and tetrahydrofuran (THF) were distilled
from sodium under nitrogen. 4-(1-Pyrenyl)butyric acid, octadecylamine,
carbonyldiimidazole, and N,N-azobis-isobutyronitrile (AIBN) were
obtained from Aldrich Chemical Company Inc. N-acryloxy-succinimide
(NASI) was obtained form Acros Chemicals. Di-tert butylcresol and glycine
ethyl ester hydrochloride were purchased from Sigma. N-isopropylamine
(NIPAM) was a gift from Kohjin Company Ltd. (Lot # H021225). It was
recrystallized from a toluene:hexane (1:1, v/v) mixture. Thin layer
chromatography (TLC) was performed with silica plates (Merck) eluted
with CHZC12/MeOH (9/1 v/v). N-[4-(1-Pyrenyl)butyl]-
N-n-octadecylacrylamide (2) was prepared as described previously
(Yamazaki, A. et al., 1998).
Instrumentation
Proton nuclear magnetic resonance (NMR) spectra were
recorded on Bruker 200 MHz and 500 MHz spectrometers. Infrared spectra
were recorded on a BioRad FTS-40 spectrometer. Ultra violet (UV) spectra
were measured with a Hewlett Packard 8452A photodiode array
spectrometer, equipped with a Hewlett Packard 89090A temperature
controller. Potentiometric titrations were performed using a Tanager
Scientific Systems 8901 dual pH meter and titrimeter. Gel Permeation
Chromatography (GPC) measurements were performed with a Waters 590
programmable high pressure liquid chromatography (HPLC) system
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(eluent: 0.1 M NaN03, flow rate of 0.7 mL/min, Ultrahydrogel columns
(Waters)) equipped with a Waters 486 UV detector and a Waters 410
Differential Refractometer. Dynamic light scattering was performed on a
Brookhaven BI9000 AT instrument equipped with an argon laser (~, = 514
nm, scattering angle: 90 degrees). Fluorescence spectra were recorded on a
SPEX Industries Fluorolog 212TM spectrometer equipped with a DM3000F
data analysis system The slits were set at 1.0 nm (emission) and 1.0 nm
(excitation). The excitation wavelength was 330 nm.
Example 1
Copolymerization of N-isopropylacrylamide, N-acryloxysuccinimide, and
N-[4-(1-pyrenyl)butyl]-N-n-octadecylacrylamide.
NIPAM (1.81 g, 16 mmol), NASI (0.48 g, 2.8 mmol), and
N-[4-(1-pyrenyl)butyl]- N-n-octadecylacrylamide (0.11 g, 0.02 mmol) were
dissolved in dry dioxane (40 mL) and degassed with N2 for 15 minutes.
The mixture was heated to 75°C. AIBN (0.04 g) was added to
initiate the
polymerization. After 15 hrs the mixture was cooled to room
temperature. The polymer was isolated by precipitation into diethyl ether
and purified by precipitations from dioxane into diethyl ether (800 mL).
The white, fluffy copolymer (PNIPAM-NASI-Py, 1.85 g) was dried in vacuo
for 2 hours at room temperature. 1H NMR (CDCl3) [200 MHz] 81.12 (broad,
48H, -CH-CH2-CH- & -CH3 & (CH2)15)~ 1.67 (broad, -CH2-CH-CH2- &
- N - C H 2- C H 2-), 1.93-2.20 (broad, 4H, -N-CH2-C H 2- C H 2- &
-N-(CH2)2-CH2-CH2-Py), 3.09-3.15 (broad, 4H, CO-CH2-CH2-CO), 4.46-4.52
(broad, 7H -CH-NH- & Py-CH2 & N-CHZ), 5.78-6.95 (broad, 1H, NH); FT IR
(KBr pellet) (cm-1): 3435.6 (N-H stretch), 3314.4 (interpolymeric
H-bonding), 3070.6 (vinylic, C-H stretch), 2933.2 (CH3 stretch), 2875.6 (CH2
stretch), 1737.4 (OC-N-CO), 1652.4 (CO stretch), 1459.4 (CH2 scissors mode).
Example 2
Reaction of PNIPAM-NASI-Py with glycine ethyl ester hydrochloride.
Triethylamine (0.4 mL, 2.9 mmol) and glycine ethyl ester hydrochloride
(0.21 g, 1.5 mmol) were added consecutively to a solution of
PNIPAM-NASI-Py (1.51 g) in dry THF (50 mL). The mixture was stirred at
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room temperature for 18 hours under NZ. Distilled isopropylamine (1.0
mL, 0.01 mmol) was added to quench the excess succinimide groups. The
resulting mixture was stirred for 2 hr. The polymer was isolated by
precipitation into diethyl ether and purified by two precipitations from
THF into diethyl ether; (1.51 g, white powder). 1H NMR (CDCl3) [500 MHz]
8: 0.85 (t, 3H, CH3), 1.12 (broad, 30H + 3H, (CHZ)i5 + CH3), 1.34 (d, 6H,
CH3),
1.62 (broad, 6H+2H, -CH-CH2-CH- + -N-CH2-CH2-), 1.78 (broad, 3H,
- C H 2- C H - C H 2-), 2.15 (broad, 4H, -N-CH2- C H Z- C H 2- &
-N-(CHZ)2-CH2-CH2-Py), 2.64 (s, 2H, -NH-CH2), 3.96 (broad, 7H, CH-NH &
Py-CHZ & -N-CH2), 4.15 (broad, 2H, CO-O-CH2-CH3), 7.83-8.23 (m, 9H, CH2,
Py); FT IR (KBr pellet) (cm-1): 3438.3 (N-H stretch), 3307.4 (interpolymeric
H bonding), 3057.9 (vinylic, C-H stretch), 2972.3 (CH3 stretch), 2876.1 (CH2
stretch), 1655.7 (CO stretch), 1460.0 (CH2 scissors mode).
Example 3
Deprotection of PNIPAM-Gly ester-Py. Sodium hydroxide (0.098, 2.2
mmol) dissolved in THF/H20 (4/1 v/v, 10 mL) was added to a solution of
PNIPAM-Gly ester-Py (1.12 g) in THF/H20 (4/1 v/v, 50 mL). The solution
was kept at room temperature for 15 hours. Then the pH of the solution
was adjusted to 3.35 with 0.1 N HCI. The solvent was concentrated until a
yellow oil separated from the aqueous phase. The aqueous phase was
decanted and the remaining oil was dissolved in THF (5 mL). This
polymer was isolated by precipitation into diethyl ether (800 mL) and dried
in vacuo for two hours yielding PNIPAM-Gly-Py as a white powder (0.73
g). 1H NMR (CDCl3) [500 MHz] 8: 0.86 (t, 3H, CH3),1.12 (broad, 39H, (CH2)15
& CH3), 1.62 (broad, 8H -CH-CH2-CH2 & -N-CH2-CH2), 1.80 (broad, 3H,
- C H 2 - C H - C H 2 ), 2.05 (broad, 4H, -N-CH2- C H 2- C H 2- &
-N-(CH2)2-CH2-CH2-Py), 3.98 (broad, 7H, CH-NH & Py-CH2- & -N-CH2),
6.31 (broad, 2H, NH), 7.83-8.23 (m, 9H, CH2, Py); FT IR (KBr pellet) (cm-1):
3435.5 (N-H stretch), 3309.9 (Interpolymeric H bonding), 3073.3 (vinylic,
C-H stretch), 2973.2 (CH3 stretch), 2876.1 (CHZ stretch), 1649.9 (CO stretch),
1459.5 (CH2 scissors mode).
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Example 4
Preparation of PNIPAM-Gly ester. A copolymer of NIPAM and NASI
(PNIPAM/NASI, 2.52 g) was prepared as previously described (Winnik,
F.M., 1990), starting from NIPAM (4.51 g, 40 mmol) and (NASI) (1.02 g, 6.0
mmol. This polymer (0.51 g) was dissolved in THF (15 mL).
Triethylamine (0.06 mL, 0.4 mmol) and glycine ethyl ester hydrochloride
(0.06 g, 0.4 mmol) were added in turn to the solution. The mixture was
stirred at room temperature, under N2, for 18 hours. Isopropylamine (0.5
mL, 0.008 mmol) was added to quench the unreacted hydroxysuccinimide
groups and the mixture was stirred for 2 hr. The polymer was isolated by
precipitation into hexane (700 mL). It was purified by further
precipitations from MeOH (30 mL) into diethyl ether (700 mL). The
collected fluffy, white polymer was dried in vacuo at room temperature
for 3 hours. 1H NMR (CDC13) [200 MHz] b: 1.13 (broad, 3H, CH3), 1.38 (d,
6H, CH3), 1.66 (broad, 4H, -CH-CH2-CH-), 2.15 (broad, 2H, -CH2-CH-CH2-),
3.47 (broad, 2H, CH2, diethyl ether), 3.94 (broad, 3H, -NH-CH- & -NH-CH2-);
FT IR (KBr pellet) (cm-1): 3435.7 (N-H stretch), 3293.1(H bonded), 3057.6
(vinylic, C-H stretch), 2973.4 (CH3 stretch), 2877.1 (CH2 stretch), 2362.0
(C02), 1646.2 (CO stretch),1460.3 (CH2 scissors mode).
Example 5
Deprotection of PNIPAM-Gly ester. The procedure used for the
preparation of PNIPAM-Gly-Py was followed, starting with PNIPAM-Gly
ester (0.33 g). PNIPAM-Gly was isolated as a white powder (0.07 g); 1H
NMR (CDCl3) [500 MHz] 8: 1.12 (broad, 6H, CH3), 1.60 (broad, 4H,
-CH-CH2-CH-), 1.78 (broad, 2H, -CH2-CH-CHZ-), 3.47 (s, 3H, CH3, MeOH),
3.98 (broad, 3H, -NH-CH- & -NH-CH2-), 6.19 (broad, 2H, NH); FT IR (KBr
pellet) (cm-1): 3495.8 (N-H stretch), 3073.9 (vinylic, C-H stretch), 2972.7
(CH3 stretch), 2875.8 (CH2 stretch), 1651.8 (CO stretch), 1459.5 (CH2 scissors
mode).
Cloud Point Determinations. Cloud points were determined by
spectrophotometric detection of the changes in transmittance (~, 600 nm)
of aqueous polymer solutions (1 g L-1) heated at a constant temperature
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(0.2 °C) in a magnetically stirred UV cell. The value reported is the
temperature at which transmittance of the solution decreased by 20 % of
its value at 25 °C. The following buffers were employed to prepare
solutions of controlled pH: pH 2: 0.2 M KH2P04, pH 2.8 to 4.71: 0.1 M citric
acid; 5.9: 0.2 M KH2P04; pH 7.4: 0.1 M Tris. All solutions contained 0.1 M
NaCI.
DISCUSSION OF THE RESULTS OF EXAMPLES 1 TO 5
The selection of the synthetic sequence employed was
dictated on the one hand by the need to obtain a random distribution
along the polymer chain of the three units, NIPAM, the pyrenyl-octadecyl
amide and glycine and, on the other, by the conflicting solubility
properties of the three structural motifs. While N-isopropylacrylamide
(NIPAM) is soluble in water as well as in several organic solvents, glycine
itself is only soluble in water and pyrenyl-octadecyl compounds are soluble
only in organic media. Previous work on the copolymerization of NIPAM
and aminoacid-derived acrylic monomers has taught us that
copolymerizations carried out in aqueous medium tend to lead to
copolymers with large segments of hydrophilic and hydrophobic units.
Therefore, we chose the slightly lengthy scheme depicted in Figure 1.
The target polymer, PNIPAM-Gly-Py, was obtained by
reaction of glycine ethyl ester with a copolymer of NIPAM,
N-acryloxysuccinimide (NASI) and N-4-(1-pyrenyl)-N-
(n-octadecylacrylamide), which itself was obtained by free radical
polymerization in dioxane of the corresponding acrylamides (Figure 1).
Reaction of the reactive copolymer with glycine methyl ester and
subsequent quenching of residual unreacted N-hydroxysuccinimide
groups with N-isopropylamine, led to the ethyl ester derivative of the
desired polymer. Evidence that the succinimide sites on the polymer have
reacted with the glycine ethyl ester is provided by the disappearance in the
1H NMR spectrum of a broad signal at S 3.09-3.15 ppm, attributed to the
resonance of the succinimide methylene protons, together with the
appearance of a signal at b 4.15 ppm, assigned to the methylene protons of
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the ethyl ester moiety. Also, in the IR spectrum the band at 1737 cm-1 due
to the imide carbonyl groups is replaced by a band at 1646 cm-1,
characteristic of the ester carbonyl stretch. Hydrolysis of the ester group
under mild alkaline conditions led to the desired copolymer.
Deprotection was confirmed by the loss in the 1H NMR spectrum of the
signal at 4.15 ppm. The 1H NMR spectrum of PNIPAM-Gly-Py also
exhibits signals characteristic of the NIPAM repeat units, namely a strong
singlet at 8 1.12 ppm and broad signals at 8 1.62 and 1.80 ppm attributed to
the methylene and methine protons of the polymer backbone. Evidence
for the incorporation of pyrenyl octadecyl groups is given by the presence
of a weak triplet at 8 0.86 ppm, due to the terminal methyl group of the
octadecyl chains and a multiplet (8 7.83- 8.23 ppm) attributed to the
aromatic protons of the pyrene substituents. A copolymer of NIPAM and
glycine-acrylamide, PNIPAM-Gly, was prepared by a similar route, starting
with a copolymer of NIPAM and N-acryloxysuccinimide, reacted first with
glycine ethyl ester, then with N-isopropylamine, and finally hydrolyzed
under mild alkaline conditions to deprotect the glycine ethyl ester
residues.
The physical properties of PNIPAM-Gly-Py and
PNIPAM-Gly are listed in Table 1. GPC was used to determine the
molecular of the polymer weight, calibrated against polyethylene oxide)
standards, and through the use in tandem of a UV detector and a
refractive index detector it was ascertained that PNIPAM-Gly-Py is not
contaminated with low molecular weight UV-absorbing impurities and
that all the chromophores are bound covalently to the polymer. The
glycine content was obtained by titration of the carboxylic acids, using
stepwise additions of HCl to a solution of fully ionized PNIPAM-Gly-Py.
The amount of pyrene incorporation (9.4 x 10-5 mol Py g-1 or 1 pyrene
chromophore for every 83 NIPAM units) was determined from UV
absorption data of polymer solutions in THF, using
N-[4-(1-pyrenyl)butyl]-N-octadecyl acrylamide (~344 = 37,000) as model
compound. This result was confirmed by the iH NMR spectrum of
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PNIPAM-Gly-Py, using the area of the singlet at 4.01 ppm due to the
resonance of the methine proton of the isopropyl groups of the NIPAM
units and the area of the triplet centered at 0.86 ppm attributed to the
terminal methyl protons of the octadecyl chains
Solution properties of the copolymers.
Both the hydrophobically-modified copolymer,
PNIPAM-Gly-Py, and PNIPAM-Gly, are soluble in water at or below room
temperature, independent on the pH of the solution. However, the
solutions become turbid when heated above a critical temperature,
indicating these copolymers each have a lower critical solution
temperature (LCST). As anticipated, the LCST of the NIPAM-Gly
copolymers exhibits a pronounced pH dependence. Under acidic
conditions, when the carboxylic acid groups are fully protonated, the cloud
points (or LCSTs) of PNIPAM-Gly-Py and PNIPAM-Gly each have values
of approximately 28°C (see Figure 2). These are slightly lower than the
LCST of PNIPAM (31°C), indicating a slight increase in
hydrophobicity of
the copolymers (Taylor, L.D., 1975). As the pH of the solution increases,
and the carboxylic acid groups take their ionized form, the LCST increases
and ultimately vanishes for pH values greater than 5 and 8.5 in the case of
solutions of PNIPAM-Gly-Py and PNIPAM-Gly, respectively (Figure 2).
Contrary to our expectations, the LCST of the hydrophobically-modified
copolymer was not significantly lower than that of the unmodified analog,
violating the general rule that the LCST of a copolymer decreases with
increasing hydrophobicity (Taylor, L.D., 1975). This observation is taken as
evidence that the alkyl chains are not exposed to water, but rather form
the inner core of micellar structures protected from the aqueous
environment by the PNIPAM-Gly chains (Winnik. F.M. et al., 1992).
Quasi-elastic light scattering measurements confirmed that in acidic
solutions PNIPAM-Gly-Py exist as assemblies 18 nm in diameter (pH 3.0,
0.1 M NaCI), while no micelles are formed in solutions of PNIPAM-Gly,
indicating the absence of interchain aggregation, a fact reported also in the
case of PNIPAM solutions below their LOST (Schild, H.G., et al., 1990;
Wirulik. F.M., 1990)
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Further evidence of the formation of polymeric micelles is
provided by the fluorescence spectrum of PNIPAM-Gly-Py (Figure 3) in
aqueous solution (pH 3, NaCI 0.1M). The spectrum is dominated by a
broad featureless emission centered at 490 nm due to pyrene excimers
(intensity IE) (Birks, J.B., 1970; Winnik, F.M., 1993) In addition, there is a
contribution from isolated excited pyrene (monomer emission, intensity
IM), with the [0,0] band located at 378 nm. The fact that the excimer
emission is very strong implies that the chromophores are kept in close
proximity (Winnik, F.M., 1993), incorporated into the core of the
polymeric micelles detected by quasi electric light scattering (QELS). The
influence of pH, temperature, and salt concentration on the solution
properties of the polymer are under current investigation, to unravel the
relative importance of electrostatic repulsion, hydrogen-bonding, and
hydrophobic interactions in guiding the formation and disruption of
polymeric micelles.
Outlined below are examples of the interactions of the
copolymer of the present invention with liposomes, using fluorescence
assays, a centrifugation assay, and capillary electrophoresis. The thermo-
and pH-sensitivity of the liposome/polymer complexes were monitored
by turbidity measurements. The results of these examples are outlined
with particular emphasis on the relative importance of electrostatic forces,
hydrophobic interactions, and hydrogen bonds in promoting the
formation of liposome/polymer complexes. Systems examined include
complexes of PNIPAM-Gly-Py (1) with cationic and anionic
non-phospholipid liposomes made up of n-octadecyldiethylene oxide and
cholesterol spiked with either the cationic surfactant
dioctadecyldimethylammonium chloride (DDAB) or the anionic
amphiphile dioctadecylphosphate (DP), and (2) with neutral and cationic
phospholipid liposomes. The results are interpreted in terms of the
relative importance of electrostatic interactions, hydrogen bond formation,
and hydrophobic interactions in guiding the formation and stability of the
various liposome/polymer complexes.
B. General Aspects of Materials Preparation for Examples 6 to 12
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Materials. Water was purified with a Barnstead NANOPure water
purification system. Dimethyldioctadecylammonium bromide (DDAB),
dioctadecylphosphate (DP), palmitoleoyl phosphatidylcholine (POPC),
dimyristoyl phosphatydylcholine (DMPC), and dipalmitoyl
phosphatidylcholine (DPPC) were purchased from Avanti Polar Lipids
(Alabaster, Alabama). Cholesterol, n-octadecyldiethylene oxide
(E02C18H3~), n-hexadecyldiethylene oxide (EO2C16H33), sodium dodecyl
sulfate (SDS) and calcein were obtained from Sigma (Sigma-Aldrich
Canada Ltd., Ontario, Canada). BODIPY-PC was supplied by Molecular
Probes. Tris, sodium azide (NaN3), sodium chloride (NaCI), and glycine
(Gly) were purchased from BDH Chemicals (VWR Scientific, Ontario,
Canada). A copolymer of N-isopropylacrylamide,
N-[4-(1-pyrenyl)butyl]-N-n-octadecylacrylamide, and N-glycydylacrylamide
(PNIPAM-Gly-Py), Mn 25,000, Mw/Mn 2.1 (from GPC data calibrated
against polyethylene oxide standards) was prepared as set out in Examples
1 to 5 of this specification.
Sample Preparation
Liposomes. A solution in chloroform of the desired amounts of lipids
(Table 1) was poured into glass test tubes. The solvent was evaporated
under a stream of nitrogen. The resulting lipid film was dried under high
vacuum for at least two hours. The dry lipid film was hydrated in an
aqueous solution of NaCI (150 mM) and NaN3 (0.02% w w) to give a lipid
suspension of a concentration of 20 g/L. The lipid suspension was
subjected to extrusion through polycarbonate filter membranes (200 nm
pore size, Nucleopore) using a Lipofast extruder (Avestin, Canada). The
hydration and extrusion of the non-phospholipid DMPC and
phospholipid POPC liposomes were performed at a temperature of 60 and
25°C, respectively.
Liposome-polymer mixtures. Liposome suspensions were added to
polymer solutions in the desired proportions. The mixtures were allowed
to equilibrate first at room temperature (30 minutes), then at the
temperature of the experiment (30 minutes)
Methods
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Dynamic light scattering (DLS). The mean diameter of polymer micelles
was evaluated by dynamic light scattering using a Brookhaven Instrument
Corporation Particle Sizer Model BI-90 (Holtsville, NY). The data
acquisition time in a typical experiment was 1000 s and the average size
obtained from three independent measurements was taken as the mean
diameter.
Fluorescence measurements. Fluorescence spectra were recorded with a
SPEX Fluorolog 212 spectrometer (Edison, NJ) equipped with a DM3000F
data system. The temperature of the water-jacketed cell holder was
controlled with a Neslab (Portsmouth, NH) circulating bath. The
temperature of the sample fluid was measured with a thermocouple
immersed in the sample. Emission spectra were recorded with excitation
wavelengths of 330 nm (pyrene) or 585 nm (BODIPY). They were not
corrected. In experiments aimed at monitoring the adsorption of
PNIPAM-Gly-Py on liposomes, the ratio IE/IM of the excimer emission
intensity to the monomer emission intensity was calculated as the ratio of
the emission intensity at 480 nm (excimer) to the half-sum of the emission
intensities at 377 nm and 397 nm (monomer).
Capillary Electrophoresis: Measurements were performed with an ATI
Unicam Crystal CE Model 310 instrument connected to a Waters 991
Photodiode array UV-Visible detector equipped with a microcell allowing
on-capillary detection. Data were collected and processed with the
MilleniumTM 2010 (revision 2.1) software from Waters. Bare fused silica
capillary tubing was purchased from Polymicro Technologies (100 ~.m I. D.,
365 ~.m O. D.). The distance between the anode and the on-column
detector window was 60 cm, and the distance from detector to grounded
cathode was 30 cm. Capillaries were conditioned by flushing 0.5 M NaOH
at 2000 millibar for 15 min prior to use to restore the negative charges on
the capillary walls. This treatment was followed by equilibration with the
running buffer. All experiments were conducted in the constant voltage
mode at fixed temperature. Hydrodynamic injections at a pressure of 20
millibar were performed to introduce the sample (390 nL) into the
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capillary. For each sample, three or four consecutive runs were performed
under identical conditions
Centrifugation assay. Liposomes were prepared by simple hydration of dry
lipid films prepared as described above, starting with a lipid mixture
containing a small amount of the fluorescent label BODIPY (0.1 mol%).
Liposome/polymer mixtures were prepared and equilibrated. They were
poured into Millipore UltrafreeMC ultrafiltration test tubes equipped with
inserts featuring 100 nm pore size WPP membrane. These inserts retain
the large liposomes (200 nm), but not the free polymer micelles (20 nm),
which easily diffuse through the membrane. After centrifugation (4000 g),
the insert was removed from the tubes and two fractions were separated.
The upper fraction containing the liposomes was collected, resuspended in
fresh buffer and subjected to the same centrifugation procedure. The lipid
concentration was evaluated using the fluorescence of BODIPY-PC. A
small aliquot (50-200 ~.L) of a sample containing up to 0.100 mg of lipid
was mixed with an aqueous solution of SDS (lmL, 70 mM) in order to
achieve complete solubilization of the liposomes and to ensure the
absence of dye self-quenching. The fluorescence intensity of the dye was
calibrated using various solutions of known NPL/BODIPY-PC
concentrations. The BODIPY fluorescence intensity increased linearly with
NPL concentrations up to 1.5 g/L. The polymer concentration in the
bottom fractions was evaluated using Py fluorescence. All samples were
solubilized in excess SDS to prevent pyrene excimer formation. Typically,
an aliquot (100 ~L) taken from a bottom fraction, containing up to 100 ~.g of
polymer, was mixed with an aqueous solution of SDS (1 mL, 50 mM).
Under these conditions, excimer emission did not occur in solutions of
polymer concentrations up to 500 ~.g/mL, while monomer emission
intensity at 377 nm increased linearly with polymer concentration.
The equilibrium constant for the polymer/liposome
systems was evaluated assuming that the following equilibrium takes
place:
Pf + L <-> Pb
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where Pf and Pb refer to the free and liposome-bound polymer,
respectively, and L refers to the lipid concentration. The equilibrium
constant K is given by: K = [Pb] / [Pf] [L] .
Cloud Point measurements. Changes in turbidity (~, = 600 nm) of
polymer/liposome mixtures with increasing temperature were
determined using a Hewlett Packard 8452A photodiode array spectrometer
equipped with a Hewlett Packard 89090A temperature controller and
operated under the HP Chemstation Windows-based software. The
solutions were heated at a constant rate (0.5 ~C min-1) in a magnetically
stirred cell. The value reported is the temperature corresponding to the
90% transmittance point.
Example 6 - Fluorescence studies
The fluorescence spectrum of PNIPAM-Gly-Py in water
displays two distinct emissions: a broad and strong signal, centered at 480
nm, due to pyrene excimers which originate from chromophores located
in close proximity to each other and a well-resolved emission of lower
intensity, the pyrene monomer emission, with the (0,0) band at 378 nm,
due to locally isolated excited pyrenes (Figure 5). The excimer emission is
very strong in this case, since the hydrophobic pyrene groups are found
mostly within the core of the micellar assemblies formed by the polymer
in neutral aqueous solution. The addition of PNIPAM-Gly-Py to a
suspension of liposomes triggers remarkable changes in the emission of
the labelled polymer. Most noticeable is a sharp increase of the intensity of
the monomer emission at the expense of the excimer emission, indicating
disruption of the polymeric micelles. The effect is illustrated in Figure 5,
which presents spectra of the copolymer in water and in the presence of
cationic NPL's. The decrease in excimer emission signals the
incorporation of the hydrophobic groups into the lipid bilayer, as the
originally tightly packed fluorophores diffuse away from each other
within the lipid membrane. We used the change in the ratio IE/IM of
pyrene excimer to monomer emission intensities as a measure of the
degree of polymer binding to liposomes.
xam 7
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The interactions between cationic liposomes and PNIPAM-
Gly-Py in neutral solution were studied. In this situation, the
liposome/copolymer interactions are expected to be promoted by attractive
electrostatic forces. Two types of vesicles were used: (a) non-phospholipid
liposomes consisting of a non-ionic surfactant (EO)2C1gH3~, cholesterol,
and the cationic surfactant dioctadecyldimethylammonium bromide
(DDAB) and (b) POPC liposomes rendered cationic by incorporation of
DDAB (Table 2). The measurements were carried out at constant salt
concentration (0.15 M NaCI) in a neutral buffer. Increasing amounts of
liposomes were added to a solution of constant polymer concentration (2
mg L-1). This triggered a large decrease of the pyrene excimer emission and
a concomitant increase of the pyrene monomer emission, signaling the
incorporation of the pyrene groups within the liposome membrane. As
more liposomes were added the excimer emission continued to decrease,
until it reached a constant value. These changes are recorded in a plot of
the changes in the ratio IE / IM as a function of lipid concentration
presented in Figure 6. Since unilamellar vesicles were used, the amount
of lipids corresponds well to the liposome concentration. Some level of
uncertainty is introduced by the fact that there is a distribution of the
liposome size, estimated by quasi-elastic light scattering measurements as
200 ( 20 nm. Overall, the value of IE/IM dropped from 1.1 in the absence of
liposomes to 0.1 after saturation was reached. In the case of cationic NPL's
a sharp decrease in excimer intensity upon addition of liposomes was
observed up to a polymer/lipid ratio of 1:5 (w/w) (or 8 x 10-4 mol/mol).
The cationic phospholipid liposomes exhibited the same type of
interactions with PNIPAM-Gly-Py, as judged from the decrease in IE/IM
upon addition of POPC/DDAB liposomes to a solution of PNIPAM-Gly-Py
(Figure 6). We note, however, that for similar membrane charge density,
provided by 5 mol % of DDAB, the ratio IE/IM dropped to a value of 0.6 in
the case of the phospholipid liposomes, whereas in the case of NPL's IE/IM
took a value of 0.1.
As a control experiment neutral POPC liposomes were
added to a solution of PNIPAM-Gly-Py. In this case the emission spectrum
CA 02254554 1998-11-20
of the polymer was hardly affected, implying that in the absence of
electrostatic attraction the interactions between phospholipid liposomes
and PNIPAM-Gly-Py are weak and that, consequently, a large fraction of
the polymer remains in solution in the form of unperturbed micelles. To
complete the study we tested as well the behavior of the copolymer in the
presence of negatively-charged liposomes. We used NPL's consisting of
(EO)2C18H3~, cholesterol, and dioctadecylphosphate (DP, 5 mol %). To our
surprise, the changes in IE/IM of PNIPAM-Gly-Py as a function of added
anionic NPL's concentration were identical to those observed upon
addition of cationic NPL's (Figure 7). This important finding points to the
existence of interactions between the copolymer and these NPL's,
overcoming the unfavorable electrostatic interactions anticipated to occur
between the phosphate groups on the liposomes and the carboxylate
moieties of the copolymer.
ExamEle 8
The effect of liposome charge density on the formation of
polymer/liposome complexes was studied. Cationic liposomes, either
POPC liposomes or NPL's containing increasing amounts of DDAB, from
2 to 30 weight percent, or anionic NPL's with increasing amounts of DP
were employed in these experiments. The results obtained for a constant
polymer concentration in contact with the liposomes are reported in plots
of the changes of the ratio IE/IM as a function of the amount of charged
lipid incorporated in the liposome bilayer (Figure 7). As anticipated, upon
incorporation of increasing amounts of DDAB to POPC liposomes, the
ratio IE/IM decreased continuously . Surprisingly, the charge density of the
cationic NPL's did not seem to affect the efficiency of interactions with the
polymer. The lowest value of IE/IM was reached already when the DDAB
content was as low as 2 wt %. It remained unaffected by the increase in the
liposome charge density. The most interesting situation is that presented
by the copolymer/anionic NPL's mixtures. In this case we observed a
modest increase in excimer emission with increasing charge density.
Indeed, as the charge density of the liposomes is enhanced, a point is
CA 02254554 1998-11-20
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reached where the unfavorable electrostatic forces become prevalent,
preventing adsorption of the polymer onto the liposome surface.
Example 9
Fluorescence experiments were also performed in the
absence of added salt. Increasing amounts of cationic liposomes, either
POPC liposomes containing 10 wt % DDAB or NPL's charged with 0.5 wt
DDAB, were added to a PNIPAM-Gly-Py solution in deionized water (2 mg
L-1). The changes of the ratio IE/IM were monitored as a function of total
lipid concentration. The ratio decreased sharply upon addition of
liposomes of either composition, reaching a saturation value of 0.1 for a
lipid concentration of 0.1 g L-1 (Figure 8). The decrease in Py excimer
induced by cationic NPL's was very similar to that observed when the
measurements were conducted under high salt conditions. In contrast,
the spectral changes caused by the addition of POPC liposomes to the
polymer solution in water were strikingly different; a much more severe
decrease in IE / IM occurred, implying a strong association of the
polyelectrolyte to the cationic phospholipid liposomes in the absence of
salt. This result emphasizes the role of the screening effect of salts in
controlling the electrostatic interactions between the cationic liposomes
and the copolymer. The fact that the association of NPL's and cationic
liposomes appears less sensitive to the ionic strength of the solution
suggests that forces other than electrostatic are involved in these
interactions.
Example 10 - Capillary el~phoresis measurements
Interactions between cationic NPL's and PNIPAM-Gly-Py in
the absence of added salt was confirmed by capillary electrophoresis
analysis of liposome/polymer mixtures. Electropherograms of solutions
in a neutral buffer of (a) PNIPAM-Gly-Py, (b) cationic NPL's and (c) a
mixture of cationic NPL's and PNIPAM-Gly-Py are presented in Figure 9.
The electropherogram of PNIPAM-Gly-Py (Figure 9a) consists of a broad
band eluting after the neutral marker in accordance to the expected
negative charge of the copolymer. The broad shape of this band suggests
that it corresponds to the elution of the polymeric micelles, which may
CA 02254554 1998-11-20
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vary in size and charge depending on the number of associated chains
(Spafford, M. et al., 1998). Liposomes alone elute as a single band of
retention time shorter than that of the neutral marker, as expected for
cationic samples (Figure 9B). In the electropherogram recorded for the
polymer/liposome mixture (Figure 9C), the bands corresponding to the
cationic liposomes and to the anionic polymer unimers and micelles
cannot be detected anymore. Instead, the electropherogram of the
polymer/liposome mixture presents a major band that co-elutes with the
neutral marker, and two minor bands corresponding to anionic species, as
yet unidentified. The major band was assigned to the polymer/liposome
complexes, indicating that binding of the polymer to the liposomes results
in the neutralization of their surface charge. Thus, the capillary
electrophoresis analysis confirmed that the interaction of PNIPAM-Gly-Py
with cationic NPL's is very strong and that the complexes do not dissociate
as the mixture elutes through the capillary.
Example 11- Centrifugation studies
A centrifugation assay was used to study the distribution of
PNIPAM-Gly-Py between the aqueous and the lipid phase and to estimate
the affinity of the polymer to cationic NPL liposomes surface in
salt-containing solutions. The polymer was incubated with large
multilamellar vesicles (MLV) and the resulting suspensions were
subjected to centrifugation. NPL's, unlike phospholipid liposomes, do not
form a pellet upon centrifugation, but rather float on top of the sample.
Therefore we devised a special centrifugation assay described in detail
herein. Centrifugation of polymer-NPL mixtures at 4000 g resulted in
separation of polymer-NPL complexes, floating on top, from the rest of the
solution containing free unbound polymer (P f). After removal of the
NPL's from the centrifuged mixture, the solution was subjected to a
fluorescence assay taking advantage of the emission of the pyrene labels
attached to the polymer in order to determine the concentration of
polymer. From these data and knowing the initial polymer concentration,
we constructed a binding isotherm for the system. The initial portion of
the binding curve (Figure 10) is linear, indicative of a partitioning
CA 02254554 1998-11-20
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mechanism controlling the polymer/liposomes interactions. The curve
levels off for mixtures with polymer/lipid molar ratios higher than 3 x
10-4. This may indicate saturation of the liposome surface with polymer.
Example 12 - Temperature effects
All the experiments reported previously were carried out at
25°C. Since the ultimate objective of this study is to develop
thermosensitive liposomes, it was important to examine in detail the
effects of heating/cooling cycles on the properties of the
polymer/liposome complexes. Aqueous solutions of PNIPAM-Gly-Py
exhibit a pH-dependent cloud point, reflecting the effect of the changes in
the level of ionization of the carboxylic acid groups on the hydrophobicity
of the copolymer (Taylor, L.D. et al., 1970; Priest, J.H. et al., 1987). At
neutral pH, when all the carboxylic groups are ionized, the polymer is
soluble in water at all temperatures. As the pH of the solution is lowered,
at constant temperature, the carboxylic acid groups are gradually
protonated and, as a consequence, the chains become more hydrophobic.
Their behavior will then become similar to that of PNIPAM itself. The
polymer solution will undergo phase separation at a critical pH where
expanded polymer chains collapse into tight hydrophobic globules and
aggregate. Thus, a PNIPAM-Gly-Py solution of pH 3 exhibits a cloud point
of 26.0°C, lower than PNIPAM itself (Heskins, M. et al., 1968), but
similar
in value to that of other randomly-hydrophobically modified PNIPAM
samples (Ringsdorf, H. et al., 1991).
Binding of the polymer to charged liposomes caused a
significant change in the LOST behavior of PNIPAM-Gly-Py. In mixtures
of the polymer with cationic liposomes, a temperature-induced increase in
turbidity was observed, as seen in typical heating/cooling cycles of aqueous
solutions of the copolymer in the absence of liposomes. We established
that the onset of turbidity depends on the total lipid concentration and on
the relative amount of cationic surfactant incorporated in the bilayer.
Specifically, the cloud point decreased as the lipid concentration increased
(Figure 11 ) and / or as the concentration of charged surfactant increased
(Figure 12). The decrease in cloud point reaches a limit for a 1:5 (w/w)
CA 02254554 1998-11-20
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polymer/lipid ratio. Further addition of liposomes, at constant polymer
concentration, has no effect on the cloud point of the mixture. We
attribute the temperature sensitivity of the polymer/liposome mixtures to
a gradual neutralization of the carboxylic acid groups of the polymer by the
cationic groups on the liposome surface. This charge neutralization
mechanism competes with the pH-sensitive protonation of the polymer,
allowing the control of the cloud point via both the pH of the medium
and the charge density of the liposome. This property can be exploited to
control the leakage of various materials encapsulated into PNIPAM-Gly-
Py/liposomes complexes.
Discussion of Examples 6 to 12 in respect of PNIPAM-G1J~-P~
The solution properties of PNIPAM-Gly-Py reflect its
ambivalent chemical composition. It can be viewed as a polyelectrolyte,
featuring approximately 130 ionizable carboxylic groups per chain. It is
also an amphiphilic polymer, carrying on average 10 hydrophobic
octadecyl groups per macromolecule. The interplay between the intrinsic
properties of PNIPAM and the specific physicochemical properties of the
modifying groups determines the solution properties of the copolymer
and its modus operandi with liposomes of different compositions under
various solution situations.
Taken together, the results reported here point to the
striking differences in the binding of PNIPAM-Gly-Py to phospholipid
liposomes, containing zwitterionic lipids, on the one hand and to NPL's,
containing nonionic surfactants on the other. The differences are
emphasized for liposome/polymer complexes in media of high salt
content. It is known that the head groups of neutral phospholipids, such
as the phosphatidyl choline groups, are in fact partially negatively charged
in NaCI solutions (Westman, j. et al., 1979). For example, in the 150 mM
NaCI solution used in our studies, about 10-15% of the phospholipid
molecules will be charged due to the bound Cl- ions. This occurrence
imposes a significant negative charge over the entire bilayer, resulting in
the repulsion of the anionic polyelectrolyte. However some degree of
interaction is still in effect, since the fluorescence experiments show
CA 02254554 1998-11-20
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(Figure 6) that there is a slight decrease in the ratio IE/IM when PNIPAM-
Gly-Py is mixed with POPC liposomes. This interaction is very weak
compared to the binding to similar liposomes of neutral
hydrophobically-modified PNIPAM (Ringsdorf, H. et al., 1991) The
strength of the interaction can be increased somewhat via the
incorporation in the bilayer of a low level, up to 10 mol %, of the cationic
surfactant DDAB.
Turning now to the case of NPL's composed primarily of
ethoxylated surfactant, we note that partial ionization of the
ethyleneglycol headgroups in the presence of salts cannot occur. Thus, in
the absence of added charged surfactant, the surface of the NPL's remains
neutral. There is no specific accumulation of ions from the solution
around the non-ionized surfactant. However, hydrogen bonds can be
expected to be formed between the NIPAM amide protons and the
relatively basic ether oxygen of the surfactant or the hydroxyl groups of
the surfactant and the amide group of the NIPAM units, the main
components of the liposomes and of the polymer, respectively. In
addition one can also envision that the protonated carboxylic groups of the
polymers form hydrogen bonds with the ether oxygen of the surfactant
head group (Figure 13). The cooperativity of hydrogen bonding results in
an extremely tight binding of the polymer to the liposome surface. The
remarkable fact that interactions take place between the negatively-charged
polymer and not only cationic NPL's containing DDAB, but also anionic
NPL's, in which DDAB was substituted with the anionic surfactant DP,
gives solid support to this assumption. Indeed, the importance of
hydrogen bond formation in controlling the properties of PNIPAM
derivatives has ample precedents (Chen, G. et al. 1995).
These findings, based primarily on changes in the
fluorescence of the labeled polymer, are corroborated by centrifugation
assays. Since the two techniques required different types of liposomes,
namely large unilamellar vesicles (LUV) in the fluorescence assays and
multilamellar large vesicles (MLV) in the centrifugation experiments,
their results cannot be compared directly. However, qualitatively the two
CA 02254554 1998-11-20
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studies are in a good agreement. The binding curves derived from the
centrifugation experiments clearly show the existence of a saturation limit
for the polymer adsorption onto the liposome surface.
The proposed mechanism of interactions is also consistent
with the changes in the cloud point of PNIPAM-Gly-Py in the presence of
liposomes. In the absence of liposomes the salt screening effect results in a
decrease of the polymer LCST at high salt concentrations. It appears that
cationic surfactants, incorporated into the liposome membrane have a
similar screening effect, since we observed that addition of cationic
liposomes to a PNIPAM-Gly-Py solution results in a lowering of its LCST
(Figure 10). The decrease in the polymer LOST depends on the lipid
concentration. It is interesting to note that the cloud point becomes
independent on lipid concentration when a 1:5 (w/w) polymer/lipid ratio
is reached. This ratio is exactly the same as the polymer saturation ratio
evaluated by fluorescence study (Figure 6). The same polymer saturation
ratio is observed for all DDAB-containing NPL's. It does not depend on
the DDAB amount, clearly indicating that the whole membrane surface
actively participates in the polymer binding, and not only a few isolated
positive charges. These observations provide additional support for our
concept of coverage of the NPL surface by the polymer facilitated by
hydrogen bonding between EO and NIPAM units. Once the liposome
surface is completely occupied, the formation of electrostatic bonds
between the carboxyl groups of PNIPAM-Gly-Py and DDAB can occur only
if the cationic surfactant head groups are available on the membrane
surface in close vicinity to the carboxylates of the adsorbed copolymer.
Formation of these electrostatic bonds, in turn, will result in a more
efficient screening of the PNIPAM-Gly-Py negative charges and,
consequently, in a lower LCST value.
Conclusions
Adsorption of polyelectrolytes by oppositely charged surfaces
occurs when the energy of adsorption exceeds the entropy loss associated
with the number of configurations available to the polyelectrolyte. The
conditions of polymer adsorption have been treated in several theoretical
CA 02254554 1998-11-20
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studies (Von Goeler, F. et al., 1994). Of particular relevance to the present
work, is the problem of understanding how large scale patterns on surfaces
get recognized by macromolecules. This problem has been addressed
recently by Muthukumar (J. Chem. Phys. 1995, 103, 4723), in the case of a
pattern made up of electrostatic charges and a polymer consisting of
opposite charges distributed along the chain backbone. By making the
analogy between polymer adsorption and the binding of an electron in a
potential well, he concluded that the kinetics of adsorption take place in
two distinct steps: a fast binding process without any registry of pattern,
followed by a slow process, where the system attempts to attain the fully
registered configuration. The adsorption of PNIPAM-Gly-Py onto charged
liposomes provides an experimental system that involves such a pattern
recognition process. There are three guiding factors involved: (1)
electrostatic interactions, and in the case of phospholipid-containing
liposomes, these forces are crucial in determining the binding of the
polymer to the lipid membrane, but they are of lesser importance in the
case of NPL/polymer interactions; (2) hydrogen bond formation, and this
is the predominant factor controlling polymer adsorption to NPL's,
overtaking even the electrostatic repulsion experienced by the polymer in
the presence of anionic liposomes; and (3) hydrophobic interactions
driven by the nonpolar side groups of the polymer. The last parameter,
while important, appears to be of similar importance for the two types of
liposomes. Our study demonstrates that efficient binding of the
copolymer PNIPAM-Gly-Py to a liposome surface is determined by a
combination of hydrophobic, electrostatic, and hydrogen bonding
interactions. Surprisingly, the hydrogen bonding prevails, resulting in a
particularly strong association of the copolymer with the surface of
non-phospholipid liposomes containing nonionic surfactant (EO)2C18H3~.
General Method for Examples 13 to 15
Preparation of liposomes and of liposome/polymer mixtures. A solution
in chloroform of the lipids (n-octadecyl-diethylene oxide [(EO)2C18H3~],
cholesterol (Sigma, Mississauga, Canada) and
dimethyl-di-n-octadecylammonium bromide (DDAB, Avanti Polar Lipids,
CA 02254554 1998-11-20
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Alabaster, Alabama)) was poured in glass test tubes. The solvent was
evaporated under a stream of nitrogen. The resulting lipid film was dried
under high vacuum for at least two hours, The dry lipid film was hydrated
in an aqueous solution of NaCI (150 mM - which is nearly physiological)
and NaN3 (0.02 wt %) to give a lipid suspension of a concentration of 20 g
L-1. For experiments with calcein-loaded liposomes (leakage and cell
interaction experiments) calcein (70 mM, Sigma) was added to the
hydration solution. The lipid suspension was subjected to extrusion
through polycarbonate membranes (200 nm pore size, Nucieopore) using a
Lipofast extruder (Avestin, Canada). The hydration and extrusion of the
NPL's were performed at 60°C. Liposome/polymer mixtures were
prepared by addition of an extruded liposome suspension to polymer
solutions in the desired proportions. The mixtures were allowed to
equilibrate, first at room temperature (30 minutes) then at 37°C.
Example 13
Fluorometric detection of liposome leakage. Liposome leakage was
monitored as the extent of increase in calcein emission upon rupture of
the liposome. The liposomes were prepared as described above. After
extrusion the free calcein was separated from the liposome suspension by
gel-filtration chromatography on a 1.5X30 cm column packed with
Sephadex G-75 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with a
150 mM NaCl+0.02% NaN3 solution. Fluorescence spectra were recorded
on a SPEX Fluorolog 212 spectrometer (Edison, NJ). The excitation
wavelength was set at 450 nm and the emission intensity was monitored
at a wavelength of 513 nm. The extent of leakage was calculated as 100
X(I-1o) / (In-1o), where 1, 1o and ln. are the current, original and final
fluorescence intensities of a liposome sample. To obtain the fluorescence
intensity, corresponding to 100% dye release, the liposomes were
solubilized with Lubrol to (0.25% w/w).
We designed a polymer/NPL system possessing
triggered-release properties at 37°C in a salt solution (150 mM NaCI)
that
model physiological conditions. The leakage was initiated by acidification
of the solution. We observed that the leakage becomes more efficient as
CA 02254554 1998-11-20
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the magnitude of the drop in pH increases (see Figure 15A), reaching over
95 % efficiency when the pH was reduced from about 7.4 to about 3.5. This
value was not achieved in control experiments using phospholipid
liposomes coated with PNIPAM-Gly-Py (leakage efficiency: 20 to 40%).
Note that the efficiency of the control system is similar to those reported
in studies of other polymer/phospholipid liposome systems (H. Hayashi,
K. Kono, T. Takagishi, Biochim. Biophys. Acta 1280 (1996) 127-134 and the
references cited therein). In the case of polymer coated NPL'S, a relatively
mild acidification results in leakage of 40% of the vesicle contents in less
than 5 minutes (See Figure 15B). The driving force for the leakage is
believed to be the pH-induced conformational change of the adsorbed
PNIPAM-Gly-Py chains. As the polymer contracts due to the protonation
of the glycine residues, it brings about severe distortions of the lipid
bilayer
with subsequent leakage of the liposome contents (see Figure 15B).
Example 14
Fluorometric detection of the fusion of liposomes. Fusion between
liposomes was monitored by measuring the extent of fluorescence increase
of the lipophilic probe BODIPY-PC(Molecular Probes, Eugene, OR) upon
dilution. Specifically, a batch of liposomes was labeled BODIPY-PC (2 mol
%). At this concentration the fluorescence quantum yield of BODIPY-PC is
very low (Hogland, R.P., Handbook of Fluorescent Probes and Research
Chemicals, Molecular Probes Inc.). These labeled liposomes were mixed
with unlabeled liposomes in a 1:10 proportion. Fusion of labeled and
unlabeled liposomes results in the dilution of BODIPY-PC in the bilayer
with concomitant increase of the fluorescence quantum yield. The
excitation wavelength was set at 585 nm and the emission intensity was
monitored at 595 nm. The extent of fusion was calculated as the degree of
increase in BODIPY-PC fluorescence intensity, using the relationship 100
X(I-lo)/(Iri lo), where 1, 1o and ln. are the current, original and final
fluorescence intensities. To obtain the final intensity, corresponding to
100% BODIPY dequenching, the liposomes were solubilized in Lubrol to
(0.25% w./w).
CA 02254554 1998-11-20
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Cell culture. A MCF-7 human breast cancer epithelial cell line was grown
in MEM medium (GIBCO) supplemented with 10% FBS fetal bovine
serum (Sigma) and 1% penicillin streptomycin in a humidified incubator
at 37~C under 5% C02.
Cationic NPL's are able to fuse efficiently with target
phospholipid liposomes of opposite charge, as demonstrated by a
fluorescence assay based on the recovery of emission of a probe
incorporated in the bilayer of the phospholipid liposomes upon contact
with unlabelled NPL'S. The increase of fluorescence reflects the relief of
the self-quenching experienced by the probe concentrated in the
phospholipid liposomes upon dilution with lipids from added unlabelled
NPL's as fusion of the two species occurs (Figure 16). Using the same
technique we were unable to detect fusion between neutral target
liposomes and cationic NPL'S (Figure 16). Thus, we propose, although not
wishing to be bound by any theory, that electrostatic attraction is a major
factor promoting fusion of the two species.
Example 15
Fluorescence microscopy of cells treated with liposomes. Cells grown on
cover slips were washed twice with 2 ml of a solution containing 137 mM
NaCI, 2.7 mM KC1, 1.5 mM KH2P04, 8.1 mM Na2HP04, pH 7.4,
supplemented with 0.4 mM calcium, 0.4 mM magnesium, and 5 mM
glucose (PBS-CMG). A suspension of calcein-containing liposomes
octadecyl diethylene oxide, cholesterol, didoctadecylammonium bromide
in a ratio of 7.5:2 : 0.5, respectively, in the same buffer (1.5 mL, lipid
concentration: 0.1 g/L) was added to the cover slips with cells. The cells
were incubated for 1 hr at 37°C with the liposomes. After the
incubation
the cells were washed twice with PBS-CMG and viewed with a Zeiss
LSM10 confocal microscope equipped with a Zeiss 40 objective. Images
were acquired with digital Sony 3 CCD camera and processed with
Northern Exposure software (Empix Image Inc. 1995).
The combined leakage and fusion properties of
PNIPAM-Gly-Py/NPL complexes make them ideal candidates for
cytoplasmic drug delivery. We examined this by monitoring the
CA 02254554 1998-11-20
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interaction of calcein-loaded NPL with MCF-7 cells (human breast cancer
epithelial), using either polymer-coated NPL's or uncoated NPL's of
identical composition. Figure 17 shows the fluorescence and phase
contrast micrographs of MCF-7 cells treated with NPL's containing calcein.
When the cells were incubated with uncoated liposomes, vesicular
fluorescence of calcein was observed (Figure 17A). In contrast, when the
cells are treated with PNIPAM-Gly-Py coated NPL'S, the fluorescence seen
in the cells is quite different (Figure 17B). A large number of cells
displayed the bright and diffuse green fluorescence characteristic of calcein
present in the cytoplasm. This result suggests that calcein was transferred
into the cytoplasm of the cells treated with PNIPAM- GlyPy/NPL's,
whereas when the cells were treated with bare NPL'S and notwithstanding
their positive surface charge, surprisingly calcein remained entrapped in
the vesicles attached to the cellular surface or confined in intracellular
compartments, such as endosomes or lysosomes. Further, preliminary
evaluation indicates that PNIPAM-Gly-Py promoted cytoplasmic delivery
of calcein encapsulated in POPC-DDAB liposomes, although not to the
same extent.
Discussion of Examples 13 to 15
To extend the capabilities of HM-PNIPAM's/liposome
complexes we have investigated the use of a HM-PNIPAM derivative that
incorporates Gly residues along the chain. The copolymer,
PNIPAM-Gly-Py (Figure 14) contains approximately 15 mol % of carboxylic
acid substituted units. Aqueous solutions undergo phase changes that can
be triggered by changes in temperature, pH or ionic strength. The
copolymer associates readily with liposomes containing hydroxyl
oxyethylene terminated lipids by a process that is facilitated by
electrostatic
interactions, hydrophobic interactions and by hydrogen bonding. In the
studies here we have chosen non-phospholipid liposomes (NPL)
composed of (40-75 molar %), cholesterol (20 mol%) and the cationic
surfactant didodecyldioctadecylammoniumbromide (DDAB, 5-40 mol %).
Compared to phospholipid liposomes, these vesicles possess several
advantages: they have high encapsulation capacity, outstanding chemical
CA 02254554 1998-11-20
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stability and they form complexes with PNIPAM-Gly-Py with high
efficiency (Polozova, A.; Winnik, F. M. Langmuir, submitted 1998). In
addition, their surface is covered by (EO) units, which have chemical
characteristics similar to those of the polyethylene glycol derivatives used
in Stealth~ liposomes which exhibit outstanding stability in blood
circulation (Woodle, M.C. et al., 1992; Lasic, D. et al. (1995) Therefore,
there
is a distinct possibility that liposomes composed of the non-ionic
surfactant (EO)2C18H3~ surfactant with similar functional groups will also
possess long-circulating properties in blood.
We have shown previously that aqueous suspensions of
PNIPAM-Gly-Py/NPL complexes have a cloud point, as do solutions of the
polymer alone. Therefore, once adsorbed on the liposome surface, the
polymer retains the properties imparted by its chemical structure. The
cloud point of the PNIPAM-Gly-Py/NPL suspensions depends on the
solution pH, the ionic strength, and the amount of cationic surfactant
incorporated in the liposome bilayer (Chen, G. et al., 1995). Under
physiological conditions, ionic strength and temperature are fixed. Our
system possesses a third variable, the composition of the bilayer, allowing
us to tune the cloud point of the system to its desired value. When
PNIPAM-Gly-Py is mixed with liposomes at conditions far from the cloud
point, the adsorption of copolymer onto the liposomal surface does not
disturb the liposome integrity and, consequently, all the encapsulated
material remains confined in the liposome interior. As the
liposome-polymer complexes approach the conditions of cloud point,
efficient leakage of the encapsulated material occurs. The leakage pattern
observed with a given system depends on the same variables as the cloud
point.
While the present invention has been described with
reference to what are presently considered to be the preferred examples, it
is to be understood that the invention is not limited to the disclosed
examples. To the contrary, the invention is intended to cover various
modifications and equivalent arrangements included within the spirit and
scope of the appended claims.
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All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.
CA 02254554 1998-11-20
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DETAILED FIGURE LEGENDS FOR FIGURES 5 -17
Figure 5. Fluorescence spectra of PNIPAM-Gly-Py in aqueous solution
without (top) and with cationic liposomes (bottom); ~,eX°: 346 nm;
polymer
concentration 0.002 g L-1; liposome concentration 0.1 g L-1; liposome
composition: 75% (EO)2C1gH3~, 20% cholesterol, 5% DDAB.
Figure 6. Changes in the ratio, IE / IM,,of excimer to monomer emission
intensities as a function of lipid concentration in mixtures of PNIPAM-
Gly-Py with liposomes of various types: ( - POPC liposomes; ( -
POPC/DDAB (9:1) liposomes; ( - (EO)2C18H3~/cholesterol/DDAB (7.5:2:0.5)
Polymer concentration 0.002g/L. in Tris buffer (5mM) containing NaCI
(0.15 mol L-1)
Figure 7. Changes in the ratio, IE/IM of excimer to monomer emisson
intensities as a function of the amount of charged surfactant in mixtures
of PNIPAM-Gly-Py with liposomes of different types:
(EO)2C18H3~/cholesterol/DDAB, ( -(EO)2C18H3~/cholesterol/DP, ( -
POPC/DDAB; (Polymer concentration - 0.002 g L-1; total lipid concentration
- 0.01 g L-1; Tris buffer (5mM) containing NaCI (0.15 mol L-1)).
Figure 8. Changes in the ratio IE/IM of excimer to monomer emission
intensities in PNIPAM-Gly-Py mixtures with liposomes as a function of
lipid concentration in the absence of added salt. (- POPC/DDAB (9:1)
liposomes; ( - (EO)2CigH3~/cholesterol/DDAB (7.5:2:0.5) liposomes.
Polymer concentration 0.002g/L; Tris buffer (5 mM).
Figure 9. Electropherograms of (a) PNIPAM-Gly-Py solution, (b) -
(EO)2C18H3~/cholesterol/DDAB (7.5:2:0.5) liposomes, (c) - mixture of
PNIPAM-Gly-Py with (EO)2C18H3~/cholesterol/DDAB (7.5:2:0.5) liposomes
(see experimental section for measurement conditions).
CA 02254554 1998-11-20
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Figure 10. Binding curve for mixture of PNIPAM-Gly-Py with
(EO)2C18H3~/cholesterol/DDAB (7:2.5:0.5) liposomes (P f: concentration of
free polymer, Pb: concentration of polymer bound to the liposomes); the
inset shows the curve in the low polymer concentration range.
Figure 11. Changes of the cloud point of PNIPAM-Gly-Py/liposome
mixtures as a function of lipid concentration; Liposome composition:
(EO)2C18H3~/cholesterol/DDAB (7.5:2:0.5); Polymer concentration - 0.1 g
L-1).
Figure 12. Changes of the cloud point of PNIPAM-Gly-Py/liposome
mixtures as a function of the amount of cationic surfactant; liposome
composition: (EO)2C1gH3~/cholesterol/DDAB); Polymer concentration: 0.1
g L-1; total lipid concentration: 0.5 g L-1;:( - Tris buffer (5 mM) with 150
mM
15 NaCI (0.15 mol L-1); pH=7.4; ( - Glycine buffer (50 mM) with 10 mM NaCI
(0.01 mol L-1); pH =5.
Figure 13. Idealized representation of the interactions between a cationic
NPL and PNIPAM-Gly-Py (for clarity only half a bilayer is shown and
20 cholesterol has been omitted).
Figure 15. Leakage of NPL coated with PNIPAM-Gly in response to the
solution acidification: (a) extent of leakage after 2000 sec as a function of
final solution pH; (b) time trace of leakage initiated by acidification of the
25 solution from 7.4 to 5. Original solution: 5 mM Tris + 150 mM NaCI,
pH=7.4. pH change was induced by rapid injection of 0.1-1 ml of 50 mM
Gly + 100 mM NaCI, pH=3 buffer. Experiments were performed at
T=37%C; lipid concentration - 0.01 g/L; polymer concentration 0.002 g/L.
30 Figure 16. Fusion of NPL coated with PNIPAM-Gly with target
phospholipid liposomes triggered by solution acidification. Curve 1- target
egg PC liposomes; curve 2 - target POPC-POPS (1:1) liposomes. NPL were
mixed with target liposomes at 1:10 (w/w) proportion. Acidification from
CA 02254554 1998-11-20
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pH=7.4 to 5 was achieved by rapid injection of 0.35 m] of 50 mM Gly + 150
NaCI (pH=3) buffer to 2 ml of original 5 mM Tris + 150 mM NaCI (pH=7.4)
buffer. Experiments were performed at T=37%C; lipid concentration - 0.01
g/L; polymer concentration - 0.002 g/L.
Figure 17. Images of cells treated with nonphospholipid liposomes: (a)
coated with PNIPAM-Gly (b) uncoated. The upper image in each pair
illustrates fluorescence due to calcien. The lower image in each pair is the
corresponding phase contrast image.
CA 02254554 1998-11-20
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c
a~
. .
c
0
U ~
C
X
'O
C
C
U
r:r ~? c?
N
N C O O
0
C ' x X
L ~ r r
0
a '
r- o
a~ C)
.o m
O C~ c~ o
1- +~r c- N
L
o a
z aMO oo
o c
a
3
N ~n
V ~ N N
~
N
t
a o o
0 0
0 0
0 0
0 0
0 0
c u~i o
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_a
U Q Q
>,
CA 02254554 1998-11-20
' -53-
Table 2
Composition of the liposomes prepared
Liposome type Lipid structure Weight ratio Mol °~
Neutral POPC 100
Cationic DMPC/cholesteroUDDAB 7:2:1 74.2/20.1/5.7
E02C,eH3,/cholesteroUDDAB 7.5:2:0.5 77.0/20.1/2.9
Anionic EOZC,8H3~/cholesterol/DP 7:2:1 73.3/20.4/6.3
