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Liposomes are self-assembling structures comprising one or more bilayers of amphipathic
lipid molecules each of which encloses an internal aqueous volume. The amphipathic lipid molecules which make up lipid bilayers comprise a polar (hydrophilic) headgroup region covalently linked to one or two non-polar (hydrophobic) acyl chains. It is
believed that the energetically unfavorable contact between the hydrophobic acyl chains and the aqueous solution surrounding the lipid molecules causes them to rearrange such that the polar headgroups are oriented towards the aqueous solution while the
acyl chains orient towards the interior of the bilayer. The net result is an energetically stable lipid bilayer structure comprising two opposing monolayers, in which the acyl chains are effectively shielded from coming into contact with the aqueous
medium.
Liposomes may be produced by a variety of methods. Bangham's procedure (J. Mol. Biol. 13:238-252 (1965)) produces "ordinary" multilamellar liposomes (MLVs). "Ordinary" MLVs can have unequal solute distribution amongst their aqueous
compartments and thereby, osmotic stress between compartments. Lenk et al. (U.S. Pat. Nos. 4,522,803, 5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578) and Cullis et al. (U.S. Pat. No. 4,975,282) disclose methods for producing
multilamellar liposomes having substantially equal distribution of an entrapped solute in each of their aqueous compartments, that is, substantially equal interlamellar solute distribution. Having substantially equal interlamellar solute distribution
means that there will be less osmotic stress amongst the aqueous compartments of these MLVs, which will therefore generally be more stable than ordinary MLVs. Unilamellar liposomes can be produced from MLVs by sonication (see Paphadjopoulos et al.
(1968)) or extrusion (Cullis et al. (U.S. Pat. No. 5,008,050) and Loughrey et al. (U.S. Pat. No. 5,059,421)).
Liposomes can be loaded with bioactive agents passively, that is, by solubilizing the molecule in the medium in which the liposomes are formed, in the case of water-soluble agents, or adding lipid-soluble agents to the lipid solutions from which
the liposomes are made. Ionizable bioactive agents can also be loaded into liposomes by establishing an electrochemical potential gradient across the liposomal membrane and then adding the agent to the medium external to the liposome (see Bally et al.,
U.S. Pat. No. 5,077,056).
Drugs entrapped within liposomes can have an enhanced therapeutic index by reducing toxicity, increasing efficacy, or both. Furthermore, liposomes, like other particulate matter in the circulation, are taken up by phagocytic cells of the
reticuloendothelial system in tissues having sinusoidal capillaries, and are thereby often directed to the sites of intracellular infections.
Fusion of biological membranes is a key process in a variety of cellular transport functions, including endocytosis, fertilization and the intracellular trafficking of proteins. Fusion of liposomes with cells is defined as the unification of the
outermost bilayer of the liposome with the plasma membrane of the cell (see Huang (1983)). For fusion to occur, the lipids of the outermost lipid bilayer must mix with lipids of the cell's plasma membrane. The proposed mechanism by which fusion occurs
between lipid membranes involves neutralization of the charged headgroups, resulting in an effective change in the geometry of the lipid species producing nonbilayer-preferring structures. These in turn give rise to micelles or other defects in the
bilayer which act as nucleation sites for membrane fusion (Cullis and Hope (1978)).
Studies employing liposomes have demonstrated a correlation between the tendency of liposomes to fuse and the propensity of component lipids to adopt non-bilayer phases, such as the hexagonal H.sub.II phase, leading to the suggestion that
non-bilayer structures, for example, inverted micelles or other bilayer defects, are intermediary structures in fusion events (see, for example, Cullis et al. (1978)). Lipid composition is believed to contribute to the tendency of liposomes to adopt
non-bilayer structures (see, for example, Martin and MacDonald (1976); Martin and MacDonald (1976); Weismann et al. (1977)). For example, membranes containing negatively charged phospholipids (for example, phosphatidic acid (PA), phosphatidylserine
(PS), etc.) have been shown to fuse in the presence of divalent cations such as Ca.sup.++ (Hope et al. (1983)). Synthetic cationic lipids have been shown to undergo fusion using high concentrations of negatively charged counterions (Duzgunes et al.
(1989)). Unsaturated diglycerides are also believed to be potent fusogens (Das and Rand (1986)). Furthermore, uncharged lipids are believed to be more readily able to adopt non-bilayer structures, and hence, to be more fusogenic, than are their charged
forms (see, for example, Gruner et al. (1985)).
Cationic lipids have proven useful in increasing the efficiency of mammalian cell transfection (see, for example, Malone et al. (1989); Konopka et al. (1991)). The extension of these applications to the in vivo delivery of liposomally
encapsulated materials has encountered problems of cellular toxicity, hemolysis and accelerated clotting responses caused by cationic lipids (Senior et al. (1991)). These effects appear to occur above a threshold level of positive charge, and do not
appear to be largely dependent on the nature of the cationic species. Although, the toxic effects of cationic species are being challenged by the synthesis of novel cationic head groups, they remain largely unsolved. The potential applications of these
lipids to targeted drug delivery make the reduction of the toxic side effects, by reducing cationic concentration, and the ability to control the fusogenic nature in vivo of these compounds, desirable.
Fusion between vesicle populations bearing opposite charges has also been demonstrated (Stamatatos et al. (1988)). Controlled fusion of liposomes using induced lipid asymmetry has been previously addressed (Eastman et al. (1991); Eastman et al.
(1992); Redelmeier et al. (1990)). However, these references are directed to the use of anionic ionizable lipids; the pH gradient used to transport the anionic lipid is the reverse of that for the loading of cationic drugs into liposomes (see Bally et
al., U.S. Pat. No. 5,077,056). Controlled fusion of these drug-loaded liposomes would be better served by an analogous fusogenic, cationic lipid.
Fusion of liposomes with biological membranes can deliver the contents of the liposomes into cells. The injection of the aqueous content of liposomes into the cytoplasm has been shown by the fluorescence dequenching of carboxyfluorescein
(Weinstein et al. (1977); Huang et al. (1978). Other reports have also shown that biologically active materials (for example, cAMP, ricin, actinomycin D, Ca.sup.++, mRNA, and viruses and viral genomes) incorporated into liposomes can be introduced into
the interiors of cells (see, for example, Paphadjopoulos et al. (1974); Dimitriadis and Butters (1979); Theoharides and Douglas (1978); Ostro et al. (1978); Dimitriadis (1978); Wilson et al. (1979); Fraley et al. (1980)) by way of fusion between
liposomal lipid bilayers and cellular membranes. However, other studies suggest that fusion is not a major mechanism of liposomal interactions with cells (see, for example, Szoka et al. (1980); Hagins and Yoshikami (1982); Pagano and Takeichi (1977)).
The contents of the above-cited publications are incorporated herein by reference. None of these publications disclose a liposome composition containing a liposome having an ionizable, cationic lipid and a transmembrane pH gradient, nor do they
disclose use of such a gradient to control the transbilayer distribution, and hence, fusogenic potential, of the ionizable lipid.
SUMMARY OF THE INVENTION
This invention provides a liposome composition which comprises a liposome having: (i) an outermost lipid bilayer comprising a neutral, bilayer-preferring lipid and a fusion-promoting effective amount of an ionizable lipid having a protonatable,
cationic headgroup and an unsaturated acyl chain; and (ii) a compartment adjacent to the outermost lipid bilayer comprising an aqueous solution having a first pH. The composition also comprises an aqueous solution external to the liposome having a
second pH, wherein the first pH is less than the pK.sub.a of the ionizable lipid in the outermost lipid bilayer and the second pH is greater than the pK.sub.a of the ionizable lipid in the outermost lipid bilayer, whereby there is a pH gradient across
the outermost lipid bilayer and whereby the ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer.
The liposome can be a unilamellar liposome; the unilamellar liposome is preferably a large unilamellar liposome. The liposome can also be a multilamellar liposome; preferably, the multilamellar liposome comprises a solute entrapped in its
aqueous compartments, wherein the concentration of the solute in each of the aqueous compartments of the multilamellar liposome is substantially equal. The internal aqueous solution in the liposome is preferably an aqueous buffer, more preferably, an
aqueous buffer having a pH of about 4.0. Preferably, the aqueous pH 4.0 buffer is a citrate buffer.
The fusion-promoting effective amount of the ionizable lipid is typically an amount sufficient to establish a concentration of the ionizable lipid in the outermost lipid bilayer of the liposome of from about 1 mole percent of the ionizable lipid
to about 20 mole percent; preferably within this range, the preferred fusion-promoting effective amount of the ionizable lipid in the outermost lipid bilayer is an amount sufficient to establish a concentration of the ionizable lipid in the outermost
lipid bilayer of from about 5 mole percent to about 10 mole percent of the ionizable lipid.
The cationic headgroup of the ionizable lipid is preferably an amino group, and the unsaturated acyl chain is preferably an oleic acid chain. In a presently preferred embodiment of the invention, the cationic headgroup is an amino group, the
unsaturated acyl chain is an oleic acid chain and the ionizable lipid is 1-N,N-dimethylamino dioleoyl propane (AL-1). The ionizable lipid can also be selected from the group consisting of .+-.-oleoyl-2-hydroxy-3-N,N-dimethylamino propane (AL-2),
asymmetric .+-.-1,2-diacyl-3-N,N-dimethylamino propane (AL-3-AL-5) and .+-.-1,2-didecanoyl-1-N,N,-dimethylamino propane (AL-6). More preferably, presently, the ionizable lipid is 1-N,N-dimethylamino dioleoyl propane (AL-1).
The aqueous solution external to the liposome is an aqueous buffer having a pH which is greater than the pK.sub.a of the ionizable lipid in the outermost lipid bilayer. Preferably, when the ionizable lipid is AL-1, the pH of the external aqueous
buffer is about 7,5. The liposome can further comprise a neutral, non-bilayer-preferring lipid, for example, dioleoyl phosphatidylethanolamine (DOPE). The liposome can comprise a biologically active agent, which is typically a nucleic acid, an
antimicrobial agent, an anticancer agent or an anti-inflammatory agent. The aqueous solution external to the liposome can be a pharmaceutically acceptable aqueous solution, and the liposome composition can be a pharmaceutical composition.
This invention also provides a dehydrated liposome having an outermost lipid bilayer comprising a neutral, bilayer-preferring lipid and a fusion-promoting effective amount of an ionizable lipid having a cationic headgroup and unsaturated acyl
chains, wherein the ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer.
Further provided herein is a method of controlling the fusion of a liposome to a second lipid bilayer. The method comprises preparing the liposome in an aqueous solution, wherein the liposome comprises: (1) an outermost lipid bilayer comprising
a neutral, bilayer-preferring lipid and a fusion-promoting effective amount of an ionizable lipid having a protonatable, cationic headgroup and an unsaturated acyl chain; and (2) a compartment adjacent to the outermost lipid bilayer comprising the
aqueous solution. The pH of the aqueous solution is less than the pK.sub.a of the ionizable lipid in the outermost lipid bilayer. The pH of the aqueous solution external to the liposome is then raised above the pK.sub.a of the ionizable lipid in the
outermost lipid bilayer, thereby establishing a pH gradient across the outermost lipid bilayer. The ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer in response to the pH gradient. The pH gradient is degraded when
fusion of the liposome to the second lipid bilayer is to occur, so that the liposome fuses to the second lipid bilayer.
The liposome used in the method of this invention can comprise a biologically active agent, which is typically a nucleic acid, antimicrobial agent, anticancer agent or anti-inflammatory agent. The second lipid bilayer to which the liposome is
fused in a controlled manner is preferably the plasma membrane of a cell, most preferably, the plasma membrane of a mammalian cell.
This invention provides a method of introducing a biologically active agent into a cell which comprises preparing a liposome comprising the agent in an aqueous solution so that the aqueous solution is internal and external to the liposome. The
liposome comprises an outermost lipid bilayer which comprises a neutral, bilayer-preferring lipid and an ionizable lipid having a cationic headgroup and unsaturated acyl chains. The pH of the internal aqueous solution is less than the pKa of the
ionizable lipid in the outermost lipid bilayer. The pH of the external aqueous medium is increased above the pKa of the ionizable lipid in the outermost lipid bilayer so that there is a pH gradient across the outermost lipid bilayer and the ionizable
lipid is then accumulated in the inner monolayer of the outermost lipid bilayer. The pH of the internal aqueous solution is increased above the pK.sub.a of the ionizable lipid in the outermost lipid bilayer prior to contacting the cell with the
liposome. Preferably, the cell is a mammalian cell
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. (A) Calibration of TNS fluorescence for EPC/Chol liposomes containing 0-10 mole % AL-1. X-axis: Mole % AL-1; y-axis: fluorescence. Error bars are smaller than symbols where not indicated. (B) Fluorescence traces showing the effect of
a pH gradient and 10 mole % AL-1 on TNS fluorescence of EPC/Chol (55:45)liposomes. Liposomes were prepared with 0 mole % AL-1 (lower traces) or 10 mole % AL-1 (upper traces). Internal buffer was either 300 mM citrate, pH 4.0 (lower traces) or 20 mM
HEPES buffer, pH 7.5 (upper trace). x-axis: Time (seconds); y-axis: fluorescence.
FIG. 2. Effect of pH on the fluorescence of EPC/Chol (55:45) Liposomes. Data shown represent mean values from the duplicate experiments, and error bars are smaller than symbols where not otherwise indicated. The dashed line is the inverted
first derivative of the titration curve and its maximum indicates a pK.sub.a of 6.7 for AL-1 in the lipid bilayer. x-axis: pH; y-axis: %.DELTA.F/.DELTA.F.sub.max.
FIG. 3. Effect of 0 and 10 mole % AL-1 on the fusion of EPC/Chol (55:45) liposomes and EPC/DOPE/Chol (30:25:55)liposomes by RET fluorescent probe dilution. Data shown are different traces of runs with, and without, dissipation of the pH
gradient, where .DELTA.F.sub.max was determined by the addition of Triton X-100 to a final concentration of 0.8 mM. x-axis: time (seconds); y-axis: fluorescence.
FIG. 4. Effect of AL-1 Concentration (0-20 Mole %) on Fusion of EPC/Chol (55:45, mole/mole) Liposomes by RET Fluorescent Probe Dilution. x-axis: Time (seconds); y-axis: fluorescence.
FIG. 5. .sup.2 H-NMR spectra of MLVs prepared with 10 mole % AL-1-d.sub.4 in EPC/Chol (55:45) in 100 mM ammonium acetate at pH 4.0 and pH 7.5. x-axis: Frequency (kHz).
FIG. 6. Effect of DOPE Concentration on Fusion Rates of Liposomes Containing AL-1. Fusion assays were carried out as described above x-axis: Time (seconds); y-axis: %.DELTA.F/.DELTA.F.sub.max.
FIG. 7. Fusion of Liposomes Consisting of 0 and 5 mole % AL-1 and EPC/Chol/DOPE (35:20:45, mole/mole). x-axis: Time (seconds); y-axis:: %.DELTA.F/.DELTA. F.sub.max.
FIG. 8. A: .sup.2 H-NMR (x-axis: frequency (kHz)), B: .sup.31 P-NMR (x-axis: .delta./ppm) spectra of freeze-thawed MLVs prepared with 5 mole % AL-1-d.sub.4 in EPC/DOPE/Chol Liposomes, buffered with 20 mM HEPES, 20 mM sodium acetate, 150 mM NaCl,
at pH 4.0 and pH 7.5.
FIG. 9. Effect of Aminolipid Structure on Fusion of EPC/Chol/DOPE (35:20:45) Liposomes by RET Fluorescence Probe Dilution. Traces (uppermost-lowermost): AL-1, AL-5, AL-4, AL-3, AL-6, AL-2 and blank. x-axis: Time (seconds); y-axis:
%.DELTA.F/.DELTA.F.sub.max.
FIG. 10. Synthetic Aminolipid Structures.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a liposome composition which comprises a liposome having: (i) an outermost lipid bilayer comprising a neutral, bilayer-preferring lipid and a fusion-promoting effective amount of an ionizable lipid comprising a
protonatable, cationic headgroup and an unsaturated acyl chain; (ii) a compartment adjacent to the outermost lipid bilayer which comprises an aqueous solution (the "internal aqueous solution") having a first pH. The composition also comprises an aqueous
solution external to the liposome having a second pH. The first pH is less than the pKa of the ionizable lipid in the outermost lipid bilayer and the second pH is greater than the pKa of the ionizable lipid in the outermost lipid bilayer, whereby there
is a pH gradient across the outermost lipid bilayer, and the ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer in response to the pH gradient.
Liposomes are self-assembling structures comprising one or more lipid bilayers, each of which surrounds a compartment comprising an aqueous solution. Each bilayer of the liposome is formed by the association of amphipathic lipid molecules such
that their polar, hydrophilic headgroups are oriented towards the surrounding aqueous solution while the hydrophobic acyl chains are oriented towards the interior of the bilayer, and away from the surrounding aqueous phase. Consequently, lipid bilayers
have both inner and outer monolayers of lipid molecules.
A unilamellar liposome has one lipid bilayer; multilamellar liposomes have more than one lipid bilayer. The liposome used in the liposome composition of this invention can be a unilamellar liposome, preferably, a large unilamellar liposome
(LUV). An LUV is a unilamellar liposome having a diameter of greater than about 50 nm. The liposome can also be a multilamellar liposome (MLV), preferably, the MLV comprises a solute entrapped in its aqueous compartments, wherein the concentration of
the solute in each of the aqueous compartments is substantially equal. Such MLVs have substantially equal interlamellar solute distribution. Typically, the liposomes of this invention have sizes, as measured by their diameters, of about 5000 nm or
less. Liposome size can be determined by techniques, for example, quasi-electric light scattering, that are well known to ordinarily skilled artisans and are readily practiced by them.
The "outermost lipid bilayer" of a unilamellar liposome is the single lipid bilayer of the liposome; in a multilamellar liposome, the "outermost lipid bilayer" is the lipid bilayer in contact with the aqueous solution external to the liposome
(the "external aqueous solution"). Liposomes can be prepared by a variety of methods (see, for example, Cullis et al. (1987), Bangham et al. (1965), Lenk et al. (U.S. Pat. Nos. 4,522,803, 5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No.
4,588,578) and Cullis et al. (U.S. Pat. No. 4,975,282)).
The liposome has an outermost lipid bilayer which comprises a neutral, bilayer-preferring lipid comprising a neutral (uncharged, non-cationic/non-anionic, nonprotonatable) headgroup and bilayer-preferring acyl chains. These acyl chains, which
can be symmetric or asymmetric (of unequal length, or number of carbon atoms), saturated (no double bonds between adjacent carbon atoms) or unsaturated (one or more double bonds between adjacent carbon atoms), are believed to inhibit or prevent phase
separation of nonbilayer-preferring lipids in the bilayer, generally by adopting compatible acyl chain packing with the acyl chains of the other lipids incorporated in the bilayer. Bilayer-preferring lipids can form stable lipid bilayers on their own,
as well as in connection with other bilayer-preferring lipids and with nonbilayer-preferring lipids. Bilayer-preferring lipids generally have a substantial similarity between the surface areas of their headgroups and the cross-sectional area of their
acyl chains. The acyl chains of bilayer-preferring lipids generally are in about parallel orientation with respect to each other in bilayers. Bilayer-preferring lipids generally adopt bilayer-compatible structures, and generally are not involved in
establishing bilayer defects. The neutral bilayer-preferring lipid can be a phosphatidylcholine, such as egg phosphatidylcholine, or other neutral, bilayer-preferring lipids.
The outermost lipid bilayer of the liposome also comprises a fusion promoting effective amount of an ionizable lipid having a protonatable, cationic headgroup, that is, a headgroup which can accept a proton, and is then positively charged, and
which can give up the proton such that is neutral. The ionizable lipid also comprises an unsaturated acyl chain, preferably, two unsaturated acyl chains. The cationic headgroup is preferably an amino group such as a dimethylamino or trimethylamino
group, but can also be other groups which can be protonated, and positively charged, and deprotonated, and neutral.
The unsaturated acyl chain is generally nonbilayer preferring. Nonbilayer-preferring acyl chains generally do not adopt conformations in bilayers in which the chains are in parallel orientation. The cross-sectional areas of such acyl chains
typically, and unlike bilayer-preferring acyl chains, are not equal to each other and to the headgroup surface area, because, it is believed, of their non-parallel orientation. Bilayers containing nonbilayer-preferring acyl chain-containing lipids
generally exhibit a lower T.sub.M or temperature at which the transition from the gel to fluid state occurs, in comparison to bilayers containing bilayer-preferring acyl chain lipids of the same length (number of carbon atoms). It is believed that
incorporation of nonbilayer-preferring lipids into bilayers generally increases the tendency of the bilayer to form defects and to have less stability than if bilayer-preferring lipids were used. Such defects are generally believed to be involved in the
fusion of lipid bilayers. Without intending in any way to be limited by theory, it is believed that bilayer destabilization, and hence, fusion, requires a substantial imbalance between the size of the headgroup of the fusogenic lipid and the size of
area occupied by its acyl chains.
Preferably, the unsaturated acyl chains are oleic acid chains, which are acyl chains having eighteen carbon chains having a double bond between the ninth and tenth carbon atoms. However, the unsaturated acyl chains can be other acyl chains as
Well, such as those typically having between 12 and 24 carbon atoms and 1-4 double bonds, for example, palmitoleate (16 carbons/1 double bond) or arachidonate (20 carbon atoms/4 double bonds) chains, as long as these generally anchor the lipid in the
bilayer and are non-bilayer-preferring. Preferably, the cationic headgroup is an amino group, the unsaturated acyl chain is an oleic acid chain, the ionizable lipid comprises two such chains, and is 1-N,N-dimethylamino dioleoylpropane (AL-1). The AL-1
can be optically active or racemic, that is, an optically inactive mixture of isomers; preferably, the AL-1 is racemic.
Other suitable ionizable lipids are those with titratable headgroups, that is, headgroups which do not have a permanent positive charge, but rather, headgroups which can be protonated and deprotonated in response to changes in the surrounding pH,
and non-bilayer preferring acyl chains. These include, but are not limited to: .+-.-oleoyl-2-hydroxy-3-N,N-dimethylaminopropane (AL-2), asymmetric .+-.-1,2-diacyl-3-N,N-dimethylaminopropane (AL-3-AL-5) and .+-.-1,2-didecanoyl-1-N,N,-dimethyl
aminopropane (AL-6). The fusogenic capacity of each of these lipids can be compared, for example, by preparing liposomes with the same amount of each of the lipids and then comparing the relative rates of fusion of the liposomes. Liposome fusion can be
monitored by a number of means, for example, by fluorescent probe dilution experiments (see, for example, Example 4 hereinbelow). FIG. 9 presents data comparing the relative fusion rates of liposomes containing the ionizable lipids AL-1, AL-2, AL-3,
AL-4, AL-5 and AL-6.
The liposome has a compartment adjacent to its outermost lipid bilayer which comprises an aqueous solution (the "internal aqueous solution") having a first pH. The liposome is suspended in an aqueous solution (the "external aqueous solution")
having a second pH. Typically, the liposome is prepared in an aqueous solution, which is both entrapped by the liposome, and in which the liposome is suspended. Accordingly, the internal and external aqueous solutions generally initially have the same
composition, and also, the same pH. Their pH is less than the pKa of the ionizable lipid in the liposome's outermost lipid bilayer.
A compound's pK.sub.a, that is, its acid dissociation constant, is the pH at which the compound is half-dissociated, that is, the pH at which about half of the molecules of the compound present in solution are deprotonated. pK.sub.a can be
defined by the formula: log ([HA]/[H.sup.+ ] [A.sup.- ]), where HA is the protonated compound and A.sup.- is the deprotonated compound. The Henderson-Hasselbach equatin (pH=pK.sub.a + log ([A-]/HA])) describes the relationship between the pH of a
solution and the relative concentrations of the protonated and deprotonated forms of a compound present in solution. At a pH greater than its pK.sub.a in a lipid bilayer, more than half of the ionizable lipid present in the bilayer will be deprotonated,
and hence, neutral. Determination of an ionizable lipid's pKa can be accomplished by well known and readily practiced means, for example, by TNS fluorescence titrations.
The ionizable lipid is substantially protonated when the pH of both the internal and external aqueous solutions are less than its pK.sub.a in the bilayer, and is generally about evenly distributed between the inner and outer monolayers of the
outermost lipid bilayer, that is, about 50% of the ionizable lipid present is in the inner monolayer, and 50% is in the outer monolayer.
A pH gradient is established across the outermost lipid bilayer by increasing the pH of the external aqueous solution so as to obtain an external aqueous solution with a second pH which is greater than the first pH, and is greater than the
pK.sub.a of the ionizable lipid in the bilayer. The protonated, charged ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer in response to the pH gradient. "Accumulate," as used herein, means that greater than about 50%
of the ionizable lipid present in the outermost bilayer is in its inner monolayer when there is a pH gradient across the bilayer; preferably, between about 75% and about 100%, more preferably, between about 90% and about 100%, and most preferably, about
100% of the protonated ionizable lipid is in the inner monolayer in response to the pH gradient.
For example, in a preferred embodiment of the invention, the ionizable lipid is AL-1. Liposomes can be prepared with AL-1 and, for example, egg phosphatidylcholine (EPC) and cholesterol (Chol), or EPC, Chol and dioleoyl phosphatidylcholine
(DOPE). As EPC, Chol and DOPE are neutral lipids, the pK.sub.a of AL-1 in EPC/Chol or EPC/Chol/DOPE bilayers is about 6.7. Accordingly, the first pH, the pH of the internal aqueous solution, is less than 6.7 in connection with AL-1/EPC/Chol or
AL-1/EPC/Chol/DOPE liposomes; preferably, the first pH in connection with such AL-1 containing liposomes is about 4.0. Typically, the internal aqueous solution is an aqueous buffer; presently, the preferred aqueous buffer is a citric acid buffer.
The second pH, that is, the pH of the external aqueous solution, is greater than 6.7 when the liposome comprises AL-1/EPC/Chol or AL-1/EPC/Chol/DOPE; preferably, the second pH is about 7.5. However, incorporation of other lipids into
AL-1/EPC/Chol or AL-1/EPC/Chol/DOPE liposomal bilayers can affect the pKa of AL-1 therein. Ionizable lipids other than AL-1 can have different pKa's than AL-1. Accordingly, the first and second pH's may vary from the preferred pH values when the
composition of the liposome is altered.
When the first pH is less than the ionizable lipid's pKa in the outermost lipid bilayer, the second pH is greater than the pKa, and the ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer, the positive charge of
the ionizable lipid is substantially absent from the outer monolayer, and is shielded from exposure to the external environment. When administered to animals, positively charged lipid can cause toxic side effects and can promote opsonization, the
binding of plasma proteins to the liposome's outer surface, thereby promoting clearance of liposomes from the animal's circulation. Accumulating the positive charge in the inner monolayer minimizes the potential for toxic side effects and for
opsonization.
Furthermore, the pH gradient which induces the ionizable lipid to accumulate in the inner monolayer can also be used to load cationic, lipophilic biologically active agents, for example, the anthracycline antineoplastic agent doxorubicin, into
the liposomes (see, for example, Bally et al., U.S. Pat. No. 5,077,056, the contents of which are incorporated herein by reference)).
Degradation of the pH gradient leads to an increase of the internal pH above the ionizable lipid's pK.sub.a. This leads to substantial deprotonation of the accumulated ionizable lipid. The substantially deprotonated, neutral ionizable lipid is
then about evenly distributed between the inner and outer monolayers of the outermost lipid bilayer. The neutral ionizable lipid is "fusogenic" when exposed, in the outer monolayer, to other lipid bilayers, that is, it can promote the fusion of the
liposome to the other lipid bilayers. Using an ionizable lipid having a pK.sub.a in the bilayer which is less than about 7 means that the pH gradient can be degraded at physiological pH, for example, in an animal, such that the ionizable lipid is
substantially deprotonated and fusogenic. The internal pH of the liposome does not generally need to be raised above physiological pH to induce substantial deprotonation of the ionizable lipid. Ionizable lipid's having pK.sub.a 's substantially above
physiological pH may not be fusogenic when administered to animals as they may not be substantially deprotonated in the animal.
The ionizable lipid is present in the outermost lipid bilayer in a "fusion promoting effective amount." Fusion of liposomes with other lipid bilayers can be defined as fusion of the outermost lipid bilayer of the liposome with the other lipid
bilayer, for example, a cell membrane (see, for example, Huang (1983)). For fusion to occur, the lipids of the outermost lipid bilayer must mix with the lipids of the other lipid bilayer. Without intending in any way to be limited by theory, it is
believed that for fusion to occur, the charged headgroups on lipids have to be neutralized, resulting in an effective change in the geometry of the lipids, which gives rise to nonbilayer-preferring structures.
For the purpose of this invention, a "fusion-promoting effective amount" of an ionizable lipid is an amount of the ionizable lipid effective to promote fusion of the liposome to another lipid bilayer when the ionizable lipid is deprotonated and
neutral. Fusion-promoting effective amounts of an ionizable lipid are generally effective to form a sufficient degree of defects in a bilayer to promote fusion of the bilayer to another bilayer. Typically, the "fusion promoting effective amount" of the
ionizable lipid is an amount sufficient to establish a concentration of the ionizable lipid in the outermost lipid bilayer of from about 1 mole percent to about 20 mole percent. Desirably, the fusion-promoting effective amount of the ionizable lipid is
an amount sufficient to establish a concentration of the ionizable lipid in the outermost lipid bilayer of between about 5 mole percent and about 10 mole percent.
The liposome can also comprise a neutral, nonbilayer-preferring lipid. As used herein, a "neutral, nonbilayer-preferring lipid" is an amphipathic lipid having a non-cationic, non-anionic headgroup and nonbilayer-preferring acyl chains.
Nonbilayer preferring acyl chains generally are not arranged in parallel orientation in bilayers, and generally do not have similar areas occupied by their headgroup surfaces and a cross-section of their acyl chains. The most favorable packing
conformation for such acyl chains is generally in non-bilayer structures. Bilayers containing nonbilayer-preferring lipids generally exhibit a lower T.sub.m, or temperature at which the transition from the gel to fluid state occurs, in comparison to
bilayers containing bilayer-preferring acyl chain lipids of the same length (number of carbon atoms). Accordingly, nonbilayer preferring lipids generally facilitate a bilayer's transition from the gel to the fluid state, and thereby generally accelerate
fusion of the bilayer to another bilayer. In a presently preferred embodiment of the invention, the neutral, nonbilayer-preferring lipid is dioleoyl phosphatidylethanolamine (DOPE).
The liposome can also comprise proteins and other lipids, for example, cholesterol and its derivatives, incorporated into the liposome in amounts, and for reasons, well known to ordinarily skilled artisans, or readily determinable by them without
undue experimentation.
The liposome can comprise a biologically active agent, a term that includes traditional pharmaceuticals, and related biologically active compounds or compositions of matter, having biological activity in an animal or on an animal's cells in
vitro. Bioactive agents include, but are not limited to: antibacterial agents, antiviral agents, antifungal agents, anti-parasitic agents, tumoricidal agents, anti-metabolites, carbohydrates, polypeptides, peptides, proteins, toxins, enzymes, hormones,
neurotransmitters, glycoproteins, lipoproteins, immunoglobulins, immunomodulators, vasodilators, dyes, radiolabels, radio-opaque compounds, fluorescent compounds, polysaccharides, cell receptor binding molecules, antiinflammatory agents, mydriatic
compounds, local anesthetics, narcotics, anti-glaucomic agents, vitamins, nucleic acids, polynucleotides, nucleosides, nucleotides, MRI and radio contrast agents.
The liposome composition can be administered to animals, preferably mammals, and more preferably, humans, to deliver biologically active agents entrapped in, or associated with, the liposome to the cells of the animal. When the composition is
administered to animals, the external aqueous solution is tolerable, that is, substantially non-toxic, to the animals; accordingly, the external aqueous solution is a pharmaceutically acceptable solution or "carrier". Pharmaceutically acceptable
carriers are generally selected with regard to the intended route of administration and standard pharmaceutical practice. For parenteral administration or injection via intravenous, intraperitoneal, intramuscular, subcutaneous, or intra-mammary route,
sterile solutions of the liposome composition are prepared; the total concentration of solutes may be controlled to render the preparation isotonic. Typical carriers used for parenteral administration include, but are not limited to, aqueous
dextrose-containing solutions such as D5W (5% weight by volume dextrose in water) and physiologically acceptable saline solutions. Pharmaceutically acceptable carriers can also include alcohols, gum arabic, benzyl alcohols, gelatin, carbohydrates, such
as lactose, amylose or starch, magnesium stearate, talc, silic acid, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like. They can also contain components, for example, preservatioves, anti-oxidants, and the like, in amounts, and for reasons,
well within the purview of the ordinarily skilled artisan to determine.
This invention also provides a dehydrated liposome having an outermost lipid bilayer comprising a neutral, non-bilayer-preferring lipid and a fusion-promoting effective amount of an ionizable lipid having a protonatable, cationic headgroup and an
unsaturated acyl chain, wherein the ionizable lipid is accumulated in the inner monolayer of the outermost lipid bilayer. Liposomal dehydration can enable the liposomes to be stored for extended periods of time; dehydrated liposomes can then be
reconstituted on an as-needed basis. Liposomes can be dehydrated, with freezing, using standard freeze-drying equipment, or its equivalents. Freeze-drying drying is preferably carried out after incorporating one or more protective sugars into liposome
preparations in accordance with the procedures described in Schneider et al. (U.S. Pat. No. 4,229,360) and Janoff et al., (U.S. Pat. No. 4,880,635), the contents of which are incorporated herein by reference). The protective sugar can be omitted if
the dehydration is conducted without freezing, and sufficient water is left remaining in the liposomal preparation to maintain the integrity of a substantial portion of the liposomal bilayers through the dehydration-rehydration process.
This invention provides a method of controlling the fusion of a liposome to a second lipid bilayer which comprises preparing the liposome in an aqueous solution, wherein the liposome comprises an outermost lipid | | |
