Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1330193
PATENT
TLC 147
SPONTANEOUS VESICULATION OF MULTILAMELLAR LIPOSOMES
BACKGROUND OF THE INVENTION
The present invention is directed to a method of forming unilamellar
vesicles without the use of homogenization, filtration, sonication, or
extrusion techniques, or other techniques that require energy input to the
system, or exposure of lipids to harsh environments. Such environments
include for example detergent or extreme pH environments.
Liposomes (vesicles) are completely closed lipid bilayer membranes
containing an entrapped aqueous volume. Liposomes may be unilamellar
vesicles (possessing a single membrane bilayer) or multilamellar vesicles
(onion-like structures characterized by multiple membrane bilayers, each
separated from the next by an aqueous layer). The bilayer is composed of two
lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head"
region. The structure of the membrane bilayer is such that the hydrophobic
(nonpolar) "tails" of the lipid monolayers orient towards the center of the
bilayer while the hydrophilic "heads" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965,
12:233-252 involves suspending phospholipids in an organic solvent which is
then evaporated to dryness leaving a phospholipid film on the reaction
vessel. Next, an appropriate amount of aqueous phase is added, the mixture
1 30 is allowed to "swell," and the resulting liposomes which consist of
¦ multilamellar vesicles ~MLVs) are dispersed by mechanical means. MLVs so
formed may be used in the practice of the present invention.
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Another class of multilamellar liposomes that may be used as the starting
liposomes of this invention are those characterized as having substantially
equal lamellar solute distribution. This class of liposomes is denominated
as stable plurilamellar vesicles (SPLV) as defined in U.S. Patent No.
4,522,803 to Lenk, et al., reverse phase evaporation vesicles ~REV) as
described in U.S. Patent No. 4,235,871 to Papahadjopoulos et al., monophasic
vesicles as described in U.S. Patent No. 4558,579 to Fountain, et al., and
frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are
exposed to at least one freeze and thaw cycle; this procedure is described in
Bally et al., PCT Publication No. 87/00043, January 15, 1987, entitled ~ ~ -
~'Multilamellar Liposomes Having Improved Trapping Efficienciesn. ~ ~
Liposomes are comprised of lipids; the term lipid as used herein shall
;
mean any suitable material resulting in a bilayer such that a hydrophobic -
`~ 15 portion of the lipid material orients toward the interior of the bilayer
while a hydrophilic portion orients toward the aqueous phase. The lipids ~ -
which can be used in the liposome formulations of the present invention are ~-
the phospholipids such as phosphatidylcholine (PC) and phosphatidylglycerol ; -
(PG), more particularly dimyristoylphosphatidylcholine (DMPC) and
20 dimyristoylphosphatidylglycerol (DMPG). Liposomes may be formed and -~
vesiculated using DMPG, or DMPG mixed with DMPC in, for example, a 3:7 mole
ratio, respectively.
During preparation of the liposomes, organic solvents may be used to
; 25 suspend the lipids. Suitable organic solvents are those with intermediate
polarities and dielectric properties, which solubilize the lipids, and
` ~ include but are not limited to halogenated, aliphatic, cycloaliphatic, or
aromatic-aliphatic hydrocarbons, such as benzene, chloroform, methylene
chloride, or alcohols, such as methanol, ethanol, and solvent mixtures such
30 as benzene:methanol (70:30). As a result, solutions (mixtures in which the
lipids and other components are uniformly distributed throughout) containing
the lipids are formed. Solvents are generally chosen on the basis of their ~ :
biocompatability, low toxicity, and solubilization abilities.
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The starting multilamellar liposomes and resulting unilamellar liposomes
of the present invention may contain lipid soluble bioactive agents. Such
agents are typically associated with the lipid bilayers of the liposomes. As
used in the present invention, the term bioactive agent is understood to
include any compound having biological activity; e.g., lipid soluble drugs
such as non steroidal antinflammatory drugs such as ibuprofen, indomethacin,
sulindac, piroxicam, and naproxen, antlneoplastic drugs such as doxorubicin,
vincristine, vinblastine, methotrexate and the like, and other therapeutic
agents such as anesthetics such as dibucaine, cholinergic agents such as
pilocarpine, antihistimines such as benedryl, analgesics such as codeine,
anticholinergic agents such as atropine, antidepressants such as imiprimine,
antiarrythmic agents such as propranolol, and other lipophilic agents such as
dyes, therapeutic proteins and peptides such as immunomodulators, radio~
opaque agents, fluorescent agents, and the like. Additiona].ly, the vesicles
- made by the process of this invention may contain bilayer-associated markers
or molecules such as proteins or peptides.
The liposomes of the invention may be used in a liposome-drug delivery ~ ~ -
sysCem. In a liposome-drug delivery system, a bioactive agent such as a drug
,~ is associated with the liposomes and then administered to the patient to be
~ treated. For example, see Rahman et al., U.S. Patent No. 3,993,754; Sears,
l;~ U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871;
Schnieder, U.S. Patent No. 4,224,179; Lenk et al., U.S. Patent No. 4,522,803;
and Pountain et al., U.S. Patent No. 4,588,57a.
r Many drugs that are useful for treating disease show toxicitiès in thepatient; such toxicates may be cardiotoxicity, as with the antitumor drug
doxorubicin, or nephrotoxicity, as with the aminoglycoside or polyene
antibiotics such as amphotericin B. Amphotericin B is an extremely toxic
antifungal polyene antibiotic with the single most reliability in the
treatment of life-threatening fungal infections (Taylor et al., Am. Rev. ~ -
Respir. Dis., 19823, 125:610-611). Because amphotericin B is a hydrophobic
: ~
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1330199 :-
drug, it is insoluble in aqueous solution and is commercially available as a
colloidal dispersion in desoxycholate, a detergent used to suspend it which
in itself is toxic. Amphotericin B methyl ester and amphotericin B have also
been shown to be active against the HTLV-III/LAV virus, a lipid-enveloped
retrovirus, shown in the etiology of acquired immuno-defiency syndrome (AIDS)
(Schaffner et al., Biochem, Pharmacal., 1986, 35:4110-sll3;). In this study,
amphotericin B methyl ester ascorbic acid salt (water soluble) and
amphotericin B were added to separate cultures of HTLV-III/LAV infected cells
and the cells assayed for replication of the virus. Results showed that
amphotericin B methyl ester and amphotericin B protected target cells against
the cytopathic effects of the virus, similar to that demonstrated for the
herpes virus (Stevens, et al., Arch. Virol, 1975, 48:391).
Reports of the use of liposome-encapsulated amphotericin B have appeared
in the literature. Juliano et al. (Annals N.Y. Acad. Sci., 1985, 446:390-
402) discuss the treatment of systemic fungal infections with liposomal
amphotericin B. Such liposomes comprise phospholipid, for example
dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol
(DMPG) in a 7:3 mole ratio, and cholesterol. Acute toxicity studies (LD50s)
and }n vitro assays comparing free and liposome-entrapped amphotericin B
showed lower toxicites using the liposomal preparations with substantially
unchanged antifungal potency. Lopez-Berestein et al., ~J. Infect. Dis., -
1986, 151:704-710) administered liposome-encapsulated amphotericin B to
patients with systemic fungal infections. The liposomes comprised a 7:3 mole - -
25 ratio of DMPC:DMPG, and the drug was encapsulated at a greater than 90% .efficiency. As a result of the liposomal-drug treatment at 5mol ~
' ~ amph'otericin B, 66~ of the patients treated responded favorably, with either
partial or complete remission of the fungal infection. Lopez-Berestein et
al. (J. Infect. Dis., 1983, 147:939-945), Ahrens et al., (S. Jour. Med. Vet
Mycol., 1984, 22:161-166), Panosian et al., (Antimicrob. Agents Chemo., 1984,
25:655-656), and Tremblay et al. (Antimicrob. Agents Chem., 1984, 265:170- `~
173) also tested the comparat}~e efficacy of free versus liposomal
amphotericin B in the treatment of candidiasis. In all cases, it was found '
..
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133019~
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that much higher dosages of amphotericin B may be tolerated when this drug is
encapsulated in liposomes. The amphotericin B-liposome formulations had
little to no effect in the treatment of leishmaniasis.
The ability of liposomes to buffer the toxicity of entrapped drugs with
little or no decrease in efficacy is becoming increasingly well established.
Therefore, there is an increasing need to be able to form liposomes of all
types which have these qualities. Uniiamellar vesicles are clearly preferred
for certain types of ~n vivo drug delivery over multilamellar vesicles, as
well as for studies of membrane-mediated processes. As used as in vivo
delivery vehicles, for example, unilamellar vesicles are cleared more slowly
from the blood than are MLVs, and exhibit an enhanced distribution to the
lungs and possibly bone marrow. Up to the time of the present invention, the
methods known for producing these type vesicles relied upon harsh treatment
IS of multilamellar vesicles, such as extrusion through filters, or other
physically damaging processes requiring energy input such as sonication,
homogenization or milling. Chemical treatment techniques employing harsh
detergents or solutions at high or low pH to form unilamellar vesicles have `.
also been employed. The present invention advances the art in that it allows
formation of unilamellar vesicles from multilamellar vesicles without the
heretofore harsh treatments required, but through the incubation of the
liposomes in low ionic strength media at selected temperatures.
Additionally, the unexpected simplicity of preparation of these systems
25 i9 complemented by the highly defined conditions under which they may be
formed. The fact that vesiculation of these lipids occurs only around about ~-
the lipid phase transition temperature (Tc) and under low ionic strength
incubations gives one a high degree of control over vesicle formation. In
addition, the characteristic bilayer instability of these systems would be ;expected to favor interaction cf the bilayer with hydrophobic compounds such
as drugs, or enhance insertion of membrane proteins or peptides.
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133019~
SUMMARY OF THE INVENTION
The present invention discloses a method for spontaneously forming
unilamellar vesicles from multilamellar vesicles (MLVs). Such MLVs comprise
lipids, and unilamellar vesicles are formed by incubating the multilamellar
vesicles in low ionic strength medium at neutral pH, around about the
transition temperature of the lipids. Preferably the lipids comprise
phospholipids, specifically phosphatidylglycerol alone or in combination with
phosphatidylcholine, more specifically dimyristoylphosphatidylcholine and
dimyristoylphosphatidylglycerol, in a 7:3 mole ratio.
To form the unilamellar vesicles of the invention, the liposomes are
incubated at about 22-26C, preferably about 24C in a medium of between
about 0 mM and 25 mM salt. More preferably, the medium comprises about 0 ~
10mM salt at pH of about 7.0 to about 8.0, preferably pH 7.6 and incubation
time is about 15 minutes to about 24 hours.
.:, ~"`~''''.
BRIEF DESCRIPTION OF THE DRAWING ; ~ -
- ~ ~-' "
FIGURE 1 demonstrates vesiculation of DMPC:DMPG (7:3) MLVs as a function
of ionic strength of the incubation medium. DMPC:DMPG (lOmM) was hydrated at
2S 4C in the media shown below and incubated at 24C (see Examples 1 and 2). -
Sample media were H2O (open circles); 2mM HEPES (closed squares); 10mM NaCl, -
2mM HEPES, pH 7.6, (open triangles); or 25 mM NaCl, 2 mM HEPES, pH 7.6
~closed triangles). -
FIGURE 2 are 31P-NMR spectra of DMPC:DMPG. Lipid (10 mM) was hydrated in
H2O at 4C and its spectrum was recorded at 30C ~A). The same lipid mixture
was then incubated at 24C for 1 hour (~3) and 12 hours (C). DMPC:DMPG (7:3 ~
~,
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X ' ~:
1330~99
mole ratio) hydrated in 150 mM NaCl, 10 mM HEPES, pH 7.6 and incubated at
24C for 12 hours is shown in (D).
FIGURE 3 are 31P-NMR spectra for mixtures of phosphatidylcholine with
S phosphatidylglycerol. Lipid (10 mM) was hydrated in 150 mM NaCl, 10 mM
HEPES, pH 7.6 (A,B,C,) or 2 mM HEPES, pH 7.6 (D,E,F,G,H,J) and incubated at
24C (A,B,C,G,H,J) or lCC (D,E,F) for 16 hours.
DETAILED DESCRIPTION OF THE INVENTION
.
The unilamellar liposomes of this invention are formed by the exposure of
multilamellar liposomes to conditions of low ionic strength media at neutral -
pH, and incubation temperatures around about the gel-to-liquid crystalline
transition temperature (Tc). Under such incubation conditions, MLVs
vesiculate to form unilamellar vesicles. Prior art techniques requiring
acidic and alkaline pH variations are not needed in the present method, as
`~ vesiculation takes place in a narrow range around neutral pH. The liposomes
of the present invention are preferably comprised of phospholipids,
specifically dimyrlstoylphosphatidylglycerol (DMPG) or with
~; dimyristoylphosphatidylcholine (DMPC). Various mole ratios of DMPC and DMPG
are suitable for liposome vesiculation, however, the rate of vesiculation
decreases with decreasing DMPG concentration.
2S
Upon hydration most naturally occurring phospholipids generally adopt
` either the bilayer organization or the hexagonal HII phase (Cullis and deXruijff, 1979, Biochim. 8iophys. Acta, 559:339; Cullis et al., 1935, ln
Phospholipids and Cellular Regulation, J. F. Ruo, Ed., CRC Press, Boca Raton,
Florida). In both instances the macromolecular structures formed are large
(several microns) and are stable, such that even transitions between these
two polymorphic pha9e9 do not generate small vesicles. One exception is the
case of cardiolipin which in the presence of calcium adopts the hexagonal HII
;
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133019,~ ~
phase. If this mixture is dialyzed against EDTA, small vesicles are
generated (Vail et al., 1979, Biochim. Biophys. Acta, 551:74). However, this
is presumably due to the removal of calcium from cardiolipin at the exterior
of the cylindrical HII arrays and the consequent ~blebbing-off~ of bilayer
vesicles. While large multilamellar vesicles are useful membrane models for
investigating the structural and motional properties of lipids, many areas of
membrane research and drug delivery require or favor, respectively, the use -
of unilamellar vesicle systems. Two categories of unilamellar vesicles can
be defined. These are small unilamellar vesicles (S W s) of diameter less
10 than about 50 nm, and large unilamellar vesicles (L W s) which generally -
encompass vesicles S0 nm to 1 micron in diameter (Hope et al., 1986, Chem. -~
Phys. Lip., 40:89).
, .
The absence of multiple internal aqueous compartments and the relatively
high trapped volumes obtained with LWS make them useful in a variety of
¦ research areas including membrane fusion (Wilschut et al., 1980,
Biochemistry, 19:6011) and the in vivo delivery of biologically active -: -
compounds (Poznansky et al., 1984, Pharmacol. Rev., 36:227). While MLVS :
formed by the simple hydration of dry lipid are under osmotic stress due to
non-equilibrium solute distribution (Gruner et al., 1985, Biochemistry,
24:2833; Mayer et al., 1986, Biochim. Biophys. Acta, 858:161), they are
nevertheless stable structures. The formation of LWs or S W s from MLVs
usually requires aggressive disruption, for example, by sonication (Huang,
1969, Biochemistry, 8:344) or extrusion through polycarbonate filters (Hope
25 et al., 1985, Biochim. Biophys. Acta, 812, 55), as mentioned above.
While the formation of LWS from mixtures of phosphatidylcholine with
either charged sini31e chain detergents (Hauser et al., 1986, Biochemistry,
25:2126) or short chain phospholipids (Gabriel et al., 1984, Biochemistry,
30 23:4011) has been described, the only reported instance of MLVS composed
~ solely of bilayer-forming phospholipids spontaneously vesiculating concerns
f mixtures of acidic phospholipids and phosphatidylcholine transiently exposed
~ to an alkaline pH ~Hauser et al., 1982, Proc. Natl. Acad. Sci USA, 79:1683;
.~
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133019~
Hauser, V.S. Patent No. 4,619,794, issued October 28, 1986, Hauser et al.,
1986, Biochemistry, 25:2126; Gains et al., 1983, Biochim. Biophys. Acta,
731:3i; Li et al., 1986, Biochemistry, 25:7477).
Since the exposure of membrane lipids to alkaline pH may result in
degradation of the lipids and/or any bioactive agent present, and leakage of
the vesicle contents, this technique has severe shortcomings in the field of
drug delivery employing liposomes. We disclose here that formation of
unilamellar vesicles can surprisingly occur at around neutral pH for
saturated phosphatidylglycerol and mixtures of saturated phosphatidylcholine
and phosphatidylglycerol. Unexpectedly, vesiculation is rapid only at
temperatures around the gel to liquid-crystalline phase transition (the
transition temperature or Tc, about 22C to about 26OC, most preferably about
24C), and when hydration or incubation media of low ionic strength are used.
When incubation media of high ionic strength (higher than about 50 mM salt)
are used, vesiculation occurs at a decreased rate, or not at all.
Vesiculation occurs as a function of lowering the ionic strength of the
incubation medium. MLVs vesiculate spontaneously when exposed to low ionic -.- strength incubation media (about 10 mM ionic strength and less) when
incubated around about the Tc of the lipid. Any ionic species solutions may
,,
~ be used as incubation media, such as the salts sodium chloride, potassium
;~ chloride, and others. While a range, therefore, of about 0-25 mM salt in the
incubation medium will promote vesiculation, the optimum conditions are
around about 0-10 ~M salt.
Vesiculation of MLV systems may be determined by incubating the liposomes
` in Iow ionic strength medium for 15 minutes to several hours, at around the
gel-to-liquid crystalline transition temperature of the lipids used. Whether
~ vesiculation has occurred may be measured by the size of the resulting ~ ~
;~ 30 liposomes using quasi-elastic light scattering, (unilamellar versus , ~-
multilamellar), visualisation of the resulting vesicles using freeze-fracture
electron microscopy, and 3lP-NMR analysis of lineshape and spectrum width.
For example, narrow spectrum width and isotropic signal is indicative of
~ ~9~
133~1 99
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~ ,.
unilamellar vesicle structure, while a low field shoulder and high field
peaks are indicative of larger vesicles. -
'' ' ' ' ' '
Liposomes incorporating a bioactive agent, such as a drug, such as for
S example, amphotericin B may be formed according to the processes of the
invention as follows. Amphotericin B is suspended in an aqueous solution,
for example distilled water, by sonication. The suspended drug is then
admixed with a suspension of lipid in aqueous solution, such as distilled
water or sodium chloride solution. The mixture is incubated at or above the
transition temperature of the lipid employed, with the resultant formation of
vesicles.
Where dimyristolyphosphatidylglycerol (DMPG) is used alone or in ~;
combination to form the liposomes, and when the lipid has been admixed with ~;
an aqueous solution having an ionic strength of about 0 mM to about 25 mM
salt, and incubated at about the transition temperature (Tc) of the lipid
,
(i.e., at about 22-24C), the liposomes spontaneously vesiculate, forming
large unilamellar vesicles (L W s). This method for formation of L W s,
employs no harsh treatment of the vesicles such as exposure to chemicals,
20 detergents, or extreme pH -
,~, ,,,
For example, DMPG can be used alone or, for example, with other lipid
such as with DMPG, e.g., in a 3:7 mole ratio of DMPC:DMPG. These lipids can
be co-lyophilized from a 70:30 v/v solution of benzene:methanol, and stored
at -20C until use. MLVs are prepared by hydrating the lipid (for example, a
total of lipid of 13.5 umoles/ml) in aqueous solution such as distilled water
or buffer at 4C. When formation of amphotericin B-containing L W s is
desired, the lipid is hydrated in an aqueous solution of ionic strength of ; ;
I about ) mM to about 25 mM salt, and incubated at about 23C. Amphotericin B
! 30 dispersed in di9tilled water by bath sonication, at a concentration of about
0.98 umoles/ml is then added to the hydrated lipid and incubated at about 23
C for about one hour, resulting in L W s containing amphotericin B. These
-10- ; ,,
1330~99
proportions of lipid and amphotericin B result in about a 7 mole ~ ratio of
amphotericin B.
The lipids of the present invention may be hydrated to form liposomes
using any available aqueous solutions, for example, distilled water, saline,
or aqueous buffers. Such buffers include but are not limited to buffered
salines such as phosphate buffered saline (nPBS"), tris-(hydroxymethyl)-
aminomethane hydrochloride Intrisn) buffers, and preferably N-2-hydroxyethyl
piperazine-N-2-ethane sulfonic acid (nHEPESn) buffer. Such buffers are
preferably used at pH of about 7.0 to about 8.0, preferably about pH 7.6. If
required, the ionic strength of the medium may be adjusted to physiological
values following the vesiculation procedure.
The liposomes of the present invention may be dehydrated either prior to
or following vesiculation, thereby enabling storage for extended periods of
time until use. Standard freeze-drying equipment or equivalent apparatus may - ~?
be used to lyophilize the liposomes. Liposomes may also be dehydrated simply
by placing them under reduced pressure and allowing the suspending solution
to evaporate. Alternatively, the liposomes and their surrounding medium may
be frozen prior to dehydration. Such dehydration may be performed in the
presence of one or more protectants such as protective sugars, according to
the process of Janoff et al., PCT 86/01103, published February 27, 1986, and
incorporated herein by reference.
- ,. : .,, - :- .
2S The liposomes resulting from the processes of the present invention can -
be used therapeutically in mammals, including man, in the treatment of -~
infections or conditions which benefit from the employment of liposomes which
give for example, sustained release, reduced toxicity, and other qualities ;~
which deliver the drug in its bioactive form.
The mode of administration of the preparation may determine the sites and
cells in the organism to which the compound will be delivered. The liposomes
of the present invention can be administered alone but will generally be ~ ~
.: ~. :
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1330199 ; ~ ~
administered in admixture with a pharmaceutical carrier selected with regard
to the intended route of administration and standard pharmaceutical practice.
The preparations may be injected parenterally, for example, intra-arterially
or intravenously. The preparations may also be administered via oral,
subcutaneous, or intramuscular routes. For parenteral administration, they
can be used, for example, in the form of a sterile aqueous solution which may
contain other solutes, for example, enough salts or glucose to make the
solution isotonic. Other uses, depending upon the particular properties of
the preparation, may be envisioned by those skilled in the art.
For the topical mode of administration, the liposomes of the present ~ ~ -
invention may be incorporated into dosage forms such as gels, oils,
emulsions, and the like. Such preparations may be administered by direct
application as a cream, paste, ointment, gel, lotion or the like.
-
For the oral mode of administration, the liposomes of this invention
encapsulating a bioactive agent can be used in the form of tablets, capsules, ~ .
losenges, troches, powders, syrups, elixirs, aqueous solutions and ;
suspensions, and the like. In the case of tablets, carriers which can be -
used include lactose, sodium citrate and salts of phosphoric acid. Various
disintegrants such as starch, and lubricating agents, such as magnesium - -
stearate, sodium lauryl sulfate and talc, are commonly used in tablets. ~or
oral administration in capsule form, useful diluents are lactose and high
molecular weight polyethylene glycols. When aqueous suspensions are required --
~ 25 for oral use, the active ingredient is combined with emulsifying and
; suspending agents. If desired, certain sweetening and/or flavoring agents
can be added.
.: -
The following examples are given for purposes of illustration only and
not by way of limitatio= on thl~ scope oE the invention.
X -12-
133~
E~AMPI,E 1
,
':
- DMPC:DMPG ~7:3 M ratio) was lyophilized from benzene:methanol (70:30
v/v). The lipid was hydrated to 10 mM with distilled water pH 7.6, at gC,
forming MLVs. The suspension was then incubated at 24C for 15 minutes.
QELS studies showed the resulting liposomes to be about 200 nm in diameter,
- corresponding to L W s. -~
The above proceclure was followed using 2 mM HEPES buffer as the hydrating
solution. QELS measurements revealed L W s. - ~ -
This Example demonstrates the formation of unilamellar liposomes by the
incubation of a 7:3 M ratio of DMPC:DMPG in low ionic strength medium ~,
(distilled water, 0 mM salt), at neutral pH. Unilamellar liposomes formed
spontaneously when the preparation was incubated at 24C.
EXAMPLE 2 ~ -
The procedures and mater~als of Example 1 were employed using 150 mM
NaCl, 2 mM HEPES buffer as the hydrating solution. QELS measurements
revealed~no change in liposome size (no vesiculation) after incubation. ~-
2S Plgure 1 demonstrates vesiculation by plotting the vesicle diameter
(obtained by quasi elastic light scattering, QELS) as an indication of MLY or --`-
L W against time of incubation, and shows that the rate of vesiculation at
24C is directly related to the ionic strength of the hydration medium.
Figure 2 demonstrates the vesiculation by 3lP-NMR spectra of the suspensions;
the vesiculated samples (3 and C, at low ionic strength incubation)
demon9trate the characteristic narrow spectrum and isotropic lipid motion
poak which would be expected for vesicles smaller than 400 nm. Figure 2 A
and ~ demonstrate the characteristic bilayer lineshape with,low field
~:
1330193
shoulder and two high field peaks. Plots A and D were recorded from samples
incubated under conditions where vesiculation does not occur; at temperatures
above the Tc, and hydration media of high ionic strength, respectively.
Freeze fracture electron microscopy confirmed the QELS and 3lP-NMR data
by allowing visualization of the multilamellar or unilamellar vesicles.
EXAMPLE 3
:
DMPG tlO mM) was hydrated with 10 mM NaCl, 2 mM HEPES at 4C, pH 7.6,
forming MLVs. These MLVs were incubated at 24C for 15 minutes, and the
sample analyzed by QELS. The resulting liposomes were unilamellar (L W s).
-
This Example may be compared with Example 13, where liposomes made of a ~
3:7 M ratio of DMPC:DMPG incubated in lOmM NaCl (Example 13) only approach -
the 200 nm diameter vesicles of Example 3 after 5 hours incubation.
: .~ ' -
A 7:3 M ratio of dry DMPC:DMPG was equilibrated at 32C in a water-
saturated atmosphere for 60 minutes, and then the procedures and materials of
Example 1 were followed to make MLVs (10 mM lipid), using 2 mM HEPES as
.
hydration medium and an incubation temperature of 32~C. After 6 hours
incubation, no vesiculation had occurred as QELS measurements revealed the
liposomes had a mean diameter of greater than 2 microns.
.~, j j ,
`~ The above preparation was then incubated at 24C and QELS measurements
revealed that the liposomes had vesiculated, resulting in unilamellar
vesicles.
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.
This Example is a control for the incubation of the liposome systems
around about the Tc of the lipid; it shows this incubation parameter is an
important requirement of the invention. -
:, .
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EXAMPLE 5
- '.:
- The procedures and materials of Bxample 4 were employed using 2 mM HEPES
as the hydration medium and an incubation temperature of 15C. After 6 hours
10 incubation, no vesiculation had occurred as QELS measurements revealed the -
liposomes had a mean diameter greater than 2 microns.
- ~,: . ;, ' "-'.' ,. - ':,.
- ~ The above preparation was then incubated at 24C and QELS measurements -
;-~ revealed that the liposomes had vesiculated, resulting in unilamellar
vesicles.
!. ~ . .. ...
This Example serves as a further control for Tc being an important . ~
~ incubation parameter. No vesiculation occurred at this incubation ~ -
r~ ; temperature. However, when this system was incubated at 24C, the liposomes ~ ; .;
rapidly vesiculated.
EXAMPLE 6 ~-
2S A 7:3 M ratio of DOPC:DOPG was hydrated with 2 mM HEPES buffer and ;~
~` incubated for 24 hours at 24C. Samples were analyzed using 31P-NMR
' spectroscopy which had a spectrum consistent with bilayer phase lipid
organization (Figure 6K), and the vesicles had a diameter greater than about ~ -
400 nm. ; ~
'~ -15~
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~ ,, r~ ~
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1330193 --
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:
EXAMPLE 7
The procedures and materials of Example 1 were employed, using a 7:3 Mratio of DOPC:DMPG. The lipid was hydrated with 2 rnM HEPES and incubated at
24C for 16 hours.
:, . .
31P-NMR spectroscopy revealed little or no vesiculation.
EXAMPLE 8
The procedures and materials of Example 7 were employed, using a 7:3 M
ratio of DMPC:DOPG. The lipid was hydrated with 2 rnM HEPES and incubated at
24C for 16 hours.
~- 15
~ 31P-NMR spectroscopy revealed little or no vesiculation.
5, ~. " EXAMPLE 9
The procedures and materials of Example 7 were employed, using a 7:7:3:3
M ratio of DOPC:DMPC:DOPG:DMPG. The lipid was hydrated with 2 TdM HEPES and
incubated at 24C for 16 hours.
31P-NMR spectroscopy revealed little or no vesiculation.
I . .~ ~
In this Example, when the gel and liquid-crystalline domains contain both
~- phospholipid species, e.g., DMPC:DOPC:DMPG:DOPG (7:7:3:3), only very limited ;
breakdown of MLV structure is apparent. In these systems the presence of
dioleoyl phospholipids stabilizes MLV structure. This Example demonstrates ~ -
~` the atability of oleoyl-containing systems. Even when phosphatidylglycerol
is present, the dioleoyl species stabilizes mixtures of 7:3 M ratio DOPC:DOPG
.
,
~ X -16-
,:
r~ .,
1330~99
so that incubation at 24C in low ionic strength buffer does not induce
vesiculation; the systems remain multilamellar.
Further, the stabilizing nature of dioleoyl chains is observed in
Examples 7-12 where no vesiculation is observed even when domains of both gel
phase lipid (i.e.: dimyristoyl chains) and liquid crystalline phase lipid
(i.e.: dioleoyl groups) are present. Figure 3 (A-J) demonstrates the 31P-NMR -;~
spectra for such samples incubated at either 10C or 24C. All spectra are
characteristic of large vesicles in the bilayer phase (M~Vs); the samples did ~ -
, . .-~, . . .
~ 10 not vesiculate.
s EXAMPLE 10
The procedures and materials of Example 7 were employed, using a 7:3 M
ratio of DOPC:DMPG. The lipid was hydrated with 150 mM NaCl, 2 mM HEPES and
incubated for 16 hours at 24C.
31P-NMR spectroscopy revealed little or no vesiculation. -
~" ~ 20 ~ i
XAMPLB 11
The procedures and materials of Example 7 were employed, using a 7:3 M
2S ratio of DMPC:DOPG. The lipid was hydrated with 150 mM NaCl, 2 M HEPES and
incubated for 16 hours at 24C. -
3lP-NMR spectroscopy revealed little or no vesiculation. -
}~
J~
i".i ~
1';~;' .` ~
-17-
133~99 j
~XAMPLE 12 ~;
The procedures and materials of Example 7 were employed, using a 7:7:3:3
M ratio of DOPC:DMPC:DOPG:DMPG. The lipid was hydrated with 150 mM NaCl, 2
mM HBPES and incubated for 16 hours at 24C. `
3lP-NMR spectroscopy revealed litt;e or no vesiculation.
EXAMPLE 13
....
The procedures and materials of Example 3 were employed, using a 3:7 M
15 ratio of DMPC:DMPG. The lipid was hydrated in 10 mM NaCl, 2 mM HEPES at pH ~ :-
~ ~7.6 at 4DC, forming MLVs. The suspension was then incubated for 1 hour at
b '. j~24C. QELS measurements revealed that vesiculation of the MLVs had formed L W s.
EXAMPLE 14
`~ ~Lipid tl4.8 umol/ml, 7:3 mol ratio of DMPC:DMPG) was hydrated in
.~ ~distilled water and incubated at 4C. The resulting MLVs were extruded
i ~ ~
~Y~ 25 through two stacked polycarbonate filters ten times using the LUVET
procedure.
;~
Amphotericin B was dispersed in distilled water using a bath sonicator at
` a concentration of 10.8 umol/ml. The amphotericin B dispersion was added to
the lipid suspension to a final lipid and amphotericin B concentration of
13.5 umol/ml and 0.98 umol/ml, respectively. To remove unincorporated
amphotericin B, 20 ml of the sample were centrifuged at 15,000 X g for 30
minutes in a Ti60 or SW 27 rotor (Beckman) at 22C in a Beckman L8-60
"~
i:
-18-
` ~ ~` . ' ~ ';'
133~199 :~
ultracentrifuge. The supernatant free amphotericin B was removed without
disturbing the liposome pellet. The resulting liposomes were measured by
quasi-elastic light scattering to be larger than 1.0 um in diameter.
The above procedure employing incubation conditions of 23C were repeated
employing 150 mM NaCl, 10 mM Na2P04, pH 7.4 to hydrate the lipids. The
resulting liposomes were measured by quasi elastic light scattering to be
larger than 1.0 um in diameter. Rate of amphotericin B uptake by liposomes
was highest when the ionic strength of the medium was low (distilled water
vs. 150 mM NaCl).
EXAMPLE 15 :
The materials and procedures of Example 14 were employed, but wherein the
lipid suspended in distilled water was incubated with the amphotericin B at
;~ 22C. The resulting liposomes were unilamellar and measure at about 0.1
0.2 um in mean diameter by quasi elastic light scattering.
- . :,
: '~,: ~.. '~i.
:,~; : . :-`..
-19-
, ~ ,,..... .... ........ .:
