U.S. patent application number 11/359999 was filed with the patent office on 2006-11-16 for methods for preparation of lipid-encapsulated therapeutic agents.
This patent application is currently assigned to The University of British Columbia. Invention is credited to Pieter R. Cullis, Norbert Maurer, Kim F. Wong.
Application Number | 20060257465 11/359999 |
Document ID | / |
Family ID | 36821671 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257465 |
Kind Code |
A1 |
Maurer; Norbert ; et
al. |
November 16, 2006 |
Methods for preparation of lipid-encapsulated therapeutic
agents
Abstract
Fully lipid-encapsulated therapeutic agent particles of a
charged therapeutic agent are prepared by combining a lipid
composition containing preformed lipid vesicles, a charged
therapeutic agent, and a destabilizing agent to form a mixture of
preformed vesicles and therapeutic agent in a destabilizing
solvent. The destabilizing solvent is effective to destabilize the
membrane of the preformed lipid vesicles without disrupting the
vesicles. The resulting mixture is incubated for a period of time
sufficient to allow the encapsulation of the therapeutic agent
within the preformed lipid vesicles. The destabilizing agent is
then removed to yield fully lipid-encapsulated therapeutic agent
particles. The preformed lipid vesicles comprise a charged lipid
which has a charge which is opposite to the charge of the charged
therapeutic agent and a modified lipid having a steric barrier
moiety for control of aggregation.
Inventors: |
Maurer; Norbert; (Vancouver,
CA) ; Wong; Kim F.; (Vancouver, CA) ; Cullis;
Pieter R.; (Vancouver, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
The University of British
Columbia
Vancouver
CA
|
Family ID: |
36821671 |
Appl. No.: |
11/359999 |
Filed: |
February 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10019199 |
Dec 20, 2001 |
7094423 |
|
|
PCT/CA00/00843 |
Jul 14, 2000 |
|
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11359999 |
Feb 22, 2006 |
|
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60143978 |
Jul 15, 1999 |
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Current U.S.
Class: |
424/450 ;
435/458; 514/44R; 977/907 |
Current CPC
Class: |
A61K 9/5089 20130101;
A61K 9/127 20130101; A61K 9/1278 20130101; A61K 31/7088 20130101;
A61P 43/00 20180101; A61K 31/711 20130101; A61K 31/7105 20130101;
A61K 9/1272 20130101 |
Class at
Publication: |
424/450 ;
435/458; 514/044; 977/907 |
International
Class: |
A61K 9/127 20060101
A61K009/127; C12N 15/88 20060101 C12N015/88; A61K 48/00 20060101
A61K048/00 |
Claims
1.-12. (canceled)
13. A therapeutic agent particle comprising a charged therapeutic
agent, a charged lipid having a charge which is opposite to that of
the charged therapeutic agent, and a modified lipid having a steric
barrier moiety, wherein the therapeutic agent is fully encapsulated
and wherein the therapeutic agent particle comprises two or more
lipid bilayers.
14. The therapeutic agent particle of claim 1, wherein the
therapeutic agent is anionic and the charged lipid is cationic.
15. The therapeutic agent particle of claim 2, wherein the
therapeutic agent is a polynucleotide.
16. The therapeutic agent particle of claim 14, wherein the
cationic charged lipid is selected from the group consisting of:
DODAC, DOTMA, DDAB, DOTAP, DC-Chol, DMRIE, DOSPA, DOGS, DODMA, and
DODAP.
17. The therapeutic agent particle of claim 1, wherein the
therapeutic agent is cationic and the charged lipid is anionic.
18. The therapeutic agent particle of claim 1, wherein the modified
lipid having a steric barrier moiety is a PEG-lipid or a polyamide
oligomer lipid.
19. The therapeutic agent particle of claim 1, wherein the lipid
composition of the particle comprises 10-40 mol % charged lipid and
0.5 to 15 mol % modified lipid.
20. The therapeutic agent particle of claim 1, further comprising a
neutral lipid and a sterol.
21. The therapeutic agent particle of claim 20, wherein the lipid
composition of the particle comprises 10-40 mol % charged lipid,
25-45 mol % neutral lipid, 35-55 mol % sterol, and 0.5-15 mol %
modified lipid.
22. The therapeutic agent particle of claim 1, wherein the particle
size is 25-250 nm diameter.
23. A pharmaceutical composition comprising therapeutic agent
particles of any of claims 13-22.
24. The composition of claim 23, wherein at least 50% of the
therapeutic agent particles in the composition comprise two or more
lipid bilayers.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/019,199, filed Dec. 20, 2001, which is a
national stage application filed under 35 U.S.C. .sctn.371 of
International Application No. PCT/CA00/00843, accorded an
International Filing Date of Jul. 14, 2000, which claims priority
to U.S. Provisional Application No. 60/143,978, filed Jul. 15,
1999.
FIELD OF THE INVENTION
[0002] This invention relates to a novel method for making
particles of lipid-encapsulated therapeutic agents, and in
particular, lipid-encapsulated therapeutic nucleic acid particles
which may be useful in antisense therapy or gene therapy.
BACKGROUND OF THE INVENTION
[0003] The concept of using lipid particles as carriers for
therapeutic agents has been considered by numerous people.
Formulations have relied on complexation of therapeutic agent to
the outside of the lipid particle, or actual entrapment of the
therapeutic agent, although the ability to make formulations of
either type depends on a matching of the characteristics of the
lipids and the therapeutic agent, as well as the methods employed
to make the particle. In the case of particles with entrapped
therapeutic agents, the entrapment method may be passive, i.e., the
lipid particles are assembled in the presence of the therapeutic
agent, some of which happens to get trapped; or active, i.e, the
therapeutic agent is drawn or forced into the interior of a lipid
particle as a result of an induced gradient of some type.
Notwithstanding the many efforts to utilize lipid particles as
carriers, there remain problems which may limit actual applications
of lipid-entrapped therapeutic agents. These include low levels of
therapeutic agent incorporation on a drug/lipid basis, low
efficiency's of capture of the therapeutic agent, and lack of a
suitable procedure for larger scale manufacturing of the
lipid-encapsulated therapeutic agent particles.
[0004] Large scale manufacturing of fully lipid-encapsulated
therapeutic agent particles has not been achieved where there is a
significant electrostatic interaction between the lipid and the
therapeutic agent. A basic problem is aggregation. Aggregation
normally results when charged lipid is mixed with oppositely
charged therapeutic agent, resulting in a solution containing a
milky flocculent mass which is not useable for further processing,
let alone for therapeutic use. The aggregation problem has
prevented the development of therapeutic compositions which could
be of great utility.
[0005] Bench scale formulations using charged lipid and oppositely
charged therapeutic agent have been successfully achieved using
cationic lipids and anionic nucleic acids in a passive
encapsulation process described in U.S. Pat. No. 5,705,385 to Bally
et al. (PCT Applic. No. WO 96/40964; See also U.S. patent
applications Ser. Nos. 08/484,282; 08/485,458; 08/660,025; and
09/140,476) and PCT patent Applic. No. WO 98/51278 to Semple et al.
(See also U.S. patent application Ser. No. 08/856,374) all assigned
to an assignee of the instant invention and incorporated herein by
reference. See also Wheeler et al. (1999) Stabilized plasmid-lipid
particles: Construction and characterization. Gen. Ther. 6:271-281.
These techniques employ an aggregation preventing lipid, such as a
PEG-lipid or ATTA-lipid (disclosed in co-pending U.S. patent
application Ser. No. 08/996,783 which is incorporated herein by
reference), which effectively prevents complex aggregate formation.
Resulting fully lipid encapsulated therapeutic agent particles have
excellent pharmaceutical characteristics, such as controlled size
(in the 30-250 nm range), full encapsulation (as measured by
nuclease resistance, for example) and stability in serum.
[0006] W098/51278 describes a bench scale procedure for the
preparation of the lipid-encapsulated therapeutic agent particles
using passive entrapment. This known method employs the two basic
steps of lipid hydration and liposome sizing. In the lipid
hydration step, a cationic lipid solution (95% EtOH solvent) is
added dropwise into an agitated reservoir containing polynucleotide
therapeutic agent in citrate buffer (pH 3.8) to a final composition
of 40% EtOH, 9.9 mg/ml lipid and 2.0 mg/ml polynucleotide. Lipid
particles resulting from this hydration step are typically 400 nm
diameter and greater, which is too large for general use as a
therapeutic. Because of this, extensive post-formulation processing
such as high temperature extrusion (at 65.degree. C.) and
optionally freeze-thawing (from liquid nitrogen to 65.degree. C.
waterbath) is required to obtain suitably-sized lipid particles.
The efficiency of encapsulation using this is fairly high (60-90%)
in terms of recovered final drug:lipid ratio, but the absolute
efficiency of incorporation of starting polynucleotide into the
final particle formulation is sub-optimal (25-45%).
[0007] Commercial large scale manufacturing of these particles is
not efficiently achieved using traditional methods employed in the
liposome field. These problems exist notwithstanding the great deal
of art on the manufacturing of liposome/drug formulations that has
emerged since the first description of liposome preparation by
Bangham, A D. et al. (1965) "The action of steroids and
streptolysin S on the permeability of phospholipid structures to
cations", J. Mol. Biol. 13,138-147.
[0008] Known large scale manufacturing techniques for lipid
particles can be broadly classified into the following categories:
1) Lipid Film Hydration (i.e. Passive entrapment); 2) Reverse Phase
Evaporation; 3) High-Pressure extrusion; 4) and Solvent injection
(dilution) (see for example U.S. Pat. Nos. 4,752,425 and 4,737,323
to Martin et al). Particular instruments for lipid particle
manufacturing disclosed in the art include: U.S. Pat. Nos.
5,270,053 and 5,466,468 to Schneider et al; Isele, U. et al. (1994)
Large-Scale Production of Liposomes Containing Monomeric Zinc
Phthalocyanine by Controlled Dilution of Organic Solvents. J.
Pharma. Sci. vol 83(11) 1608-1616; Kriftner, R W. (1992) Liposome
Production: The Ethanol Injection Technique, in Bruan-Falco et al.,
eds, Liposome Derivatives, Berlin, Springer-Verlag, 1992, pp.
91-100; Kremer et al. (1977) Vesicles of Variable Diameter Prepared
by a Modified Injection Method. Biochemistry 16(17): 3932-3935;
Batzri, S. and Korn, E D. (1973) Single Bilayer Liposomes Prepared
Without Sonication, Bioch. Biophys. Acta 298: 1015-1019.
[0009] None of the above noted methods or instruments are suitable
for scale up of formulations of charged lipid and oppositely
charged therapeutic agents with the excellent pharmaceutical
characteristics of Bally et al., supra, and Semple et al., supra.
The manufacturing techniques set out in Bally et al., supra, and
Semple et al., supra were developed only for 1-100 ml preparations,
and are cumbersome and lead to unsustainable inefficiencies in
large scale manufacturing (i.e. at the scale of 20-200 litres).
[0010] The instant invention provides, for the first time, methods
for the large-scale preparation of fully encapsulated
lipid-therapeutic agent particles where the lipid and therapeutic
agent are oppositely charged. These particles are useful as
therapeutic compositions and for experimentation and otherwise. It
is an object of this invention to provide such methods.
SUMMARY OF THE INVENTION
[0011] In accordance with the present invention, fully
lipid-encapsulated therapeutic agent particles of a charged
therapeutic agent are prepared by combining a lipid composition
comprising preformed lipid vesicles, a charged therapeutic agent,
and a destabilizing agent to form a mixture of preformed vesicles
and therapeutic agent in a destabilizing solvent. The destabilizing
solvent is effective to destabilize the membrane of the preformed
lipid vesicles without disrupting the vesicles. The resulting
mixture is incubated for a period of time sufficient to allow the
encapsulation of the therapeutic agent within the preformed lipid
vesicles. The destabilizing agent is then removed to yield fully
lipid-encapsulated therapeutic agent particles. The preformed lipid
vesicles comprise a charged lipid which has a charge which is
opposite to the charge of the charged therapeutic agent and a
modified lipid having a steric barrier moiety for control of
aggregation. The modified lipid is present in the preformed
vesicles in an amount effective to retard, but not prevent,
aggregation of the preformed vesicles. In a preferred embodiment of
the invention, effective to provide efficient formation of lipid
particles on large scale (for example 20-200 liters), a therapeutic
agent solution comprising nucleic acids (for example antisense
oligodeoxynucleotides) is combined with preformed lipid vesicles in
a 25-40% solution of aqueous ethanol. Incubation of this mixture of
a period of about 1 hour is sufficient to result in the spontaneous
production of fully encapsulated therapeutic agent particles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a possible model of the mechanistic steps of
the method of the invention;
[0013] FIG. 2 depicts encapsulation efficiencies as a function of
ethanol concentration for liposomes containing 10 mol %
PEG-Cer;
[0014] FIG. 3A depicts the release of calcein entrapped at
self-quenching concentrations in DSPC/Chol/PEG-CerC.sub.14/DODAP
liposomes as a function of ethanol concentration (closed circles)
together with the encapsulation efficiencies obtained using
liposomes of the same lipid composition (open circles);
[0015] FIG. 3B illustrates rapid exchange of lipids during the
formation of lipid entrapped nucleic acids using the method of the
invention;
[0016] FIG. 4 shows entrapment efficiencies and calcein leakage
data plotted as a function of temperature;
[0017] FIGS. 5A, B and C show NMR spectra of lipid-associated
oligonucleotides;
[0018] FIG. 6 shows a graph of entrapment efficiency plotted as a
function of the initial oligonucleotide-to-lipid ratio; and
[0019] FIG. 7 shows encapsulation efficiency for several species of
antisense oligodeoxynucleotides and for plasmid DNA (pDNA).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0020] While the terms used in the application are intended to be
interpreted with the ordinary meaning as understood by persons
skilled in the art, some terms are expressly defined to avoid any
ambiguity. Thus, as used in the specification and claims of this
application the term:
[0021] charged lipid refers to a lipid species having either a
cationic charge or negative charge or which is a zwitterion which
is not net neutrally charged, and generally requires reference to
the pH of the solution in which the lipid is found.
[0022] destabilization refers to modification of the properties of
a lipid membrane as a result of interaction with a solvent. When
the membrane is destabilized, the fundamental morphology of the
original lipid membrane is preserved. However, the leakage rate of
low molecular weight solutes increases and lipids can "flip-flop"
across the membrane and exchange rapidly with other lipid
particles. Destabilization of a lipid membrane is observed in the
invention, for example, at ethanol concentrations of 25-40%.
Solvents which achieve destabilization but not disruption of lipid
vesicles are referred to herein as destabilizing solvents.
[0023] disruption refers to modification of the properties of a
lipid membrane such that the fundamental morphology of the original
membrane is lost. Disruption of a lipid membrane is observed, for
example, at ethanol concentrations of >60%.
[0024] fully encapsulated refers to lipid particles in which the
therapeutic agent is contained in the lumen of a lipid vesicle such
as a liposome, or embedded within a bilayer of a lipid particle
such that no part of the therapeutic agent is directly accessible
to the external medium surrounding the lipid particle. Lipid
particles in which the therapeutic agent is fully encapsulated are
distinct from particles in which a therapeutic agent is complexed
(for example by ionic interaction) with the exterior of the
particle, or from particles in which the therapeutic agent is
partially embedded in the lipid and partially exposed to the
exterior medium. The degree of encapsulation can be determined
using methods which degrade available therapeutic agent. In the
case of a polynucleotide, these methods include S1 Nuclease
Digestion, Serum Nuclease, and Micrococcal Nuclease analysis.
Alternatively, an OliGreen.TM. assay can be employed. In a
quantitative sense, a "fully encapsulated" therapeutic agent is one
where less than 10% of the therapeutic agent, and preferably less
than 5% of the therapeutic agent in a lipid particle is degraded
under conditions where greater than 90% of therapeutic agent is
degraded in the free form. It should further be noted that
additional therapeutic agent(s) may be associated with the lipid
particle by complexation or another manner which is not fully
encapsulated with out departing from the present invention.
[0025] hydration refers to a common process by which lipid
particles, including liposomes, are formed. In this process, the
amount of water in the solvent surrounding the lipids is increased
from a concentration of around 5% or less (at which concentration
the lipid molecules are generally individually solvated) to a
concentration of 40-60% or greater (at which lipids spontaneously
form into membranes, micelles or particles).
[0026] lipid refers to a group of organic compounds that are esters
of fatty acids and are characterized by being insoluble in water
but soluble in many organic solvents. They are usually divided in
at least three classes: (1) "simple lipids" which include fats and
oils as well as waxes; (2) "compound lipids" which include
phospholipids and glycolipids; and (3) "derived lipids" such as
steroids. A wide variety of lipids may be used with the invention,
some of which are described below.
[0027] preformed vesicle refers to the starting lipid composition
used in the method of the invention which contains lipid vesicles.
These vesicles have a self-closed structure of generally spherical
or oval shape formed from one or more lipid layers and having an
interior lumen containing a part of the solvent. The vesicles may
be unilamellar, oligolamellar or multilamellar structures.
[0028] The invention disclosed herein relates to a novel method for
making lipid-encapsulated therapeutic agent particles which is
particularly applicable to the large-scale manufacture of such
particles when the lipid and therapeutic agent are oppositely
charged, such as found in formulations of cationic lipid and
anionic polynucleotides. This invention relies upon the surprising
and unexpected observation that combining preformed lipid vesicles
with a solution of therapeutic agent can result spontaneously in
the formation of particles of fully lipid-encapsulated therapeutic
agent of a therapeutically useful size. Thus, fully
lipid-encapsulated therapeutic agent particles are formed in
accordance with the invention by a method comprising the step of
combining a lipid component comprising preformed lipid vesicles and
a solution of the therapeutic agent and incubating the resulting
mixture for a period of time to result in the encapsulation of the
therapeutic agent in the lipid vesicles. The lipid component
further comprises a solvent system which is effective to
destabilize the membrane of the lipid vesicles without disrupting
the vesicles.
[0029] The method of the invention has several important
characteristics which make it of substantial utility to the art.
First, it is a large-scale method which can be used to make
substantial quantities (e.g. >100 g) of the encapsulated
therapeutic agent in a single batch. Second, the size of the
preformed lipid vesicles is substantially maintained, such that
processing of the lipid particles after introduction of the
therapeutic agent to obtain particles of therapeutically useful
size is not necessary. Third, the efficiency of encapsulation is
high. Fourth, the amount of therapeutic agent loaded into the
particles is high.
[0030] The lipid particles used in the present invention are formed
from a combination of several types of lipids, including at least
(1) a charged lipid, having a net charge which is opposite to the
charge of the therapeutic agent; and (2) a modified lipid including
a modification such as a polyethylene glycol substituent effective
to limit aggregation. In addition, the formulation may contain a
neutral lipid or sterol. In formulating the lipid particles using
all of the above-mentioned components, the following amounts of
each lipid components are suitably used: 10 to 40 mol % charged
lipid; 25 to 45 mol % neutral lipid, 35-55 mol % sterol; and 0.5 to
15 mol % modified lipid. Specific lipid components may be selected
from among the following non-limiting examples.
[0031] Charged Lipids
[0032] A wide variety of charged lipids and oppositely charged
therapeutic agents may be used with the invention. Examples of such
compounds are available and known to persons skilled in the art.
The following lists are intended to provide illustrative,
non-limiting examples.
[0033] Cationic charged lipids at physiological pH include, but are
not limited to, N,N-dioleyl-N,N-dimethylammonium chloride
("DODAC"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride ("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide
("DDAB"); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride ("DOTAP");
3.beta.-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol") and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids are available which can be used in
the present invention. These include, for example, Lipofectin.TM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE"), from GIBCO/BRL,
Grand Island, N.Y., USA); Lipofectamine.TM. (commercially available
cationic liposomes comprising
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoroacetate ("DOSPA") and DOPE from GIBCO/BRL); and
Transfectam.TM. (commercially available cationic lipids comprising
dioctadecylamidoglycyl carboxyspermine ("DOGS") in ethanol from
Promega Corp., Madison, Wis., USA).
[0034] Some cationic charged lipids are titrateable, that is to say
they have a pKa at or near physiological pH, with the significant
consequence for this invention that they are strongly cationic in
mild acid conditions and weakly (or not) cationic at physiological
pH. Such cationic charged lipids include, but are not limited to,
N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride ("DODMA")
and 1,2-Dioleoyl-3-dimethylammonium-propane ("DODAP").
[0035] Anionic charged lipids at physiological pH include, but are
not limited to, phosphatidyl inositol, phosphatidyl serine,
phosphatidyl glycerol, phosphatidic acid, diphosphatidyl glycerol,
poly(ethylene glycol)-phosphatidyl ethanolamine,
dimyristoylphosphatidyl glycerol, dioleoylphosphatidyl glycerol,
dilauryloylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol,
distearyloylphosphatidyl glycerol, dimyristoyl phosphatic acid,
dipalmitoyl phosphatic acid, dimyristoyl phosphatidyl serine,
dipalmitoyl phosphatidyl serine, brain phosphatidyl serine, and the
like.
[0036] Some anionic charged lipids may be titrateable, that is to
say they would have a pKa at or near physiological pH, with the
significant consequence for this invention that they are strongly
anionic in mild base conditions and weakly (or not) anionic at
physiological pH. Such anionic charged lipids can be identified by
one skilled in the art based on the principles disclosed
herein.
[0037] Neutral Lipids and Sterols
[0038] The term "neutral lipid" refers to any of a number of lipid
species which exist either in an uncharged or neutral zwitterionic
form a physiological pH. Such lipids include, for example,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and
diacylglycerols.
[0039] Modified Lipids
[0040] Certain preferred formulations used in the invention include
aggregation preventing lipids such as PEG-lipids or polyamide
oligomer-lipids (such as an ATTA-lipid), and other steric-barrier
or "stealth"-lipids. Such lipids are described in U.S. Pat. No.
4,320,121 to Sears, U.S. Pat. No. 5,820,873 to Choi et al., U.S.
Pat. No. 5,885,613 to Holland et al., WO 98/51278 (inventors Semple
et al.), and U.S. patent application Ser. No. 09/218,988 relating
to polyamide oligomers, all incorporated herein by reference. These
lipids prevent precipitation and aggregation of formulations
containing oppositely charged lipids and therapeutic agents. These
lipids may also be employed to improve circulation lifetime in vivo
(see Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or
they may be selected to rapidly exchange out of the formulation in
vivo (see U.S. Pat. No. 5,885,613). Particularly useful
exchangeable lipids are PEG ceramides having shorter acyl chains
(i.e, C14 or C18, referred to herein as PEG-CerC14 and PEG-CerC18)
or PEG-PE having a C14 acyl chain.
[0041] Some lipid particle formulations may employ targeting
moieties designed to encourage localization of liposomes at certain
target cells or target tissues. Targeting moieties may be linked to
the outer bilayer of the lipid particle during formulation or
post-formulation. These methods are well known in the art. In
addition, some lipid particle formulations may employ fusogenic
polymers such as PEAA, hemagluttinin, other lipopeptides (see U.S.
patent applications Ser. Nos. 08/835,281, and 60/083,294, all
incorporated herein by reference) and other features useful for in
vivo and/or intracellular delivery.
[0042] The preformed lipid vesicles may be prepared in a solution
of ethanol or other organic solvent using a simple lipid hydration
step. The percentage of ethanol or other organic solvent must be
selected such that the lipid particles do not disassemble or
redissolve into the solvent (generally at >60% ethanol) but
provide conditions which permit the spontaneous encapsulation
process of the invention (approx. 5%-50% ethanol, more preferably
25-40% ethanol). Alternatively, additional components such as
detergents may be included in the lipid vesicle solution which
contribute to the destabilization of the membrane. For purpose of
this specification, "organic solvent" means either a completely
organic solvent (i.e. 100% ethanol) or a partially organic solvent
(such as ethanol in water, ie. 20% ethanol, 40% ethanol, etc.). A
wide variety of water miscible organic solvents may be used
including ethanol or other alcohols, acetonitrile,
dimethylformamide, DMSO, acetone, other ketones, and the like.
Solvents with greater or lesser polarity may be useful in some
cases. Detergent solutions include .beta.-D-glucopyranoside, Tween
20 and those set out in WO 96/40964 and U.S. patent application
Ser. No. 09/169,573, both incorporated herein by reference, and any
other detergent or steric barrier compound that can provide the
same solubility features, and/or can prevent particle aggregation
during mixing of oppositely charged lipid and therapeutic agent.
Preferably all organic solvents or detergent solutions are
pharmaceutically acceptable in trace amounts, or greater, in order
that the formulation process does not preclude patient
administration.
[0043] Anionic therapeutic agents include any therapeutic agent
with a net negative charge, or having a negatively charged group
that is able to interact with a cationic lipid without being
blocked by other cationic charge groups of the therapeutic agent.
Such therapeutic agents include any known or potential therapeutic
agent, including all drugs and compounds such as, but not limited
to, oligonucleotides, nucleic acids, modified nucleic acids
(including protein-nucleic acids and the like), proteins and
peptides with negative charge groups, conventional drugs such as
plant alkaloids and analogues having negative charge groups, and
the like. Therapeutic agents which are not inherently anionic may
be derivatized with anionic groups to facilitate their use in the
invention. For example, paclitaxel can be derivatized with a
polyglutamic acid group linked to the 2' carbon.
[0044] Cationic therapeutic agents include any therapeutic agent
with a net positive charge, or having a positively charged group
that is able to interact with a negative lipid without being
blocked by other negative charge groups of the therapeutic agent.
Such therapeutic agents include any known or potential therapeutic
agent, including all drugs and compounds such as, but not limited
to modified nucleic acids linked to cationic charges, proteins and
peptides with positive charge groups, conventional drugs such as
plant alkaloids and analogues having positive charge groups, and
the like. Therapeutic agents which are not inherently cationic may
be derivatized with cationic groups to facilitate their use in the
invention.
[0045] Typically, charged therapeutic agents are initially provided
in buffered aqueous solution, generally containing some amount of
ethanol or other organic solvent. Salt concentration can strongly
effect the self assembly process (see U.S. patent application Ser.
No. 09/169,573 incorporated herein by reference) employed in the
invention, so the buffered salts employed need to be carefully
selected. Further, all buffers must be pharmaceutically acceptable,
as traces may remain in the final formulation. A suitable buffer is
300 mM citrate buffer for phosphorothioate oligodeoxynucleotides.
For phosphodiester-based oligodeoxynucleotides and plasmid DNA
which have lower binding affinities, a buffer of lower ionic
strength is appropriate. For example, typical citarte
concentrations are between 25 and 150 mM, with maximum entrapment
occurring at around 50 mM. The amount of ethanol or other organic
solvent which may be included is controlled by the solubility of
the therapeutic agent in the aqueous organic mixture, and also by
the desired characteristics of the final mixture of therapeutic
agent and preformed lipid vesicles.
[0046] The selection of lipids, destabilizing solvent and
therapeutic agents are made to work in concert to provide fully
lipid-encapsulated compositions. Thus, if the therapeutic agent is
a polyanionic oligonucleotide, the lipid components should be
selected to include lipids which are cationic under conditions in
the stabilizing solvent. Conversely, if the therapeutic agent is
cationic, the lipids components should be selected to include
lipids which are anionic under the conditions in the destabilizing
solvent. This does not mean that all of the lipids included in the
lipid solution must be charged, nor does it exclude the
incorporation of some quantity of like-charged lipids or of
zwiterrionic lipids. It merely means that the lipid solution should
include lipids which have a net charge which is opposite to the net
charge of the therapeutic agent.
[0047] The method of the invention employs relatively dilute
solutions of lipid particles and therapeutic agent. In general, the
therapeutic agent solution will have a concentration of 1 to 1000
mg/ml, preferably 10-50 mg/ml of the therapeutic agent, to yield a
final concentration (after mixing with the preformed lipid
vesicles) in the range of 0.2-10 mg/ml, preferably about 1-2 mg/ml.
Preformed lipid vesicles are combined with the therapeutic agent
solution such that the resulting lipid concentration (after mixing
with therapeutic agent solution) is about 1.5-30 mg/ml (about 2-40
mM), preferably 10 mg/ml. A preferred composition for preformed
vesicles for use with polynucleotide therapeutic agent is made at
the standard lipid ratios (PEG-cerC.sub.14:DODAP:DSPC:Chol (molar
ratios 5:25:25:45). This solution, in 100% ethanol, is diluted to
5-50% ethanol, preferably 40% ethanol by mixing with aqueous
buffer, for example 300 mM citrate, pH 4.0.
[0048] Encapsulation results upon stirring the lipid solution and
the oligonucleotide solution together until well mixed, and then
incubating with no mixing or gentle mixing for a period of from
about 1 to 2 hours. The resulting solution is then dialyzed to
remove ethanol or other material which destabilizes the lipid
particle membrane. pH adjustments may be used to neutralize surface
charges (in the case that the charged lipid is titratable) in order
to release therapeutic agent which may be complexed with the
exterior of the particle.
[0049] At the end of the incubation, the method of the invention
results in spontaneously-formed fully-encapsulated therapeutic
agents particles having a size which is acceptable for therapeutic
use and which can be predicted based on the starting side of the
preformed lipid vesicles. Thus, in general, a sizing step of the
type known in the art is not necessary after the addition of the
therapeutic agent. This is advantageous because there is no
requirement for application of mechanical stress to the lipid
vesicles after incorporation of the therapeutic agent, and thus no
risk of loss of or damage to the therapeutic agent. Should further
sizing of the product particles be desired, however, an optional
step for sizing of the resulting lipid particles may be employed.
Further, a sizing step may be employed as part of the preparation
of the preformed vesicles prior to the introduction of the
therapeutic agent in order to obtain starting vesicles of the
desired size.
[0050] There are several methods for the sizing of lipid particles,
and any of these methods may generally be employed when sizing is
used as part of the invention. The extrusion method is a preferred
method of liposome sizing. see Hope, M J et al. Reduction of
Liposome Size and Preparation of Unilamellar Vesicles by Extrusion
Techniques. In: Liposome Technology (G. Gregoriadis, Ed.) Vol. 1. p
123 (1993). The method consists of extruding liposomes through a
small-pore polycarbonate membrane or an asymmetric ceramic membrane
to reduce liposome sizes to a relatively well-defined size
distribution. Typically, the suspension is cycled through the
membrane one or more times until the desired liposome size
distribution is achieved. The liposomes may be extruded through
successively smaller pore membranes to achieve gradual reduction in
liposome size.
[0051] A variety of alternative methods known in the art are
available for reducing the size of a population of liposomes
("sizing liposomes"). One sizing method is described in U.S. Pat.
No. 4,737,323, incorporated herein by reference. Sonicating a
liposome suspension either by bath or probe sonication produces a
progressive size reduction down to small unilamellar vesicles less
than about 0.05 microns in diameter. Homogenization is another
method; it relies on shearing energy to fragment large liposomes
into smaller ones. In a typical homogenization procedure,
multilamellar vesicles are recirculated through a standard emulsion
homogenizer until selected liposome sizes, typically between about
0.1 and 0.5 microns, are observed. The size of the liposomal
vesicles may be determined by quasi-electric light scattering
(QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng.,
10:421-450 (1981), incorporated herein by reference. Average
liposome diameter may be reduced by sonication of formed liposomes.
Intermittent sonication cycles may be alternated with QELS
assessment to guide efficient liposome synthesis.
[0052] Preferred sizes for liposomes made by the various liposome
sizing methods will depend to some extent on the application for
which the liposome is being made, but will in general fall within
the range of 25 to 250 nm. Specific examples of suitable sizes are
set out in the Examples below.
[0053] In studying the lipid particles made in accordance with the
invention, it was surprisingly found that large empty unilamellar
vesicles (LUV), were converted into multilamellar vesicles with
entrapped therapeutic agent. While not intending to be bound by any
particular mechanism, it is believed that the process which is
occurring is as shown in FIG. 1, where a cationic charged lipid and
an anionic therapeutic agent are assumed. The process starts with a
unilamellar vesicle 10 which as a result of the inclusion of
cationic lipids has positive surface charges on the inside and
outside surfaces of the bilayer wall. Addition of anionic
therapeutic agent, such as antisense oligodeoxynucleotides 11
results in the formation of an intermediate complex 12 in which the
therapeutic agent molecules 11 are bound by an ionic/electrostatic
mechanism to the oppositely charged lipids on the surface of the
LUV.
[0054] The next step in the process appears to be an aggregation
step, in which aggregates 13 of the LUV/therapeutic agent complexes
are formed. This aggregation step is very complex and is apparently
dependent on the amount of destabilizing agent (for example
ethanol) and the amount of modified lipid in the preformed
vesicles, as well as being mediated by the charged therapeutic
agent. Some limited knowledge is provided in the art about these
processes, but they neither predict nor explain the phenomenon
which is the basis of the present invention. It is known that
cationicliposome/DNA complexes exhibit a large variety of different
structures including clusters of aggregated liposomes with flat
double-bilayer diaphragms in the areas of contact, liposomes coated
with DNA and (aggregated) multilamellar structures, where DNA is
sandwiched between lipid bilayers (Gustafsson et al., 1995; Lasic,
1997; Lasic et al., 1997; Huebner et al., 1999; Xu et al., 1999).
The latter structures can be flat stacks of bilayers or liposomes,
which frequently exhibit open bilayer segments on their outer
surface. Similar structures have been observed following binding of
Ca.sup.2+ to negatively charged liposomes (Papahadjopoulos, 1975;
Miller and Dahl, 1982; Rand et al., 1985; Kachar et al., 1986). The
structural transformations occurring in these systems were
attributed to adhesion-mediated processes such as bilayer rupture
and fusion (Rand et al., 1985; Kachar et al., 1986; Huebner et al.,
1999). First, liposomes aggregate crosslinked by DNA or Ca.sup.2+.
Rapid spreading of the contact area deforms the liposomes as they
flatten against each other. This places the bilayer under increased
tension. If the tension (adhesion energy) is high enough, the
stress imposed on the lipid membrane can be relieved either by
fusion (increase in area/volume ratio) and/or rupture (volume
loss). Most bilayers rupture when the area is increased by about 3%
(Evans and Parsegian, 1983). Upon bilayer rupture, vesicles
collapse flattening against each other to form multilamellar
stacks. Membrane-destabilizing agents such as ethanol can modulate
the structural rearrangements occurring upon interaction of
cationic liposomes with DNA or oligonucleotides.
[0055] In the method of the present invention, the formation of
multilamellar liposomes from unilamellar vesicles in the presence
of ethanol also points to an adhesion-mediated process for their
formation. However, the process differs in some way from the
complexes with their terminated membranes, since the product in
this case is concentric bilayer shells. While ethanol or a
comparable destabilizing agent is required for the latter
structures to form it is not clear how it affects these structural
rearrangements. These rearrangements correlate with the loss of the
membrane permeability barrier fr smaller moeclues and rapid lipid
exchange, as well as lipid flip-flop (which correlates with alcohol
concentration). In addition, the exchange out of the modified lipid
from the LUV may be a significant factor in to reorganization of
the lipid vesicles. In any event, by some mechanism, the aggregates
13 rearrange to form multilamellar vesicles 14 with the therapeutic
agent entrapped between the lamellae and on the inside of the
vesicle. This rearrangement is dependent not only on the nature of
the aggregates formed, but also on the temperature at which the
aggregates are incubated. Some of the therapeutic agent may also
remain associated with charges on the exterior of the multilamellar
vesicle, and, these may be removed by charge neutralization (for
example with acid or base in the case of a titratable charged
lipid), or by ion exchange.
[0056] Several characteristics of the lipid vesicles and the
destabilizing solvent were found experimentally to be of importance
to the characteristics of the final products, and the selection of
these characteristics can be used to control the characteristics of
the product multilamellar vesicles. These characteristics
include:
[0057] (1) the inclusion of a charged lipid in the preformed lipid
vesicles with a charge opposite that of the therapeutic agent;
[0058] (2) the inclusion of a modified lipid in an amount
sufficient to retard aggregation, but not enough to prevent
aggregation. In the case of PEG-CerC.sub.14, this amount was found
to be on the order of 2.5 to 10%;
[0059] (3) the inclusion in the destabilizing solvent of a
destabilizing agent (such as ethanol or detergent) in an amount
that destabilizes but does not disrupt the preformed lipid
vesicles; and
[0060] (4) performing the assembly of the fully lipid-encapsulated
therapeutic agent particles at a temperature where the aggregation
and the entrapment step are not decoupled. In general this will
require operation in a temperature range of room temperature
(.about.20.degree. C.) or above, depending on the concentration of
destabilizing agent and the lipid composition.
[0061] The method of the invention can be practiced using
conventional mixing apparatus. For large scale manufacture,
however, it may be desirable to use a specifically adapted
apparatus which is described in a concurrently filed PCT
application, entitled "Methods and Apparatus for Preparation of
Lipid Vesicles", Serial No. Not yet assigned, filed 14 Jul. 2000,
(Attorney Docket No. 80472-6) which is incorporated herein by
reference.
[0062] The method of the invention will now be further described
with reference to the following, non-limiting examples.
EXAMPLES
[0063] Materials Used in the Following Examples are Supplied as
Follows:
[0064] The phosphorothioate antisense oligodeoxynucleotides and
plasmid DNA used in this study were provided by Inex
Pharmaceuticals (Burnaby, BC, Canada). The mRNA targets and
sequences of the oligonucleotides are as follows: [0065] human
c-myc, 5'-TAACGTTGAGGGGCAT-3' (Seq ID No. 1); [0066] human
ICAM-1,5'-GCCCAAGCTGGCATCCGTCA-3' (SEQ ID No. 2); and [0067]
FITC-labeled human EGFR, 5'-CCGTGGTCATGCTCC-3' (SEQ ID No. 3).
[0068] 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was
purchased from Northern Lipids (Vancouver, BC, Canada) and
1,2-dioleoyl-3-dimethylammoniumpropane (DODAP),
1,2-dioleoyl-sn-glycero-3-phosphoserine-N-(7-nitro-2-1,3-benzoxadiazol-4--
yl) (NBD-PS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine b sulfonyl) (LRh-PE) as well as
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadia-
zol-4-yl) (NBD-PE) from Avanti Polar Lipids (Alabaster, Ala.).
1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine
(Py-HPC) and the oligonucleotide-binding dye OliGreen were obtained
from Molecular Probes (Eugene, Oreg.).
1-O-(2'-(.omega.-methoxypolyethylene-glycol)succinoyl)-2-N-myristoylsphin-
gosine (PEG-CerC.sub.14), radioactively labeled
[.sup.3H]-PEG-CerC.sub.14 as well as
1-O-(2'-(.omega.-methoxypolyethylene-glycol)succinoyl)-2-N-dodecanoylsphi-
ngosine (PEG-CerC.sub.20) were provided by INEX Pharmaceuticals
(Burnaby, BC, Canada). Cholesterol (chol), n-octyl
.beta.-D-glucopyranoside (OGP), Triton X-100, calcein,
dichlorodimethylsilane, sodium hydrosulfite (dithionite),
2-p-toluidinylnaphthalene-6-sulfonate (TNS) and
polyanetholesulfonic acid (PASA) were obtained from Sigma
(Oakville, ON, Canada). All materials for transmission electron
microscopy including osmium tetroxide, lead citrate, maleic acid,
sodium cacodylate and the embedding resin Embed 812 were purchased
from Electron Microscopy Sciences (Fort Washington, Pa.) and low
melting point (L.M.P.) agarose from Life Technologies (Burlington,
Ontario). Cholesterol (CHOL) was purchased from Sigma Chemical
Company (St. Louis, Mo., USA). PEG-ceramides were synthesized by
Dr. Zhao Wang at Inex Pharmaceuticals Corp. using procedures
described in PCT WO 96/40964, incorporated herein by reference.
[.sup.3H] or [.sup.14C]-CHE was purchased from NEN (Boston, Mass.,
USA). All lipids were >99% pure. Ethanol (95%), methanol,
chloroform, citric acid, HEPES and NaCl were all purchased from
commercial suppliers.
[0069] Analytical Methods: Assays employed to determine if a
lipid-therapeutic agent is "encapsulated" such as being "fully
encapsulated" are set out in WO 98/51278, and incorporated herein
by reference. Such methods include S1 Nuclease Digestion, Serum
Nuclease, and Micrococcal Nuclease analysis.
[0070] The Oligreen Assay was used to quantify the amount of
oligonucleotide loaded into the vesicles. A fluorescent dye binding
assay for quantifying single stranded oligonucleotide in aqueous
solutions was established using a Biolumin.TM. 960 fluorescent
plate reader (Molecular Dynamics, Sunnyvale, Calif., USA). Briefly,
aliquots of encapsulated oligonucleotide were diluted in HEPES
buffered saline (HBS; 20 mM HEPES, 145 mM NaCl, pH 7.5). A 10 .mu.L
aliquot of the diluted sample was added to 100 .mu.L of a 1:200
dilution of Oligreen.TM. reagent, both with and without 0.1% of
Triton X-100 detergent. An oligo standard curve was prepared with
and without 0.1% Triton X-100 for quantification of encapsulated
oligo. Fluorescence of the Oligreen.TM.-antisense complex was
measured using excitation and emission wavelengths of 485 nm and
520 nm, respectively. Surface associated antisense was determined
by comparing the fluorescence measurements in the absence and
presence of detergent.
[0071] Dynamic light scattering. Sizes were determined by dynamic
light scattering using a NICOMP 370 particle sizer (Nicomp Particle
Sizing Inc., Santa Barbara, Calif.). Throughout the application,
number-averaged sizes are presented, which were obtained by a
cumulant fit from the experimental correlation functions. The
polydispersity is expressed as the half-width at half-height of a
monomodal Gaussian size distribution. The viscosity of the
ethanol/citrate buffer was determined using an Ubelohde-type
viscometer (Cannon 50). The viscosity of ethanol/300 mM citrate
buffer (40/60 (v/v)) at 23.degree. C. measured relative to water at
the same temperature was found to be 2.674 mPa*s. The zeta
potential was determined by electrophoretic light scattering using
a Coulter light scattering instrument (DELSA, Coulter Electronics
Inc., FL).
[0072] Lipid flip-flop. Lipid flip-flop was determined by chemical
reduction of the fluorescent lipid, NBD-PS, to a nonfluorescent
compound with sodium dithionite (McIntyre and Sleight, 1991; Lentz
et al., 1997). Liposomes were prepared at 20 mM lipid by extrusion
in the presence of 1 mol % NBD-PS. Only NBD-PS located in the outer
monolayer is accessible to the reducing agent, dithionite, added to
the external medium. Its redistribution from the inner monolayer to
the outer can be followed after reduction of NBD-PS in the outer
membrane leaflet. A 1 M sodium dithionite solution was freshly
prepared in 1 M TRIS. NBD-PS in the outer monolayer was reduced by
addition of a 100-fold molar excess of sodium dithionite relative
to NBD and incubation for 10 min. The completion of the reaction
was checked by measuring the dithionite fluorescence at 520 nm
before and after reduction exciting at 465 nm. Excess dithionite
was subsequently removed by size exclusion chromatography on a
Sephadex G50 column. The liposomes were incubated in the presence
of 40% ethanol and aliquots corresponding to a final lipid
concentration of 150 .mu.M removed for measurement at different
time points.
[0073] Leakage experiments. Ethanol-induced permeabilization of
LUVs was measured at different temperatures and as a function of
the size (MW) of the entrapped solute. Calcein was used as a low
molecular weight marker for leakage and FITC-dextran (MW 19500) as
a high molecular weight marker. Leakage of calcein entrapped at
self-quenching concentrations was followed by monitoring the
dequenching of the calcein fluorescence. LUVs were prepared by
hydration of a lipid film with an aqueous solution containing 75 mM
calcein and 5 mM HEPES adjusted to pH 7.5 by addition of sodium
hydroxide, followed by 5 freeze/thaw cycles and extrusion through 2
stacked 100 nm filters (10 passes). In the case of
DSPC/chol/PEG-CerC.sub.14/DODAP extrusion was performed at
60.degree. C. Unentrapped calcein was exchanged against an
isoosmotic HBS buffer by anion exchange chromatography on a DEAE
Sepharose CL6B column. The liposome stock solution was diluted to a
lipid concentration of 3 .mu.M in HBS containing varying amounts of
ethanol pre-equilibrated at 25, 40 or 60.degree. C. The
fluorescence at 520 nm was measured (excitation wavelength 488 nm,
long-pass filter at 430 nm) with a Perkin Elmer LS50 Fluorimeter
(Perkin Elmer) after 5 min of incubation at the corresponding
temperature. The value for 100% leakage (maximum dequenching) was
obtained by addition of a 10% Triton X-100 solution to a final
concentration of 0.05%. Calcein leakage was calculated according to
% leakage=(F.sub.s-F.sub.b)/(F.sub.Tx-F.sub.b)*100, where F.sub.s
is the fluorescence of the sample, F.sub.b the background
corresponding to calcein containing liposomes in the absence of
ethanol and F.sub.Tx the Triton X-100 value.
[0074] FITC-dextran (MW 19500) was entrapped in
DSPC/Chol/PEG-CerC.sub.14/DODAP liposomes incorporating 0.5 mol %
LRh-PE at a final concentration of 45 mg/ml. Entrapment was
performed by addition of the lipids dissolved in ethanol to the
FITC-dextran solution in HBS followed by extrusion (2 stacked 100
nm filters, 2 passes) and subsequent removal of ethanol by
dialysis. Unentrapped FITC-dextran was removed by size exclusion
chromatography on a Sepharose CL4B column (1.5.times.15 cm). The
loss of FITC-dextran from liposomes exposed to 40% ethanol was
determined after removal of released FITC-dextran by size exclusion
chromatography on a Sepharose CL4B column (1.5.times.15 cm). The
FITC/LRh-PE ratio was measured before and after addition of
ethanol. FITC and LRh-PE fluorescence were measured at 515 nm and
590 nm with the excitation wavelength set to 485 nm and 560 nm,
respectively.
[0075] Lipid mixing. Ethanol-induced lipid mixing/exchange was
followed by the loss of resonance energy transfer, occuring between
a donor, NBD-PE, and an acceptor, LRh-PE, which are in close
proximity, upon dilution of the probes into an unlabeled target
membrane (Struck et al., 1981). LUVs contained 0.75 mol % of both
NBD-PE and LRh-PE. Labeled and unlabeled liposomes were prepared in
HBS pH 7.5 by extrusion at lipid concentrations of 20 mM. Ethanol
was added to labeled and unlabeled liposomes to a final
concentration of 40% (v/v). Subsequently, the ethanolic dispersions
of labeled and unlabeled liposomes were mixed at a molar lipid
ratio of 1:5 and incubated at the appropriate temperatures.
Aliquots were withdrawn at given time-points and added to 2 ml of
HBS to give a final lipid concentration of 150 .mu.M. Emission
spectra of NBD and LRh were measured in the region from 505 to 650
nm with the excitation wavelength set to 465 nm (430 nm emission
long-pass filter). After background subtraction (unlabeled
liposomes at 150 .mu.M lipid) the loss of resonance energy transfer
was expressed as the increase in NBD/LRh ratio.
[0076] Pyrene-HPC assay. Pyrene-HPC forms excited state dimers at
high concentrations, which fluoresce at a different wavelength than
the monomers. Excimer formation is a diffusion-controlled process
and requires two molecules to come together to form a dimer. Lipid
mixing (target membrane) as well as a decrease in the lateral
mobility of pyrene-HPC in the membrane can result in a decrease in
pyrene excimer fluorescence (Hoekstra, 1990; Duportail and Lianos,
1996). Lateral phase separation usually results in an increase in
pyrene excimer fluorescence (Duportail and Lianos, 1996). The
rationale of this experiment was to look at the effect of
oligonucleotide binding on the liposomal membrane. The pyrene-HPC
fluorescence of liposomes entrapping oligonucleotide was compared
to empty control liposomes before and after depletion of the
transmembrane pH gradient. Increasing the internal pH to 7.5
results in the release of membrane-bound oligonucleotides.
Liposomes incorporating pyrene-HPC at a concentration of 7 mol %
were prepared by addition of lipids dissolved in ethanol to pH 4
citrate buffer. An aliquot was removed and oligonucleotide
entrapped as described above. The remaining initial liposomes were
treated the same way in all the subsequent steps (see under
entrapment). The pH gradient was dissipated with ammonium acetate
adjusted to pH 7.5. Liposomes were diluted into the appropriate
buffer, HBS pH 7.5 or 150 mM ammonium acetate pH 7.5, to a final
lipid concentration of 2 .mu.M. Pyrene-HPC emission spectra were
recorded in the wavelength region from 365-550 nm with excitation
at 345 nm and an emission cut-off filter at 350 nm. The intensity
ratio of monomer fluorescence at 397 nm to dimer fluorescence at
478 nm was plotted for the initial liposomes as well as for the
oligonucleotide containing liposomes before and after depletion of
the pH gradient.
[0077] .sup.31P NMR Spectroscopy. .sup.31P NMR spectra were
obtained with a Bruker MSL200 spectrometer operating at 81 MHz.
Free induction decays (FIDs) corresponding to 800 or 2400 scans
were collected by using a 2.8 .mu.s 50.degree. pulse with a 3 sec
interpulse delay and a spectral width of 20000 Hz on a 2.0 ml
sample in a 10 mm probe. No proton decoupling was employed. An
exponential multiplication corresponding to 25 Hz of line
broadening was applied to the FIDs prior to Fourier transformation.
The chemical shift was referenced to external 85% phoshoric acid
(H.sub.3PO.sub.4). The spin-lattice relaxation times (T.sub.1) of
free and encapsulated oligonucleotides at pH 7.5 are essentially
the same with T.sub.1.sup.free=1.7 sec and T.sub.1.sup.enc.=2.1
sec. The T.sub.1-values were measured by an inversion-recovery
pulse sequence. The interpulse delay of 3 sec for 50.degree. pulses
allows for complete relaxation of all antisense resonances.
[0078] Ultracentrifugation. Liposomes with and without entrapped
oligonucleotides were fractionated by ultracentrifugation on a
sucrose step gradient consisting of 1%, 2.5%, 10% and 15% (w/v)
sucrose in HBS pH 7.5 with a step volume of 3.5, 3.5, 2.5 and 1.5
ml, respectively. Samples were centrifuged for 2 hrs at 36000 rpm
(RCF.sub.Rmax 221000.times.g) using a Beckmann L8-70
ultracentrifuge in combination with a SW41Ti rotor. The gradient
was either fractionated from the top or individual bands were
removed with a syringe after puncturing the tube with a needle.
[0079] Cryo-Transmission Electron Microscopy (cryo-TEM). A drop of
sample was applied to a standard electron microscopy grid with a
perforated carbon film. Excess liquid was removed by blotting with
filter paper leaving a thin layer of water covering the holes of
the carbon film. The grid was rapidly frozen in liquid ethane,
resulting in vesicles embedded in a thin film of amorphous ice.
Images of the vesicles in ice were obtained under cryogenic
conditions at a magnification of 66000 and a defocus of -1.5 micron
using a Gatan cryo-holder in a Philips CM200 FEG electron
microscope.
[0080] Freeze-Fracture Electron Microscopy. Samples were cryofixed
in the presence of 25% glycerol by plunging them into liquid Freon
22 cooled by liquid N.sub.2. The fractured surface was shadowed
unidirectionally with platinum (45.degree.) and coated with carbon
(90.degree.) employing a Balzers Freeze-Etching system BAF 400D
(Balzers, Liechtenstein). Replicas were analyzed using a JEOL Model
JEM 1200 EX electron microscope (Soquelec, Montreal, QC,
Canada).
[0081] Transmission Electron Microscopy (TEM). Vesicles were fixed
by the addition of 1 volume of 2% osmium tetroxide to 0.5 volumes
of vesicles in HBS followed by centrifugation at 17000.times.g and
4.degree. C. for 45 min. The resulting pellet was mixed with an
equal volume of 3% agarose/PBS, pipetted onto a microscope slide
and allowed to cool to 4.degree. C. The solidified agarose
containing the vesicles was cut into 1 mm pieces and transferred to
a glass tube for further processing. The blocks were washed for
3.times.5 min with 0.05 M maleic acid pH 5.2 before staining in 2%
uranyl acetate for 1 h. The tissue pieces were dehydrated through a
graded series of alcohols (50-100%), infiltrated with increasing
ratios of epoxy resin (EMbed 812):propylene oxide and embedded in
100% EMbed 812 at 60.degree. C. for 24 h. Ultrathin sections were
stained with 2% lead citrate and examined using a Zeiss EM 10C
transmission electron microscope (Oberkochen, Germany)
[0082] Phase contrast and fluorescence microscopy. Phase contrast
and fluorescence microscopy were performed on a Zeiss Axiovert 100
microscope using a Plan Apochromat 63x/1.4NA oil immersion
objective in combination with a 1.6.times. optovar lens and a XF100
filter set from Omega Optical (Brattleboro, Vermont) with the
following optical specifications: excitation 475.+-.20/dichroic
500/emission 535.+-.22.5. Images were recorded on Kodak Ektachrome
P1600 color reversal film at 1600 ISO with a Zeiss MC80 DX
microscope camera. Slides and coverglasses were siliconized with
dichlorodimethylsilane to neutralize the otherwise negatively
charged glass surface.
Example 1
[0083] Empty preformed vesicles were prepared from a lipid mixture
containing PEG-CerC.sub.14, DODAP, DPSC and CHOL in a molar ratio
of 5:25:25:45. The four lipids were dissolved in a 100% ethanol to
a total lipid concentration of 25 mg/ml (33 mM). The ethanolic
lipid was then introduced through an injection port with an orifice
diameter of 0.25 mm into a reservoir containing 300 mM citrate
buffer, pH 4.0. The reservoir and all solutions were at room
temperature. The total volume of ethanolic lipid was 6 liters, and
the flow rate for lipid introduction was 200-300 ml/min. The total
volume of citrate buffer was 9 liters. The resulting 15 liter
mixture had an ethanol concentration of 40% and 180 mM citrate.
Vesicles of 170.+-.20 nm median diameter were generated. The empty
preformed vesicles were sized to 90-120 nm median diameter by 1-3
passes through the extrusion circuit (65.degree. C.) at low
pressure (100 p.s.i., reduced from classical 500-1000 p.s.i.) using
two stacked 80 nm membranes. The empty preformed vesicles were then
pooled in a reservoir 20 and maintained at 40.degree. C. until
addition of therapeutic agent solution.
Example 2
[0084] Preformed vesicles of example 1 were used to make fully
lipid-encapsulated therapeutic agent particles using
oligonucleotide INX-6295 (Seq. ID No. 1) as the therapeutic agent.
Oligonucleotide INX-6295 in distilled water was diluted by the
addition of 100% ethanol to form a various solutions of 10, 20, 30
40 or 50 mg/ml oligonucleotide in 40% ethanol. The ethanolic
oligonucleotide was added to the preformed vesicles in reservoir 20
at 40.degree. C. with gentle mixing. The amount and volume of
ethanolic oligonucleotide was calculated to provide a final
drug:lipid ratio of 0.1 to 0.25 by weight. The mixture was then
incubated at 40.degree. C. with gentle and periodic mixing for 1
hour. After incubation, the solution was processed by diafiltration
to strip free or excess associated oligonucleotide, remove ethanol
and exchange the buffer system to phosphate buffered saline (PBS),
pH 7.4. Concentration, sterile filtration and packaging complete
the preparation of a commercial product.
Example 3
[0085] The procedure of Example 2 was repeated with changes to
various parameters to determine which might be critical to the
preparation of fully lipid-encapsulated therapeutic agent particles
in accordance with the invention. In these experiments, the total
oligonucleotide recovery (yield), the total lipid recovery (yield)
and the encapsulation efficiency were considered as indications of
the quality of the product and the process. Total oligonucleotide
recovery was calculated using the formula: final .times. .times.
oligo .times. .times. concentration .times. .times. ( mg .times. /
.times. ml ) .times. final .times. .times. volume .times. .times. (
ml ) .times. initial .times. .times. oligo .times. .times.
concentration .times. .times. ( mg .times. / .times. ml ) .times.
initial .times. .times. volume .times. .times. ( ml ) .times. 100
.times. % ##EQU1## Total lipid recovery was calculated using the
formula: final .times. .times. lipid .times. .times. concentration
.times. .times. ( mg .times. / .times. ml ) .times. final .times.
.times. volume .times. .times. ( ml ) .times. initial .times.
.times. lipid .times. .times. concentration .times. .times. ( mg
.times. / .times. ml ) .times. initial .times. .times. volume
.times. .times. ( ml ) .times. 100 .times. % ##EQU2## Encapsulation
Efficiency (E.E.) was calculated using the formula: initial .times.
.times. oligo .times. .times. ( mg .times. / .times. ml ) / initial
.times. .times. lipid .times. .times. ( mg .times. / .times. ml )
final .times. .times. oligo .times. .times. ( mg .times. / .times.
ml ) .times. final .times. .times. lipid .times. .times. ( mg
.times. / .times. ml ) .times. 100 .times. % ##EQU3## The
percentage of oligo that is encapsulated (i.e., incorporated in
bilayers or entrapped in the interior of the lipid particle) was
determined with the OliGreen assay described above.
[0086] To assess the significance of the initial drug to lipid
ratio, the experiment was conducted with two different starting
ratios. The results are summarized in Table 1. No change in the
size of the vesicles was observed in the process of loading the
oligonucleotide. TABLE-US-00001 TABLE 1 Initial Drug/ Final Lipid
Oligo Lipid Encap Vesicle Drug/Lipid Ratio Yield % Yield % Oligo %
size (nm) Ratio E.E. % 0.1 80-90 70-80 .gtoreq.90 106 0.1 100 0.2
60-78 70-75 .gtoreq.80 119 0.17-0.2 85-100
[0087] To assess the significance of incubation temperature, the
experiment was conducted at room temperatures and at two elevated
temperatures for 1 hour. The results are summarized in Table 2. As
shown, the higher temperature of 60.degree. C. begins to impair the
efficiency of the process, and to lead to an increase in particle
size. Thus, lower temperatures are preferred. TABLE-US-00002 TABLE
2 Incubation Oligo Encaps Vesicle Temp (.degree. C.) Yield % Oligo
% Size (nm) RT (20-22) 73 90 114 40 84 91 109 60 52 83 140
[0088] To assess the significance of incubation time, the
experiment was conducted at three incubation times and an
incubation temperature of 40 .degree. C. The results are summarized
in Table 3. As shown, the yield improves between 0.5 hours and 1
hour, but increased incubation time beyond an hour does not result
in a substantial improvement. Thus, the most efficient process in
the apparatus used will employ an incubation time of about 1 hour.
TABLE-US-00003 TABLE 3 Incubation Oligo Encapsulated time (hr)
Yield % Oligo % 0.5 22 92 1 60 94 2 56 95
[0089] To assess the significance of buffer concentration in the
oligonucleotide solution, the experiment was conducted at four
different concentrations of citrate buffer and an initial
drug/lipid ratio of 0.1. The results are summarized in Table 4.
TABLE-US-00004 TABLE 4 Citrate Buffer Oligo Encapsulated Vesicle
Conc (nM) Yield % Oligo % Size (nm) 50 100 94 80 100 88 90 90 200
89 91 93 300 80-90 92 106
[0090] To assess the significance of the initial ethanol
concentration during the mixing step, the experiment was conducted
with 3 different initial ethanol concentrations at each of two
initial drug to lipid ratios. The results are summarized in Table
5. There appears to be an optimum ethanol concentration which is
different for each starting oligo/lipid ratio. In an addition
experiment not reported in the Table, an initial ethanol
concentration of 50% was used with an oligo/lipid ratio of 0.1.
Significant problems of unknown cause were encountered in this
experiment and no yield of product was obtained. TABLE-US-00005
TABLE 5 Initial Initial Drug/ Oligo Encaps Vesicle Final E.E. EtOH
% Lipid Yield % Oligo % size (nm) Drug/Lipid % 33 0.2 42-47 88 115
0.12 60 40 0.2 70 82 114 0.15 75 43 0.2 64 62 105 0.19 95 36 0.1
52-66 85-89 110 nd nd 43 0.1 90-100 84-89 116 nd nd 45 0.1 90-100
90-92 108 nd nd
[0091] To assess the significance of initial oligonucleotide
concentration (and thus of the volume of therapeutic agent solution
to obtain the same initial drug to lipid ratio), stock solutions at
four different concentrations of oligonucleotide were used. The
results are summarized in Table 6. As shown, this parameter does
not appear to be critical to the results obtained using the method
of the invention. TABLE-US-00006 TABLE 6 Oligo Stock Initial Oligo
Encaps Vesicle Size mg/ml Drug/Lipid Yield % Oligo % (nm) 10 0.1 85
90 106 20 0.1 80 88 112 30 0.1 87 90 110 40-50 0.1 80-90 88-94
106
Example 4
[0092] To demonstrate the applicability of the invention to larger
therapeutic agents, plasmid pINEX L1018, a 5.5 kb plasmid encoding
the luciferase gene linked to a CMV promoter, and also carrying
SV40 enhancer elements and an AmpL gene was loaded into preformed
lipid vesicles.
[0093] Preformed lipid vesicles were prepared by slowly adding 10
mg of lipids (DSPC/Chol/.DODAP/PEG-CerC14 in a 20/45/25/10 mol %
ratio) dissolved in 100% ethanol to 25 mM citrate buffer (25 mM
citric acid, 255 mM sucrose, adjusted to pH 4 with sodium
hydroxide). Both solutions were prewarmed to 40.degree. C. before
mixing. The final ethanol concentration was 40% (v/v). The
ethanolic dispersion of lipid vesicles was extruded 2.times.
through 2 stacked 100 nm polycarbonate filters at room temperature.
0.25 mg of plasmid DNA in 40% ethanol was added to the lipid
vesicles at room temperature, followed by a 1 hour incubation of
the sample at 40.degree. C. The initial plasmid/lipid ratio was
0.025. Subsequently, the sample was dialyzed against 2L of 25 mM
saline, pH 7.5 (20 mM HEPES, 150 mM NaCl) for a total of 18-20
hours.
[0094] Trapping efficiency was determined after removing remaining
external plasmid DNA by anion exchange chromatography on a DEAE
Sepharose CL6B column. Plasmid DNA was quantified using the DNA
Binding System PicoGreen lipid by inorganic phosphate assay
according to Fiske and Subbarrow after separation from the plasmid
by a Bligh Dyer extraction. In addition, the final lipid
concentration was determined by incorporating 0.25 mol % of the
fluorescently-labeled lipid Lissamine rhodamine-PE in the lipid
vesicles.
[0095] The final plasmid lipid ratio was 0.022, which corresponds
to 88% entrapment. The resulting lipid-encapsulated therapeutic
agent particles had an average size of 100 nm and a very small size
distribution.
Example 5
[0096] Liposome preparation: Large unilamellar liposomes in
ethanol/buffer solutions were either prepared by addition of
ethanol to extruded liposomes or by addition of lipids dissolved in
ethanol to an aqueous buffer solution and subsequent extrusion.
Both methods give the same entrapment results and will be described
in greater detail in the following:
[0097] 1. After hydration of a lipid film in pH 4 citrate buffer
and 5 freeze/thaw cycles LUVs were generated by extrusion through 2
stacked 100 nm filters (10 passes). In the case of
DODAP/DSPC/Chol/PEG-CerC14 liposomes the extrusion was performed at
60.degree. C. Ethanol was subsequently slowly added under rapid
mixing. Typical liposome sizes determined after removal of ethanol
by dynamic light scattering were 90.+-.20 nm for the
DODAP/DSPC/Chol/PEG-CerC.sub.14 system. Slow addition of ethanol
and rapid mixing are important as liposomes become unstable and
coalesce into large lipid structures as soon as the ethanol
concentration exceeds a certain upper limit. The latter depends on
the lipid composition. For example, an initially translucent
DSPC/Chol/PEG-CerC.sub.14/DODAP liposome dispersion becomes milky
white if the ethanol concentration exceeds 50% (v/v).
[0098] 2. LUVs were prepared by slow addition of the lipids
dissolved in ethanol (0.4 ml) to citrate buffer at pH 4 (0.6 ml)
followed by extrusion through 2 stacked 100 nm filters (2 passes)
at RT. Dynamic light scattering measurements performed in ethanol
and after removal of ethanol by dialysis show no significant
differences in size, which is typically 75.+-.18 nm. The extrusion
step can be omitted if ethanol is added very slowly under vigorous
mixing to avoid locally high ethanol concentrations.
[0099] Entrapment procedure. An oligonucleotide solution was slowly
added under vortexing to the ethanolic liposome dispersion, which
was subsequently incubated at the appropriate temperature for 1 hr,
dialyzed for 2 hrs against citrate buffer to remove most of the
ethanol and twice against HBS (20 mM HEPES/145 mM NaCl, pH 7.5). At
pH 7.5 DODAP becomes charge-neutral and oligonucleotides bound to
the external membrane surface are released from their association
with the cationic lipid. Unencapsulated oligonucleotides were
subsequently removed by anion exchange chromatography on
DEAE-sepharose CL-6B columns equilibrated in HBS pH 7.5. When
octylglucoside was used in place of ethanol the detergent was added
to liposomes (1:1 v/v) to final concentrations ranging from 30-40
mM. All the subsequent steps were performed as described above
except for the initial dialysis step against pH 4 citrate buffer,
which was extended to 5 hrs. In the following examples, if not
otherwise mentioned, DSPC/Chol/PEG-CerC.sub.14/DODAP liposomes
(20:45:10:25 mol %), c-myc (Seq. ID No. 1), 40% (v/v) ethanol, 300
mM citrate buffer and incubation at 40.degree. C. were used.
[0100] Determination of trapping efficiencies: Trapping
efficiencies were determined after removal of external
oligonucleotides by anion exchange chromatography. Oligonucleotide
concentrations were determined by UV-spectroscopy on a Shimadzu
UV160U spectrophotometer. The absorbance at 260 nm was measured
after solubilization of the samples in chloroform/methanol at a
volume ratio of 1:2.1:1 chloroform/methanol/aqueous phase
(sample/HBS). If the solution was not completely clear after mixing
an additional 50-100 .mu.l of methanol was added. Alternatively,
absorbance was read after solubilization of the samples in 100 mM
octylglucoside. The antisense concentrations were calculated
according to: c[.mu.g/.mu.l]=A.sub.260*1 OD.sub.260
unit[.mu.g/ml]*dilution factor[ml/.mu.l], where the dilution factor
is given by the total assay volume [ml] divided by the sample
volume[.mu.l]. OD.sub.260 units were calculated from pairwise
extinction coefficients for individual deoxynucleotides, which take
into account nearest neighbor interactions. 1OD corresponds to
30.97 .mu.g/ml c-myc (Seq. ID. No 1), 33.37 .mu.g/ml h-ICAM-1 (Seq.
ID No. 2) and 34 .mu.g/ml EGFR (Seq. ID No. 3). Lipid
concentrations were determined by the inorganic phosphorus assay
after separation of the lipids from the oligonucleotides by a Bligh
and Dyer extraction (Bligh and Dyer, 1959). Briefly, to 250 .mu.l
of aqueous phase (sample/HBS) 525 .mu.l methanol and 250 .mu.l
chloroform were added to form a clear single phase (aqueous
phase/methanol/chlorofrom 1:2.1:1 vol). If the solution was not
clear a small amount of methanol was added. Subsequently, 250 .mu.l
HBS and an equal volume of chloroform were added. The samples were
mixed and centrifuged for 5-10 min at 3000 rpm. This resulted in a
clear two-phase system. The chloroform phase was assayed for
phospholipid content according to the method of Fiske and Subbarrow
(1925). If not otherwise mentioned, trapping efficiencies were
expressed as oligonucleotide-to-lipid weight ratios [w/w].
Example 6
[0101] Following the procedures of Example 5, increasing amounts of
ethanol were added to 100 nm DSPC/Chol/DODAP liposomes (no modified
lipid component) prepared by extrusion. All samples became milky
white immediately upon oligonucleotide addition, indicating
oligonucleotide-induced aggregation. Following incubation with
antisense oligonucleotides at a molar ODN/lipid ratio of 0.035 at
pH 4, ethanol and unentrapped oligonucleotides were removed. Table
7 lists encapsulation efficiencies as determined by dynamic light
scattering, together with the final sizes of the resulting
multilamellar vesicles. Increasingly more antisense oligonucleotide
becomes entrapped as the ethanol concentration is increased. The
concomitant increase in size and polydispersity reflects a
progressive reorganization of the LUVs into larger lipid
structures, which appear to be predominantly large multilamellar
liposomes. It should be noted that due to the size of these systems
some of the lipid is lost on the anion exchange column used to
remove external unentrapped oligonucleotides. The eluted fraction
corresponds to roughly 50-60% of total lipid. At ethanol
concentrations of 40% and higher the initial liposomes become
unstable and fuse to form a milky white dispersion. TABLE-US-00007
TABLE 7 % EtOH [v/v] % encapsulation Average size [nm] 0 4.4 148
.+-. 56 20 20.5 226 .+-. 104 30 32.5 470 .+-. 244 After extrusion
(no antisense) 99 .+-. 22
These results demonstrate that ethanol makes the lipid membranes
susceptible to structural rearrangements which lead lead to
entrapment of the oligonucleotide between the concentric lamellae
of large multilamellar liposomes. However, the size of resulting
liposomes cannot be readily controlled. Example 7
[0102] Liposomes were made containing 2.5 to 10 mol % of modified
lipid (PEG-Cer) and tested using the protocols of Examples 5 and 6.
In each case, the decrease in PEG-Cer concentration was made up
with an increase in DPSC levels. It was found that the
incorporation of the modified lipid into the liposomes allows the
final size of the antisense-containing liposomes to be regulated.
Liposomes were stable at higher ethanol concentrations in the
presence of PEG-Cer than in its absence. The dispersions remained
optically translucent in 40% ethanol, although a slight increase in
turbidity was noted for the sample containing 2.5 mol % PEG-Cer.
The increased stability is also reflected in the higher amounts of
ethanol required for entrapment to occur (Table 8, FIG. 2). FIG. 2
depicts encapsulation efficiencies as a function of ethanol
concentration for liposomes containing 10 mol % PEG-Cer. Maximum
entrapment was reached at 40% ethanol and ethanol concentrations in
excess of 25% (v/v) (>4.3 M) were required for entrapment to
occur. No entrapment was found in the absence of ethanol. Table 8
lists trapping efficiencies and sizes determined by dynamic light
scattering as a function of PEG-Cer content (2.5-10 mol %) at the
minimum and maximum ethanol concentrations determined from FIG. 2.
The sizes of the initial extruded liposomes are given in brackets.
The amount of ethanol required for entrapment to occur depends on
the PEG-Cer content of the liposomes. Liposomes containing 2.5 mol
% PEG-Cer entrapped approximately 15% of the oligonucleotides at
25% ethanol and 45% in the presence of 40% ethanol. In contrast, at
10 mol % PEG-Cer entrapment was virtually abolished in the presence
of 25% ethanol (.ltoreq.5%) and was 60% in 40% ethanol. In all
cases the initial oligonucleotide-to-lipid ratio was 0.037
(mol/mol). Entrapment levels increased from 45% to almost 60% in
40% ethanol when the PEG-Cer content was increased from 2.5 to 10
mol %. Liposome size and polydispersity decreased from 131.+-.40 nm
to 100.+-.26 nm. TABLE-US-00008 TABLE 8 PEG-CerC.sub.14 [mol %] %
encapsulation Average size [nm] 25% ethanol 2.5 14.1 125 .+-. 35
(108 .+-. 26) 10 5 92 .+-. 18 (93 .+-. 18) 40% ethanol 2.5 45.7 131
.+-. 40 (108 .+-. 26) 5 50.9 126 .+-. 36 (107 .+-. 223) 10 56.5 100
.+-. 26 (93 .+-. 18)
Example 8
[0103] The perturbing effect of ethanol on lipid membranes has been
mainly studied at low ethanol concentrations(<15% v/v) in
relation to changes in lipid hydration, acyl chain order, membrane
permeability to small ions and induction of chain interdigitation
in DPPC systems (Slater and Huang, 1988; Barchfeld and Deamer,
1988; Schwichtenhovel et al. 1992; Slater et al., 1993; Barry and
Gawrisch, 1994; Vierl et al., 1994; Lobbecke and Cevc, 1995;
Komatsu and Okada, 1996; Holte and Gawrisch, 1997). It was logical
to ask whether liposomes are still intact at the high ethanol
concentrations required for entrapment. FIG. 3A depicts the release
of calcein entrapped at self-quenching concentrations in
DSPC/Chol/PEG-CerC.sub.14/DODAP liposomes as a function of ethanol
concentration (closed circles) together with the encapsulation
efficiencies obtained using liposomes of the same lipid composition
(open circles). Both the encapsulation as well as the leakage
experiments were performed at 40.degree. C. Leakage of calcein, a
small molecule with a MW of 623, starts at .gtoreq.30% ethanol and
reaches a maximum around 40% ethanol. The oligonucleotide
entrapment shows a similar ethanol dependence indicating that the
entrapment is highly correlated with the destabilization of the
liposomal membrane permeability barrier. In contrast to calcein,
the release of FITC-dextran (MW 19500) was less than 10% in 40%
ethanol. This shows that the loss of the permeability barrier is MW
dependent, as has also been reported for detergents such as
octylglucoside (Almog et al., 1990). The liposomes maintained their
morphology in the presence of 40% ethanol. Phase contrast
microscopy of giant liposomes in 40% ethanol also revealed intact
liposomal structures.
[0104] Lipids are also able to exchange rapidly between liposomes
and between the inner and outer monolayers of the lipid bilayers
comprising the liposomes. As shown in FIG. 3B, lipid mixing as
detected by the NBD-PE/LRh-PE FRET assay is effectively immediate
in 40% ethanol. No increase in vesicle size was observed indicating
the lipid mixing is arising from rapid lipid exchange between
liposomes rather than liposome fusion. The results shown in FIG. 3B
also demonstrate that lipids are able to rapidly migrate
(flip-flop) from one side of the liposomal lipid bilayer to the
other, as shown by the of loss in fluorescence of NBD-PS located in
the outer lipid monolayer upon chemical reduction with sodium
dithionite.
Example 9
[0105] The increase in turbidity upon encapsulation indicates that
entrapment is preceded by an initial aggregation step (formation of
microaggregates). The aggregation step and the entrapment can be
decoupled at low temperatures. Samples become turbid upon or
shortly after addition of oligonucleotide and the turbidity
increases over time. In the absence of ethanol there is only a
slight increase in turbidity following which light transmission
remains constant. In contrast to samples prepared at 40.degree. C.,
samples incubated at 4.degree. C. become translucent again when
ethanol is removed and liposomes do not entrap oligonucleotide.
Entrapment efficiencies are plotted as a function of temperature in
FIG. 4 together with calcein leakage data. Leakage data are
presented as the ethanol concentrations required to induce 50%
calcein release. Again there is a qualitative correlation between
the destabilization of the liposomal membrane and the entrapment
efficiency.
Example 10
[0106] .sup.31P-NMR can be used to assay for oligonucleotide
entrapment. FIGS. 5A and 5B show .sup.31P NMR spectra of c-myc in
solution (FIG. 5A) and entrapped in DODAP/DSPC/Chol/PEG-CerC.sub.14
liposomes (FIG. 5B). Initially, the liposomes exhibited a
transmembrane pH gradient, where the internal pH is 4 and the
external pH 7.5. Under these conditions the entrapped
oligonucleotides are tightly associated with the positively charged
liposomal membrane. This immobilization results in the
disappearance (broadening out) of the NMR signal (FIG. 5B). Upon
dissipation of the pH gradient by addition of ammonium acetate and
adjustment of the external pH to 7.5, DODAP is deprotonated and the
oligonucleotides dissociate from the liposomal membrane. This is
demonstrated by the recovery of the NMR signal in FIG. 5C. However,
the recovery is incomplete, about 50% of the initial signal. The
signal attenuation is not due to NMR resonance saturation. It may
be attributed to two possibilities: either the amount of
encapsulated antisense exceeds its solubility so that a portion of
it precipitates, or the mobility of the antisense molecules is
spatially constrained e.g. by immobilization between two closely
apposing bilayers (see FIG. 3A). To confirm that the
oligonucleotides were encapsulated and localized in the aqueous
interior of the liposomes, 5 mM MnSO.sub.4 was added to the
external solution (FIG. 5D). Mn.sup.2+ is a membrane impermeable
paramagnetic line broadening agent and will quench the signals of
all accessible phosphate groups, phospholipids as well as
oligonucleotides. However, the oligonucleotide signal remained
unaffected and disappeared only upon solubilization of the
liposomes with OGP (FIG. 5E). The whole oligonucleotide signal is
recovered when the initial liposomes (FIG. 5B) are solubilized with
OGP in the absence of Mn.sup.2+ (FIG. 5F). These data clearly
demonstrate that the oligonucleotide is entrapped in the liposomes
and not simply associated with the external membrane. It should
also be noted that entrapped oligonucleotides were not accessible
to the oligonucleotide-binding dye OliGreen.
[0107] The NMR studies describe the interaction between
oligonucleotides and liposomes as seen from the perspective of the
oligonucleotides. Changes in lipid dynamics and membrane
organization can be probed with pyrene-labeled lipids (Duportail
and Lianos, 1996). Pyrene-labeled lipids form excited state dimers
at high concentrations, which fluoresce at a different wavelength
than the monomers. Excimer formation is a diffusion-controlled
process and requires two molecules to come together to form a
dimer. The binding of the oligonucleotides results in a dramatic
reduction of the lateral mobility of all lipid species relative to
control liposomes, which do not contain oligonucleotides. The
membrane is laterally compressed. This follows from the observed
decrease in excimer fluorescence of pyrene-HPC. The depletion of
the transmembrane pH gradient results in an increase of the excimer
fluorescence and restoration of lipid mobility.
Example 11
[0108] Both the size of the liposomes entrapping antisense as well
as the entrapment efficiency depend on the initial
antisense-to-lipid ratio. FIG. 6 shows that oligonucleotides can be
efficiently entrapped at high antisense-to-lipid ratios. The
entrapment efficiency is plotted as a function of the initial
oligonucleotide-to-lipid ratio. The binding level at maximum
entrapment is 0.16 mg oligonucleotide per mg of lipid (0.024
mol/mol). This corresponds to approximately 2250 oligonucleotide
molecules per 100 nm liposome and demonstrates the high efficiency
of this entrapment procedure. Entrapment efficiencies are about 3
orders of magnitude higher than obtained by passive
encapsulation.
[0109] Upon increasing the oligonucleotide-to-lipid ratio, the size
as well as the polydispersity of the samples increase slightly from
70.+-.10 nm for liposomes alone to 110.+-.30 for an initial
ODN-to-lipid weight ratio of 0.2. Freeze-fracture electron
microscopy showed an increase in the number of larger liposomes
with increasing initial oligonucleotide-to-lipid ratios. As an
aside it should be noted that the initially translucent liposome
dispersion becomes increasingly turbid as the antisense-to-lipid
ratio is increased.
Example 12
[0110] It would be expected that the PEG coating would inhibit
formation of the closely opposed membranes observed for the
multilamellar structures by TEM. The fate of PEG-Cer was therefore
examined by using radioactively-labeled PEG-CerC.sub.14. Antisense
oligonucleotides were encapsulated in liposomes containing trace
amounts of [.sup.3H]-PEG-CerC.sub.14 in addition to 10 mol %
unlabeled PEG-CerC.sub.14 and [.sup.14C]-cholesterol hexadecylether
(CHE) as a cholesterol marker at a [.sup.3H]/[.sup.14C] ratio of
5.9. This ratio represents an apparent PEG-Cer/chol ratio and will
be used in place of the molar PEG-Cer/chol ratio. The initial
antisense-to-lipid weight ratio was 0.29. Entrapment resulted in a
final antisense-to-lipid ratio of 0.16. Free PEG-Cer and PEG
micelles were separated from liposomes by ultracentrifugation using
a sucrose step gradient (1%, 2.5%, 10%, 15% (w/v) sucrose in HBS).
Empty liposomes band at the interface between 2.5% and 10% sucrose
with an apparent PEG-Cer/chol ratio of 5.5. This band accounts for
roughly 80% of the total lipid. The antisense-containing liposomes
show a faint band at the same location, which corresponds to less
than 9% of total lipid. However, most of the liposomal antisense
migrates down to the 15% sucrose layer or pellets at the bottom. A
complete analysis of the liposome-containing fractions of the
gradient is presented in Table 9. The results are representative
for samples prepared at high oligonucleotide-to-lipid ratios. It
can be seen that the relative PEG-Cer/chol ratios progressively
decrease towards the bottom of the gradient. More than 50% of the
PEG-Cer is lost from the bottom fraction relative to the initial
liposomes (apparent PEG-Cer/chol ratio 5.5). The DSPC/Chol ratio
does not change. 27% of the PEG-Cer can be found in the top
fractions along with 6.6% of cholesterol. Approximately the same
amount of non liposome-associated PEG-Cer was found for the empty
control liposomes.
[0111] Further analysis of the fractions of the above gradient show
that the antisense-containing liposomes show large differences in
their antisense content and size (Table 9). The
oligonucleotide-to-lipid ratios as well as the average size
increase from top to bottom. Three main populations can be
identified as distinct bands (Table 10). Their relative proportions
depended on the initial oligonucleotide-to-lipid ratio (Table 10).
First, liposomes entrapping antisense at low ODN/lipid ratio
(0.03-0.05). Secondly, liposomes with an ODN/lipid ratio of
0.14-0.15 and finally, liposomes with very high ODN/lipid ratios
(0.29 mg/mg). The latter population decreases in favor of the first
two with decreasing initial ODN/1 ratio. It is optically turbid
whereas the other two are translucent. It was attempted to
correlate the observed differences in entrapment and size to the
morphological heterogeneity seen by cryo-TEM. Antisense was
entrapped at high initial oligonucleotide-to-lipid ratio (0.28
mg/mg) and the two main fractions corresponding to fractions 15 and
17 in Table 10 viewed by cryo-TEM after removal of sucrose by
dialysis. The upper fraction consists exclusively of bilamellar
liposomes, many of which exhibit bulbs, whereas the bottom fraction
contained a mixture of bi-and multilamellar liposomes.
TABLE-US-00009 TABLE 9 Chol/ PEG- % % % DSPC CER/chol fraction
PEG-Cer Chol DSPC [mol/mol] ratio [r.u.] Size [nm] b.u. -- -- --
2.2 5.9 86 .+-. 24 1-10 26.8 6.6 -- -- -- -- 11 13.7 9.2 -- -- 8.6
89 .+-. 21 12 4.3 3.9 -- -- 6.4 -- 13 3.6 4.1 -- -- 5.0 -- 14 8.9
11.4 -- -- 4.5 83 .+-. 21 15 27.1 36.6 37.6 2.2 4.2 70 .+-. 15 16
8.5 12.8 -- -- 3.8 75 .+-. 16 17 7.1 15.4 15.5 2.2 2.6 129 .+-.
39
[0112] TABLE-US-00010 TABLE 10 High initial Low initial ODN/lipid
ratio ODN/lipid ratio Fraction % lipid % ODN ODN/1 % lipid % ODN
ODN/1 b.u. -- -- 0.156 -- -- 0.1 11 9.2 1.9 0.030 15.7 6.7 0.036 12
3.9 3.1 0.117 14.8 8.9 0.050 13 4.1 3.9 0.140 3.8 3.4 0.09 14 11.4
10.5 0.140 8.1 11.2 0.14 15 36.6 36.2 0.148 31.5 45.9 0.146 16 12.8
14.4 0.168 9.7 15.4 0.16 17 15.4 30.1 0.29 5.3 8.5 0.162
Example 13
[0113] The addition of oligonucleotides to cationic liposomes in
the presence of ethanol can give rise to domain formation. The
formation of the multilamellar liposomes seen must be preceded by
liposome adhesion. However, 10 mol % PEG-Cer completely inhibits
adhesion in the absence of ethanol. In the presence of ethanol, two
effects could contribute to liposome adhesion: first, the increase
in the amount of non membrane-incorporated PEG-Cer through rapid
lipid exchange and second, formation of small domains depleted in
PEG-Cer and enriched in antisense oligonucleotides. The latter
possibility was investigated. The effect of oligonucleotide binding
was visualized by phase contrast and fluorescence microscopy using
giant DSPC/Chol/DODAP/PEG-CerC.sub.14 liposomes in conjunction with
FITC-labeled oligonucleotides. Most of the liposomes observed were
multilamellar and displayed internal structure. In the absence of
ethanol, the giant liposomes disintegrated into irregularly-shaped
aggregates and smaller liposomes on addition of antisense. The
green FITC fluorescence revealed the location of the
oligonucleotides. A completely different picture is presented in
the presence of 40% ethanol. The initially round liposomes adopt a
pear-shaped form 5-10 min after addition of oligonucleotide with
the oligonucleotides located in a semicircle on one side of these
structures. The interior membranes are squeezed out from this
horseshoe, which detaches and collapses, in particular upon raising
the temperature, into a compact slightly irregular structure that
appears completely green in fluorescence. The segregation of the
oligonucleotides indicates that ethanol is able to stimulate domain
formation.
Example 14
[0114] The encapsulation procedure of the invention is not
dependent on a particular oligonucleotide and lipid composition,
nor is it restricted to the high citrate concentrations used.
Encapsulation is most efficient in 50 mM citrate buffer and
decreases at higher as well as at lower citrate concentrations. The
size and polydispersity increases considerably at citrate
concentrations below 25 mM. FIG. 7 shows that other
oligonucleotides than c-myc (Seq, ID No 1) as well as plasmid-DNA
can be efficiently entrapped in DSPC/Chol/DODAP/PEG-CerC.sub.14
liposomes. The initial oligonucleotide-to-lipid weight ratio was
0.1 mg/mg, 300 mM citrate buffer was used for oligonucleotide
entrapment. The pDNA entrapment was performed in 50 mM citrate
buffer at a pDNA-to-lipid weight ratio of 0.03. Unlike
phosphorothioate antisense oligonucleotides phosphodiester-based
molecules cannot be encapsulated high ionic strengths buffers such
as 300 mM citrate buffer. This probably reflects differences in
binding affinities (Semple et al., 2000). In contrast to the
efficient encapsulation of large molecules, less than 10% of ATP
could be entrapped in DSPC/Chol/DODAP/PEG-CerC.sub.14 liposomes at
an initial ATP-to-lipid ratio of 0.2 mg/mg. The ATP entrapment was
performed in 50 mM citrate buffer. Table 5 demonstrates that the
entrapment procedure can be extended to other lipid compositions
including DOPE systems. Preliminary results with negatively charged
liposomes and positively charged polyelectrolytes including
polylysines show that entrapment is a general feature of the
interaction of polyelectrolytes with oppositely charged liposomes
in ethanol.
Example 15
[0115] Octylglucoside (OGP) was used in place of ethanol. The
detergent was added to liposomes (1:1 v/v) to final concentrations
ranging from 30-40 mM. All the subsequent steps were performed as
described as in Example 5 except for the initial dialysis step
against pH 4 citrate buffer, which was extended to 5 hrs. The
oligonucleotide was shown to be protected from externally added
OliGreen, a flourescent oligo-binding dye. The initial
oligonucleotide-to-lipid ratio was 0.23 (mg/mg). Sizes represent
number-averaged sizes. DSPC/Chol/DODAP/PEG-CerC.sub.14 (20/45/25/10
mol %). The observed levels of encapsulation and final particle
size are summarized in Table 11. TABLE-US-00011 TABLE 11 OGP [mM] %
encapsulation Size [nm] 30 51 65 .+-. 12 35 57 100 .+-. 22 40 55
145 .+-. 38
[0116] Using this invention, and the teachings of this
specification, those skilled in the art will be able to identify
other methods and means for generating fully encapsulated
lipid-therapeutic agent particles, all of which are encompassed by
the claims set out below.
[0117] All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet are
incorporated herein by reference, in their entirety.
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Sequence CWU 1
1
3 1 16 DNA Homo sapiens c-myc 1 taacgttgag gggcat 16 2 20 DNA Homo
sapiens ICAM-1 2 gcccaagctg gcatccgtca 20 3 15 DNA Homo sapiens
EGFR 3 ccgtggtcat gctcc 15
* * * * *