U.S. patent application number 10/884690 was filed with the patent office on 2004-12-02 for cationic peg-lipids and methods of use.
This patent application is currently assigned to The University of British Columbia. Invention is credited to Chen, Tao, Cullis, Pieter R., Fenske, David B., Palmer, Lorne R., Wong, Kim.
Application Number | 20040241855 10/884690 |
Document ID | / |
Family ID | 33455853 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241855 |
Kind Code |
A1 |
Cullis, Pieter R. ; et
al. |
December 2, 2004 |
Cationic peg-lipids and methods of use
Abstract
The present invention provides cationic-polymer-lipid conjugates
(CPLs) such as distal cationic-poly(ethylene glycol)-lipid
conjugates which can be incorporated into conventional and stealth
liposomes or other lipid-based formulation for enhancing cellular
uptake. The CPLs of the present invention comprise a lipid moiety;
a hydrophilic polymer; and a polycationic moiety. Method of
increasing intracellular delivery of nucleic acids are also
provided.
Inventors: |
Cullis, Pieter R.;
(Vancouver, CA) ; Chen, Tao; (Richmond, CA)
; Fenske, David B.; (Surrey, CA) ; Palmer, Lorne
R.; (Vancouver, CA) ; Wong, Kim; (Vancouver,
CA) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Assignee: |
The University of British
Columbia
University Industry Liaison Office #103 - 6190 Agronomy
Road
Vancouver
CA
|
Family ID: |
33455853 |
Appl. No.: |
10/884690 |
Filed: |
July 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10884690 |
Jul 2, 2004 |
|
|
|
09553639 |
Apr 20, 2000 |
|
|
|
60130151 |
Apr 20, 1999 |
|
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Current U.S.
Class: |
435/455 ;
525/54.1 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 47/543 20170801; A61K 9/1272 20130101; C12N 15/88 20130101;
A61K 47/6911 20170801; A61K 49/0041 20130101; A61K 49/0054
20130101; A61K 49/0021 20130101 |
Class at
Publication: |
435/455 ;
525/054.1 |
International
Class: |
C12N 015/85; C08G
063/48 |
Claims
1. A compound having the general structure of Formula I: A-W-Y I
wherein: A is a lipid moiety; W is a hydrophilic polymer; and Y is
a polycationic moiety.
2. The compound according to claim 1, wherein said hydrophilic
polymer is non-immunogenic or weakly immunogenic.
3. The compound according to claim 1, wherein W is a polymer
selected from the group consisting of PEG, polyamide, polylactic
acid, polyglycolic acid, polylactic acid/polyglycolic acid
copolymers and combinations thereof, said polymer having a
molecular weight of about 250 to about 7000 daltons.
4. The compound according to claim 1, wherein Y comprises a least
one basic amino acid or derivative thereof.
5. The compound according to claim 1, wherein Y has at least 4
positive charges at a selected pH.
6. The compound according to claim 1, wherein Y has at least 8
positive charges at a selected pH.
7. The compound according to claim 4, wherein Y comprises an amino
acid selected from the group consisting of lysine, arginine,
asparagine, glutamine, derivatives thereof and combinations
thereof.
8. The compound according to claim 2, wherein A is a member
selected from the group consisting of a diacylglycerolyl moiety, a
dialkylglycerolyl moiety, a N--N-dialkylamino moiety, a
1,2-diacyloxy-3-aminopropane moiety and a
1,2,dialkyl-3-aminopropane moiey.
9. The compound according to claim 3, wherein W is PEG.
10. The compound according to claim 3, wherein W is a polyamide
polymer.
11. The compound according to claim 3, wherein W has a molecular
weight of about 250 to about 2000 daltons.
12. The compound according to claim 9, having the general structure
of Formula II: 10wherein X is a member selected from the group
consisting of a single bond and a functional group covalently
attaching said lipid moiety A to at least one ethylene oxide unit;
Z is a member selected from the group consisting of a single bond
and a functional group covalently attaching at least one ethylene
oxide unit to the polycationic moiety Y; and n is an integer having
a value of between about 6 and about 50.
13. The compound according to claim 12, wherein X is a member
selected from the group consisting of a single bond,
phosphatidylethanolamino, phosphatidylethanolamido, phosphoro,
phospho, phosphoethanolamino, phosphoethanolamido, carbonyl,
carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur
and NR, wherein R is a hydrogen or alkyl group.
14. The compound according to claim 12, wherein Z is a member
selected from the group consisting of a single bond,
phosphatidylethanolamino, phosphatidylethanolamido, phosphoro,
phospho, phosphoethanolamino, phosphoethanolamido, carbonyl,
carbamate, carboxyl, carbonate, amido, thioamido, oxygen, sulfur
and NR, wherein R is a hydrogen or alkyl group.
15. The compound according to claim 12, wherein A is a
diacylglycerolyl moiety; X is phosphoethanolamido; Z is NR, wherein
R is a hydrogen atom; and Y comprises about 1 to about 10 basic
amino acids or derivatives thereof.
16. The compound according to claim 15, where A is a
diacylglycerolyl moiety having 2 fatty acyl chains, wherein each
acyl chain is independently between 2 and 30 carbons in length and
is either saturated or has vary degrees of saturation.
17. The compound according to claim 15, wherein Y comprises an
amino acid selected from the group consisting of lysine, arginine,
glutamine, derivatives thereof and combinations thereof.
18. The compound according to claim 15, wherein A is a
diacylglycerolyl moiety having 2 fatty acyl chains, wherein each
acyl chain is a saturated C-18 carbon chain; and Y is a cationic
group comprising 4 lysine residues or derivatives thereof.
19-54. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/130,151 filed Apr. 20, 1999, the teachings
of which are incorporated herein by reference in their entirety for
all purposes.
FIELD OF THE INVENTION
[0002] This invention relates to cationic lipid conjugates, and
more particularly, to cationic polymer lipid conjugates and
lipid-based drug formulations thereof, containing one or more
bioactive agents.
BACKGROUND OF THE INVENTION
[0003] Current vectors for gene delivery and gene therapy are
comprised of viral based and non-viral based systems. Lipid-based
non-viral systems include cationic lipid plasmid DNA complexes.
Limitations of these systems include large sizes, toxicity and
instability of the complexes in the serum. Unfortunately, the
foregoing drawbacks limit the applications for these complexes.
[0004] Researchers have devoted tremendous effort to the design of
long circulation stealth liposomes that can be used for systemic
delivery (see, Papahadjopoulos, D. et al., Proc. Natl. Acad. Sci.
88:11460-11464 (1991); Klibanov, A. L. et al., J. Liposome Res.,
2:321 (1992); Woodle, M. C. et al., Biochim. Biophys. Acta.,
1113:171 (1992); Torchilin, V. P. et al., In: Stealth Liposomes.
Ed. By D. Lasic, F. Martin. CRC Press, Boca Raton, Fla., pp. 51-62
(1995); Allen, T. M. et al., Biochim. Biophys. Acta., 1237:99-108
(1995) and Zalipsky, S. et al., J. Controlled Release, 39:153-161
(1996)). In certain instances, and depending on the formulation,
stealth liposomes are often comparatively inefficient at
facilitating cellular uptake and therefore the therapeutic efficacy
is reduced.
[0005] In general, the molecular mechanism of liposomal longevity
in vivo can be attributed to steric hindrance resulting from
hydrophilic polymer surface barriers. The hydrophilic polymer
barriers prevent or reduce the rate of the adsorption of
macromolecules from the blood and sterically inhibit both
electrostatic and hydrophobic interactions between liposomes and
blood components. Thus, although the longevity of stealth liposomes
has been increased by the insertion of hydrophilic polymers, the
cellular uptake of the stealth liposomes often is inefficient.
[0006] Over the past decade, it has also become clear that
liposomal systems possessing cationic lipids are highly effective
transfection agents in vitro (Felgner, P. L. et al., Nature
337:387-388 (1989); Felgner, P. L. et al., Proceedings of the
National Academy of Sciences of the United States of America
84:7413-7417 (1987)). The addition of cationic liposomes to plasmid
DNA gives rise to large DNA-lipid complexes that possess excellent
transfection properties in vitro, but which are ineffective in vivo
due to their rapid clearance from the circulation by cells of the
reticuloendothelial system (RES). The need for a non-viral
lipid-based system capable of systemic delivery of genes to cells
led to the recent development of stabilized plasmid-lipid particles
(SPLPs) (Wheeler, J. J. et al., Gene Therapy 6:271-281 (1999)).
These particles are small (about 70 nm), contain a single copy of a
plasmid vector, possess stealth properties resulting from a surface
coating of poly(ethyleneglycol) (PEG), and protect DNA from
degradation by serum nucleases.
[0007] Enhancing intracellular delivery of liposomes and/or their
contents represents one of the major remaining problems in the
development of the next generation of drug delivery systems. In
order to optimize the delivery of drugs (conventional or genetic)
in vivo, general methods for increasing the interactions of
liposomes with cells need to be developed. To date, attempts
include the use of specific targeting information on the liposome
surface, such as an antibody (see, Meyer, O. et al., Journal of
Biological Chemistry 273:15621-15627 (1998); Kao, G. Y. et al.,
Cancer Gene Therapy 3:250-256 (1996); Hansen, C. B. et al.,
Biochimica et Biophysica Acta 1239:133-144 (1995)), vitamin--(see,
Gabizon, A. et al., Bioconjugate Chemistry 10:289-298 (1999); Lee,
R. J. et al., Journal of Biological Chemistry 269:3198-3204 (1994);
Reddy, J. A. et al., Critical Reviews in Therapeutic Drug Carrier
Systems 15:587-627 (1998); Holladay, S. R. et al., Biochimica et
Biophysica Acta 1426:195-204 (1999); Wang, S. et al., Journal of
Controlled Release 53:39-48 (1998)), oligopeptide--(see, Zalipsky,
S. et al., Bioconjugate Chemistry 6:705-708 (1995); Zalipsky, S. et
al., Bioconjugate Chemistry 8:111-118 (1997)), or the use of
oligosaccharide constructs specific for a particular membrane
protein or receptor. Unfortunately, these methods have not been
successful in achieving this goal, despite promising in vitro
results. While specific targeting of liposomes to tissues remains
an important area of research, other approaches may also provide
significant improvements in the effectiveness of liposomal
carriers.
[0008] In view of the foregoing, what is needed in the art is a
lipid-based drug formulation with increased longevity coupled with
increased cellular uptake. The present invention satisfies this and
other needs.
SUMMARY OF THE INVENTION
[0009] In certain aspects, the present invention relates to new
conjugates that can be incorporated or inserted into stabilized
plasmid lipid particles to enhance transfection efficiencies. The
conjugates of the present invention possess a lipid anchor for
anchoring the conjugate into the bilayer lipid particle, wherein
the lipid anchor is attached to a non-immunogenic polymer, such as
a PEG moiety, and wherein the non-immunogenic polymer is, in turn,
attached to a polycationic moiety, such as a positively charged
moiety. As such, the present invention provides a compound of
Formula I:
A-W-Y I
[0010] In Formula I, "A" is a lipid moiety attached to a
non-immunogenic polymer. "W," in Formula I, is a non-immunogenic
polymer, and "Y", in Formula I, is a polycationic moiety.
[0011] In certain preferred embodiments, the compounds of Formula I
contain groups that give rise to compounds having the general
structure of Formula II: 1
[0012] In Formula II, "A" is a lipid, such as a hydrophobic lipid.
In Formula II, "X" is a single bond or a functional group that
covalently attaches the lipid to at least one ethylene oxide unit,
i.e., (--CH.sub.2--CH.sub.2--O--). In Formula II, "Z" is a single
bond or a functional group that covalently attaches the at least
one ethylene oxide unit to a cationic group. In Formula II, "Y" is
a polycationic moiety. In Formula II, the index "n" is an integer
ranging in value from about 6 to about 160.
[0013] In other aspects, the present invention relates to a
lipid-based drug formulation comprising:
[0014] (a) a compound having Formula I
A-W-Y I
[0015] wherein: A, W and Y have been defined;
[0016] (b) a bioactive agent; and
[0017] (c) a second lipid.
[0018] In certain embodiments, the lipid-based drug formulation is
in the form of a liposome, a micelle, a virosome, a lipid-nucleic
acid particle, a nucleic acid aggregate and mixtures thereof. In
certain other embodiments, the bioactive agent is a therapeutic
nucleic acid or other drugs.
[0019] In yet other aspects, the present invention relates to a
method for increasing intracellular delivery of a lipid-based drug
delivery system, comprising: incorporating into the lipid-based
drug delivery system a compound of Formulae I or II, thereby
increasing the intracellular delivery of the lipid-based drug
delivery system.
[0020] Additional aspects and advantages of the present invention
will be apparent when read with the following detailed description
and the accompanying drawings.
Definitions
[0021] The term "lipid" refers to a group of organic compounds that
include, but are not limited to, esters of fatty acids and are
characterized by being insoluble in water, but soluble in many
organic solvents. They are usually divided into at least three
classes: (1) "simple lipids" which include fats and oils as well as
waxes; (2) "compound lipids" which include phospholipids and
glycolipids; (3) "derived lipids" such as steroids.
[0022] The term "vesicle-forming lipid" is intended to include any
amphipathic lipid having a hydrophobic moiety and a polar head
group, and which by itself can form spontaneously into bilayer
vesicles in water, as exemplified by most phospholipids.
[0023] The term "vesicle-adopting lipid" is intended to include any
amphipathic lipid which is stably incorporated into lipid bilayers
in combination with other amphipathic lipids, with its hydrophobic
moiety in contact with the interior, hydrophobic region of the
bilayer membrane, and its polar head group moiety oriented toward
the exterior, polar surface of the membrane. Vesicle-adopting
lipids include lipids that on their own tend to adopt a
non-lamellar phase, yet which are capable of assuming a bilayer
structure in the presence of a bilayer-stabilizing component. A
typical example is DOPE (dioleoylphosphatidylethanolamine). Bilayer
stabilizing components include, but are not limited to, polyamide
oligomers, peptides, proteins, detergents, lipid-derivatives,
PEG-lipid derivatives such as PEG coupled to
phosphatidylethanolamines, and PEG conjugated to ceramides (see,
U.S. application Ser. No. 08/485,608, now U.S. Pat. No. 5,885,613,
which is incorporated herein by reference).
[0024] The term "amphipathic lipid" refers, in part, to any
suitable material wherein the hydrophobic portion of the lipid
material orients into a hydrophobic phase, while the hydrophilic
portion orients toward the aqueous phase. Amphipathic lipids are
usually the major component of a lipid vesicle. Hydrophilic
characteristics derive from the presence of polar or charged groups
such as carbohydrates, phosphato, carboxylic, sulfato, amino,
sulfbydryl, nitro, hydroxy and other like groups. Hydrophobicity
can be conferred by the inclusion of apolar groups that include,
but are not limited to, long chain saturated and unsaturated
aliphatic hydrocarbon groups and such groups substituted by one or
more aromatic, cycloaliphatic or heterocyclic group(s). Examples of
amphipathic compounds include, but are not limited to,
phospholipids, aminolipids and sphingolipids. Representative
examples of phospholipids include, but are not limited to,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine or
dilinoleoylphosphatidylcholine. Other compounds lacking in
phosphorus, such as sphingolipid, glycosphingolipid families,
diacylglycerols and .beta.-acyloxyacids, are also within the group
designated as amphipathic lipids. Additionally, the amphipathic
lipid described above can be mixed with other lipids including
triglycerides and sterols.
[0025] The term "neutral lipid" refers to any of a number of lipid
species that exist either in an uncharged or neutral zwitterionic
form at a selected pH. At physiological pH, such lipids include,
for example, diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides and diacylglycerols.
[0026] The term "hydrophopic lipid" refers to compounds having
apolar groups that include, but are not limited to, long chain
saturated and unsaturated aliphatic hydrocarbon groups and such
groups optionally substituted by one or more aromatic,
cycloaliphatic or heterocyclic group(s). Suitable examples include,
but are not limited to, diacylglycerol, dialkylglycerol,
N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and
1,2-dialkyl-3-aminopropane.
[0027] The term "diacylglycerolyl" denotes 2-fatty acyl chains,
R.sup.1 and R.sup.2 having independently between 2 and 30 carbons
bonded to the 1- and 2-position of glycerol by ester linkages. The
acyl groups can be saturated or have varying degrees of
unsaturation. Diacylglycerol groups have the following general
formula: 2
[0028] The term "dialkylglycerolyl" denotes two C.sub.1-C.sub.30
alkyl chains bonded to the 1- and 2-position of glycerol by ether
linkages. Dialkylglycerol groups have the following general
formula: 3
[0029] The term "N--N-dialkylamino" denotes 4
[0030] The term "1,2-diacyloxy-3-aminopropane" denotes 2-fatty acyl
chains C.sub.1-C.sub.30 bonded to the 1- and 2-position of propane
by an ester linkage. The acyl groups can be saturated or have
varying degrees of unsaturation. The 3-position of the propane
molecule has a --NH-- group attached. 1,2-diacyloxy-3-aminopropanes
have the following general formula: 5
[0031] The term "1,2-dialkyl-3-aminopropane" denotes 2-alkyl chains
(C.sub.1-C.sub.30) bonded to the 1- and 2-position of propane by an
ether linkage. The 3-position of the propane molecule has a --NH--
group attached. 1,2-dialkyl-3-aminopropanes have the following
general formula: 6
[0032] The term "non-cationic lipid" refers to any neutral lipid as
described above as well as anionic -lipids. Examples of anionic
lipids include, but are not limited to, phosphatidylglycerol,
cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,
N-dodecanoyl phosphatidylethanolamines, N-succinyl
phosphatidylethanolamines, N-glutarylphosphatidylethanolamines,
lysophosphatidylglycerols, and other anionic modifying groups
joined to neutral lipids.
[0033] The term "cationic lipid" refers to any of a number of lipid
species that carry a net positive charge at a selected pH, such as
physiological pH. Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl)-N,N,N-t rimethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 3-(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.RTM.
(commercially available cationic liposomes comprising DOTMA and
1,2-dioleoyl-sn-3-phosphoethanola- mine ("DOPE"), from GIBCO/BRL,
Grand Island, N.Y., USA); LIPOFECTAMINE.RTM. (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.RTM. (commercially available cationic lipids
comprising dioctadecylamidoglycyl carboxyspermine ("DOGS") in
ethanol from Promega Corp., Madison, Wis., USA). The following
lipids are cationic and have a positive charge at below
physiological pH: DODAP, DODMA, DMDMA and the like.
[0034] The term "fusogenic" refers to the ability of a liposome or
other drug delivery system to fuse with membranes of a cell. The
membranes can be either the plasma membrane or membranes
surrounding organelles, e.g., endosome, nucleus, etc. Fusogenesis
is the fusion of a liposome to such a membrane.
[0035] The term "dendrimer" includes reference to branched polymers
that possess multiple generations. In dendrimers, each generation
creates multiple branch points.
[0036] The term "ligand" includes any molecule, compound or device
with a reactive functional group and includes lipids, amphipathic
lipids, carrier compounds, bioaffinity compounds, biomaterials,
biopolymers, biomedical devices, analytically detectable compounds,
therapeutically active compounds, enzymes, peptides, proteins,
antibodies, immune stimulators, radiolabels, fluorogens, biotin,
drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes,
micelles, immunoglobulins, functional groups, targeting agents, or
toxins. The foregoing list is illustrative and not intended to be
exhaustive.
[0037] The term "ATTA" or "polyamide" refers to, but is not limited
to, compounds disclosed in U.S. patent application Ser. No.
09/218,988, filed Dec. 22, 1998. These compounds include a compound
having the formula 7
[0038] wherein: R is a member selected from the group consisting of
hydrogen, alkyl and acyl; R.sup.1 is a member selected from the
group consisting of hydrogen and alkyl; or optionally, R and
R.sup.1 and the nitrogen to which they are bound form an azido
moiety; R.sup.2 is a member of the group selected from hydrogen,
optionally substituted alkyl, optionally substituted aryl and a
side chain of an amino acid; R.sup.3 is a member selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto,
hydrazino, amino and NR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5
are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p
is 1 to 4; and q is 0 or 1. It will be apparent to those of skill
in the art that other polyamides can be used in the compounds of
the present invention.
[0039] As used herein, the term "alkyl" denotes branched or
unbranched hydrocarbon chains, such as, methyl, ethyl, n-propyl,
iso-propyl, n-butyl, sec-butyl, iso-butyl, tertbutyl, octa-decyl
and 2-methylpentyl. These groups can be optionally substituted with
one or more functional groups which are attached commonly to such
chains, such as, hydroxyl, bromo, fluoro, chloro, iodo, mercapto or
thio, cyano, alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl,
carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the
like to form alkyl groups such as trifluoromethyl, 3-hydroxyhexyl,
2-carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl and the
like.
[0040] The term "alkylene" refers to a divalent alkyl as defined
above, such as methylene (--CH.sub.2--), propylene
(--CH.sub.2CH.sub.2CH.sub.2--- ), chloroethylene
(--CHClCH.sub.2--), 2-thiobutene (--CH.sub.2CH(SH)CH.sub-
.2CH.sub.2--), 1-bromo-3-hydroxyl-4-methylpentene
(--CHBrCH.sub.2CH(OH)CH(- CH.sub.3)CH.sub.2--), and the like.
[0041] The term "alkenyl" denotes branched or unbranched
hydrocarbon chains containing one or more carbon-carbon double
bonds.
[0042] The term "alkynyl" refers to branched or unbranched
hydrocarbon chains containing one or more carbon-carbon triple
bonds.
[0043] The term "aryl" denotes a chain of carbon atoms which form
at least one aromatic ring having preferably between about 6-14
carbon atoms, such as phenyl, naphthyl, indenyl, and the like, and
which may be substituted with one or more functional groups which
are attached commonly to such chains, such as hydroxyl, bromo,
fluoro, chloro, iodo, mercapto or thio, cyano, cyanoamido,
alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carbalkoyl,
alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form
aryl groups such as biphenyl, iodobiphenyl, methoxybiphenyl,
anthryl, bromophenyl, iodophenyl, chlorophenyl, hydroxyphenyl,
methoxyphenyl, formylphenyl, acetylphenyl,
trifluoromethylthiophenyl, trifluoromethoxyphenyl, alkylthiophenyl,
trialkylamrnmoniumphenyl, amidophenyl, thiazolylphenyl,
oxazolylphenyl, imidazolylphenyl, imidazolylmethylphenyl, and the
like.
[0044] The term "acyl" denotes the --C(O)R group, wherein R is
alkyl or aryl as defined above, such as formyl, acetyl, propionyl,
or butyryl.
[0045] The term "alkoxy" denotes --OR--, wherein R is alkyl.
[0046] The term "amido" denotes an amide linkage: --C(O)NR--
(wherein R is hydrogen or alkyl).
[0047] The term "amino" denotes an amine linkage: --NR--, wherein R
is hydrogen or alkyl or a terminal NH.sub.2.
[0048] The term "carboxyl" denotes the group --C(O)O--, and the
term "carbonyl" denotes the group --C(O)--.
[0049] The term "carbonate" indicates the group --OC(O)O--.
[0050] The term "carbamate" denotes the group --NHC(O)O--, and the
term "urea" denotes the group --NHC(O)NH--.
[0051] The term "phosphoro" denotes the group --OP(O)(OH)O--.
[0052] The term "basic amino acid" refers to naturally-occurring
amino acids as well as synthetic amino acids and/or or amino acid
mimetics having a net positive charge at a selected pH, such as
physiological pH. This group includes, but is not limited to,
lysine, arginine, asparagine, glutamine, histidine and the
like.
[0053] The term "phosphorylethanolamino" denotes the group
--OP(O)(OH)OCH.sub.2CH.sub.2NH--.
[0054] The term "phosphorylethanolamido" denotes the group
--OP(O)(OH)OCH.sub.2CH.sub.2NHC(O)--.
[0055] The term "phospho" denotes a pentavalent phosphorous moiety
--P(O)(OH)O--.
[0056] The term "phosphoethanolamino" denotes the group
--P(O)(OH)OCH.sub.2CH.sub.2NH--.
[0057] The term "phosphoethanolamido" denotes the
group-P(O)(OH)OCH.sub.2C- H.sub.2NHC(O)--.
[0058] The term "ethylene oxide unit" denotes the group
--OCH.sub.2CH.sub.2--.
[0059] The term "CPL" refers to a cationic-polymer-lipid e.g.,
cationic-PEG-lipid. Preferred CPLs are compounds of Formulae I and
II.
[0060] The term "d-DSPE-CPL-M" is encompassed by the term "CPL1"
which refers to a DSPE-CPL having one positive charge. The "d-" in
d-DSPE-CPL-M indicates that the CPL contains a fluorescent dansyl
group. It will be apparent to those of skill in the art that a CPL
can be synthesized without the dansyl moiety, and thus the term
"DSPE-CPL-M" is encompassed by in the term "CPL1" as defined
above.
[0061] The term "d-DSPE-CPL-D" is encompassed by the term "CPL2"
which refers to DSPE-CPL having two positive charges.
[0062] The term "d-DSPE-CPL-T1" is encompassed by the term "CPL3"
which refers to DSPE-CPL having three positive charges.
[0063] The term "d-DSPE-CPL-Q1" is encompassed by the term "CPL4a"
which refers to DSPE-CPL having four positive charges.
[0064] The term "d-DSPE-CPL-Q5," or, alternatively, DSPE-PEGQuad5,
or, alternatively, DSPE-CPL-4, are all encompassed by the term
"CPL4 (or CPL4b)" which refer to a DSPE-CPL having four positive
charges. By modifying the headgroup region, CPLs were synthesized
which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T), and 4
(quad, or Q) positive charges. Various Quad CPLs were synthesized,
hence these are numbered Q1 through Q5.
[0065] The abbreviations "HBS" refers to Hepes-buffered saline,
"Rho-PE" refers to rhodamine-phosphatidylethanolamine, and "LUVs"
refers to "large unilamellar vesicles."
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 illustrates a structural design of a cationic-polymer
lipid (CPL) conjugate.
[0067] FIG. 2 illustrates a synthetic scheme for the preparation of
cationic-PEG-lipid conjugates having varying amount of charged head
groups (a.) Et.sub.3N/CHCl.sub.3; (b.) TFA /CHCl.sub.3; c.
Et.sub.3N/CHCl.sub.3 N.alpha., N.epsilon.-di-t-Boc-L-Lysine
N-hydroxysuccinide ester.
[0068] FIG. 3 illustrates a CPL incorporated liposome. The large
unilamellar vesicles (LUV) have incorporated different examples of
CPLs (CPL1, CPL2, CPL4, and CPL8, respectively).
[0069] FIG. 4 illustrates a distribution of DSPE-CPL-4 between the
inner/outer leaflets of a liposomal membrane. CPL-4-LUVs
(DSPC/Chol/DSPE-CPL-4, 55:40:5 mole %) were prepared by extrusion
method as described herein. The distribution of outer leaflet CPLs
was quantified by a fluorescamine assay. For the outer leaflet
CPLs, the following assay was used. An appropriate amount of
CPL-4-LUVs was diluted with 1 M Borate buffer (pH 8.5) and cooled
in ice-water. 20 .mu.l of 10% Triton X-100 was added to the above
sample solution to solubilize the membrane, and then an additional
20 .mu.l of a cooled fluorescamine ethanol solution (10 mg/ml) was
added and then measured.
[0070] FIG. 5 illustrates a cellular uptake study of CPL-4 LUVs in
BHK cells in PBS-CMG. The controls were LUVs (DSPC/Chol, 60:40) and
CPL-4-LUVs (DSPC/Ch/DSPE-CPL-4, 55:40:5) were prepared by extrusion
as described herein.
[0071] FIG. 6 illustrates cellular uptake of CPL-4-LUVs in BHK
cells in DMEM (with 10% FBS). The control used LUVs (DSPC/Chol,
60:40). CPL-4-LUVs (DSPC/Ch/DSPE-CPL-4, 55:40:5), which were
prepared by extrusion as described herein.
[0072] FIG. 7 illustrates a cellular uptake of CPL-liposomes in BHK
cells in PBS-CMG after 4 hr incubation. LUVs (DSPC/Chol, 60:40) and
CPL-LUVs (DSPC/Chol/DSPE-CPL, 55:40:5) were prepared by extrusion
as described herein.
[0073] FIG. 8 illustrates the preparation of CPL-LUVs by detergent
dialysis. Lipids were codissolved in chloroform at the indicated
ratios, following which the solvent was removed by nitrogen gas and
high vacuum. The lipid mixture was dissolved in detergent/buffer
(OGP in HBS) and dialysed against HBS for 2-3 days. The LUVs, which
formed during dialysis, were then fractionated as shown on
Sepharose CL-4B. Panel A: Fractionation of
DOPE/DODAC/CPL4[3.4K]/PEGCerC20/Rho-PE (79.5/6/4/10/0.5); Panel B:
Fractionation of DOPE/DODAC/CPL4[1K]/PEGCerC2- 0/Rho-PE
(79.5/6/4/10/0.5); and Panel C: Fractionation of
DOPE/DODAC/CPL4[3.4K]/PEGCerC20/Rho-PE (71.5/6/12/10/0.5).
[0074] FIG. 9 Panel A illustrates the insertion of DSPE-CPL-Q5 into
DOPC LUVs (100 mm). DOPC LUVs (2.5 .mu.mol lipid) were incubated
with 0.214 mmol DSPE-CPL-Q5 (total volume 300 .mu.L) at 60.degree.
C. for 3 hours, following which the sample was applied to a column
of Sepharose CL-4B equilibrated in HEPES-buffered saline. 1 mL
fractions were collected and assayed for dansyl-labelled CPL and
rhodamine-PE as described herein. Panel B illustrates the insertion
of DSPE-CPL-Q5 into LUVs (100 nm) composed of
DOPE/DODAC/PEG-Cer-C20 (84/6/10). LUVs (5 .mu.mol lipid) were
incubated with 0.43 .mu.mol DSPE-CPL-Q5 (total volume 519 .mu.l) at
60.degree. C. for 3 hours, following which the sample was applied
to a column of Sepharose CL-4B equilibrated in HEPES-buffered
saline. The elution of free CPL is also shown, demonstrating a
straightforward method for isolation of the CPL-LUV. 1 mL fractions
were collected and assayed for dansyl-labelled CPL and rhodamine-PE
as described herein. Panel C illustrates retention of DSPE-CPL-Q5
in LUVs (100 nm) composed of DOPE/DODAC/PEG-Cer-C20 (84/6/10). The
main LUV fraction from FIG. 9 Panel B was re-applied to a column of
Sepharose CL-4B equilibrated in HEPES-buffered saline. 1 mL
fractions were collected and assayed for dansyl-labelled CPL and
rhodamine-PE as described.
[0075] FIG. 10 illustrates the effect of time and temperature on
the insertion of d-DSPE-CPL-Q1 into DOPE/DODAC/PEG-Cer-C20 LUVs.
For each of the 3 temperatures, 3 .mu.mol lipid was combined with
0.17 .mu.mol CPL (total volume 240 .mu.l). At 1, 3, and 6 hours, 1
.mu.mol of lipid was withdrawn and cooled in ice to halt insertion
of CPL. The samples were passed down a column of Sepharose CL-4B to
remove excess CPL, and assayed for CPL insertion.
[0076] FIG. 11 illustrates the effect of initial CPL/lipid ratio on
final CPL insertion levels. Initial CPL/lipid molar ratios were
0.011, 0.024, 0.047, 0.071, 0.095, and 0.14. Final mol % inserted
were 0.8, 1.8, 3.4, 5.0, 6.5, and 7.0. Right-hand axis is
%-insertion.
[0077] FIG. 12 illustrates the insertion of DSPE-CPL-Q1 and
DSPE-CPL-Q5 into neutral vesicles. The initial CPL/lipid molar
ratio was 0.065 for Q1 (2.5 .mu.mol lipid and 0.21 .mu.mol CPL) and
0.034 for Q5. Samples were incubated at 60.degree. C. for 3 hours.
The DOPC and DOPC/Chol LUVs were prepared by extrusion, while the
others were prepared by detergent dialysis. As described herein,
the presence of 4% methanol in the Q5 samples appear to account for
the higher insertion observed for this sample. Sample compositions
were as follows: DOPC/Chol (55/45), DOPC/PEG-Cer-C20 (90/10),
DOPC/Chol/PEG-Cer-C20 (45/45/10).
[0078] FIG. 13 illustrates the effect of chain length of PEG-Cer on
mol-% CPL inserted. LUVs composed of DOPE/DODAC/PEG-Cer-C20
(84/6/10), DOPE/DODAC/PEG-Cer-C14 (84/6/10), and
DOPE/DODAC/PEG-Cer-C8 (79/6/15) were incubated in the presence of
between 2-8.6 mol % d-DSPE-CPL-Q1 at 60.degree. C. for 3 hrs.
[0079] FIG. 14 illustrates the effect of PEG-Cer-C20 content on
insertion of d-DSPE-CPL-Q5. Vesicles composed of
DOPC/DODAC/PEGCerC20, with the latter lipid ranging from 4-10 mol
%, were incubated in the presence of CPL-Q5 (initial CPL/lipid
molar ratio=0.071).
[0080] FIG. 15 illustrates the uptake of CPL-LUVs incubated in
PBS/CMG on BHK cells. Approximately 105 BHK cells were incubated
with 20 nmol of DOPE/DODAC/PEGCerC20 (84/6/10) LUVs containing (1)
no CPL, (2) 8% DSPE-CPL-D, (3) 7% DSPE-CPL-T 1, and (4) 4%
DSPE-CPL-Q5. Incubations were performed at 4.degree. C. and
37.degree. C., the former giving an estimate of cell binding, and
the latter of binding and uptake. By taking the difference of the
two values, an estimate of lipid uptake at 37.degree. C. was
obtained.
[0081] FIG. 16 Panel A illustrates a structure of the CPL.sub.4.
Panel B illustrates a protocol for the insertion of CPL.sub.4 into
the SPLP system.
[0082] FIG. 17 Panel A illustrates a model for
DOPE/DODAC/PEG-Cer-C20 LUVs, i.e., a standard liposome containing a
PEG-lipid (or "stealth" lipid); Panel B illustrates the same LUVs
with CPL.sub.4 (i.e. long chain) inserted. "Long chain" refers to
the polymer W being the same length or greater length than the
polymer component of the PEG-lipid. Thus, the charged group of the
CPL1 is immediately exposed to the outside environment; and Panel C
illustrates the same LUVs with CPL.sub.4 with a short chain
inserted. A "short chain" CPL, wherein polymer W is shorter than
the corresponding polymer of the PEG-lipid.
[0083] FIG. 18 Panel A illustrates a time-course for the uptake of
SPLP system (.circle-solid.) compared to DOPE:DODAC (1:1) liposomes
complexed to pLuc (.box-solid.) on BHK cells. Lipid concentration
was 20 .mu.M. Panel B illustrates transfection efficiencies of 1.5
.mu.g/mL pLuc obtained using the SPLP system compared to those
obtained using complexes after 4 hour (.box-solid.) or 8 hour
(.box-solid.) incubations.
[0084] FIG. 19 illustrates a column profile, following insertion of
3.5 mol %.sub.initial (3 mol %.sub.final) CPL.sub.4 into SPLP, for
the separation of CPL-SPLP from free CPL. Profiles for lipid
(.circle-solid.), CPL (.quadrature.), and DNA (.diamond.) with
respect to the total amount applied to a Sepharose CL-4B column are
shown. Panel B shows the column profile for Fraction #9 from Panel
A.
[0085] FIG. 20 illustrates a time course for the insertion of
CPL.sub.4 (15 nmol) into SPLP (200 mmol).
[0086] FIG. 21 illustrates a time course for the uptake of 20 .mu.M
of SPLP possessing 0% (.box-solid.), 3% (.diamond.), or 4%
(.circle-solid.) CPL.sub.4 in BHK cells.
[0087] FIG. 22 illustrates transfection of BHK cells by SPLP (2.5
.mu.g/mL pLuc) following insertion of various mol % of the
CPL.sub.4 compared to SPLP alone (0% CPL). Transfections were
carried out by incubating the samples on top of the cells for 4 or
9 hours and replacing with complete media for a complete 24 hours
incubation (see also FIG. 33).
[0088] FIG. 23 tabulates CPL insertion results.
[0089] FIG. 24 also tabulates CPL insertion results.
[0090] FIG. 25 illustrates the post-insertion method for
preparation of CPL-containing liposomes. The preformed liposomes
were made of DSPC/Chol (55:45, mol:mol). The CPL was incubated with
the preformed liposomes at 60.degree. C. for 2 hour. Panel A
illustrates separation of free CPL and CPL-LUVs by gel filtration
after post-insertion. Panel B illustrates elution of fraction 10
(Panel A) on a Sepharose CL-4B column.
[0091] FIG. 26 illustrates cellular uptake of the stealth liposomes
containing DSPE-CPLs in BHK cells in DMEM (10% FBS). Control LUVs
(DSPC/Chol/PEG-PE, 56:40:4) and CPL-LUVs (DSPC/Chol/PEG-PE/CPL,
55.5:40:2:2) were prepared by extrusion as described herein.
[0092] FIG. 27 illustrates cellular uptake of stealth liposomes
containing DSPE-CPLs in BHK cells in PBS-CMG. Control LUVs
(DSPC/Chol/PEG-PE, 56:40:4) and CPL-LUVs (DSPC/Chol/PEG-PE/CPL,
55.5:40:2:2) were prepared by extrusion as described herein.
[0093] FIG. 28 Panel A: Chemical structures of various CPLs; Panel
B: Chemical structures of various CPLs. Note that CPL.sub.4 (Panel
A) is identical to CPL.sub.4b (Panel B); and Panel C: Chemical
structures of various CPLs.
[0094] FIG. 29 illustrates a synthetic embodiment to generate
compounds of the present invention.
[0095] FIG. 30 illustrates a structure of dansylated CPL.sub.4.
CPL.sub.4 possesses four positive charges at the end of a
PEG.sub.3400 molecule which is attached to a DSPE molecule. The
CPL.sub.4 is dansylated by incorporation of a dansylated
lysine.
[0096] FIG. 31 illustrates an effect of cation concentration on the
deaggregation of SPLP-CPL.sub.4. The mean diameter and standard
deviation of the particles in the presence of increasing [Cation],
Ca.sup.2+ (.circle-solid.) and Mg.sup.2+ (.box-solid.), from 0 mM
to 70 mM, was measured using quasi-elastic light scattering (QELS).
To .about.180 nmol of SPLP-CPL.sub.4 in 400 mL in a Nicomp tube was
added small quantities of either CaCl.sub.2 or MgCl.sub.2 (500 mM
stock solutions). Measurement of the mean diameter.+-.standard
deviation of the particles in the presence of differing amounts of
the cation were made using a Nicomp Model 270 Submicron Particle
Sizer. The diameters of the particles do not dramatically change,
however, the Gaussian distributions do get broader. Thus, the
standard deviations were used as a measure of deaggregation with
smaller deviations indicating less aggregation.
[0097] FIG. 32 illustrates uptake of SPLP containing various
percentages of CPL.sub.4. Panel A. Time course for the uptake of 20
.mu.M SPLP possessing 0 mol % (.circle-solid.), 2 mol %
(.box-solid.), 3 mol % (.tangle-solidup.), or 4 mol %
(.diamond-solid.) CPL.sub.4 and DOPE:DODAC complexes
(.tangle-soliddn.) by BHK cells. The insertion of the CPL.sub.4
into SPLP and the preparation of complexes was performed as
described herein The mol % of CPL.sub.4 in the SPLP-CPL.sub.4 was
also determined, as described herein. BHK cells were plated in
12-well plates at 1.times.10.sup.5 cells/well. To 200 .mu.L of
sample (containing SPLP-CPL.sub.4 or complex+CaCl.sub.2) was added
800 .mu.L of DMEM+10% FBS. The resulting CaCl.sub.2 concentration
was diluted to 20% of the original. Following incubation periods of
2, 4, 6 and 8 hours, the cells were lysed with 600 mL of lysis
buffer and the rhodamine fluorescence and BCA assays were measured
for the lysate, as described herein (see FIG. 21).
[0098] FIG. 33 illustrates tansfection of BHK cells by SPLP (5.0
.mu.g/mL pLuc) following insertion of various mole percentages of
CPL.sub.4 (2, 3, and 4 mol %). The CPL.sub.4 was inserted into
SPLPs using the procedure described herein. As a comparison, SPLP
(0 mol % CPL) and DOPE:DODAC (1:1) complex transfections were also
performed. BHK cells were plated at 1.times.10.sup.4 in 96-well
plates. Transfections were carried out by incubating the samples
[20 .mu.L (SPLP-CPL.sub.4+CaCl.sub.2)+80 .mu.L of complete media]
on the cells for 4 hours followed by a 24 hour complete incubation.
The CaCl.sub.2 concentration again is diluted to 20% of the
original concentration. Following the 24 hour incubation, the cells
were lysed with lysing buffer and the luciferase and BCA assays
were performed (see FIG. 22).
[0099] FIG. 34 illustrates the effect of [Cation], Ca.sup.2+
(.circle-solid.) and Mg.sup.2+ (.box-solid.), on the transfection
of SPLP-CLP.sub.4 (5.0 .mu.g/mL pLuc) on BHK cells.
SPLP-CPL.sub.4+CaCl.sub.- 2 or MgCl.sub.2 was mixed with DMEM+10%
FBS and the mixtures were applied to 1.times.10.sup.4 BHK cells
plated in a 96-well plate. Following a complete 48 hour incubation,
the transfection media was removed and the cells were lysed with
lysing buffer and the luciferase activity and protein content were
measured as described earlier.
[0100] FIG. 35 illustrates the effect of [Cation], Ca.sup.2+
(.circle-solid.) and Mg.sup.2+ (.box-solid.), on the lipid binding
and uptake of 80 .mu.M SPLP-CPL.sub.4 on BHK cells. The samples
possessing varying concentrations of the cation (0-14 mM final
concentration) were incubated on 1.times.10.sup.5 BHK cells for 4
hours at which time the cells were lysed and the rhodamine
fluorescence and protein content were measured.
[0101] FIG. 36 illustrates transfection of SPLP-CPL.sub.4, SPLP and
complexes (each containing 5.0 .mu.g/mL pCMVLuc) at longer time
points. Transfection of SPLP-CPL.sub.4 (4 mol % CPL.sub.4)+40
mM.sub.initial CaCl.sub.2 (.circle-solid.), SPLP
(.tangle-soliddn.), DOPE:DODAC complexes (.box-solid.), and
Lipofectin complexes (.diamond-solid.) was performed on
1.times.10.sup.4 BHK cells. The transfection media was incubated on
the cells for 4, 8 or 24 hours, after which the transfection media
was replaced by complete media for the 4 and 8 hour timepoints.
Then at a total incubation time of 24 hours (20, 16, and 0 hours,
respectively, after removal of the transfection media), the cells
were lysed and the luciferase activity and protein content were
measured.
[0102] FIG. 37 illustrates transfection potency and toxicity of
SPLP-CPL.sub.4 compared to Lipofectin complexes. A. Transfection
activity for SPLP-CPL.sub.4+CaCl.sub.2 (.circle-solid.), SPLP
(.box-solid.), and Lipofectin (.diamond-solid.) on 1.times.10.sup.4
BHK cells incubated for 24 and 48 hours followed by immediate cell
lysis, and measurement of luciferase activity and protein content.
B. Measurement of the cellular survival following 24 and 48 hour
incubations of the SPLP-CPL.sub.4+CaCl.sub.2 (.circle-solid.),
Lipofectin (.diamond-solid.), and DOPE/DODAC (1:1) complexes on
1.times.10.sup.4 BHK cells. Following incubation, the cells were
lysed and the protein content from the BCA assay was used as a
measure of protein survival.
[0103] FIG. 38 illustrates the transfection of BHK cells using both
long and short chained CPLs. The presence of the short chained PEG
in the CPL results in a decrease by a factor of about 4 compared to
the transfection by the long chained CPL.
[0104] FIG. 39 illustrates the transfection of Neuro-2a cells.
SPLP+4 mol % CPL4-1k produces 4 orders of magnitude of gene
expression more than SPLP alone in Neuro-2a cells.
[0105] FIG. 40 illustrates in vivo pharmacokinetics of SPLP
containing a short chain CPL.sub.4.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0106] A. Compounds and Synthesis
[0107] In certain aspects, the present invention provides
cationic-polymer-lipid conjugates (CPLs), such as distal
cationic-poly(ethylene glycol)-lipid conjugates that can be
incorporated into conventional and stealth liposomes for enhancing,
inter alia, cellular uptake. The CPLs of the present invention have
the following architectural features: (1) a lipid anchor, such as a
hydrophobic lipid, for incorporating the CPLs into the lipid
bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol,
for linking the lipid anchor to a cationic head group; and (3) a
polycationic moiety, such as a naturally occurring amino acid, to
produce a protonizable cationic head group. As such, the present
invention provides a compound of Formula I:
A W-Y I
[0108] wherein A, W and Y are as previously defined.
[0109] With reference to Formula I, "A" is a lipid moiety such as
an amphipathic lipid, a neutral lipid or a hydrophobic lipid that
acts as a lipid anchor. Suitable lipid examples include
vesicle-forming lipids or vesicle adopting lipids and include, but
are not limited to, diacylglycerolyls, dialkylglycerolyls,
N-N-dialkylaminos, 1,2-diacyloxy-3-aminopropanes and
1,2-dialkyl-3-aminopropanes.
[0110] "W" is a polymer or an oligomer, such as a hydrophilic
polymer or oligomer. Preferably, the hydrophilic polymer is a
biocompatable polymer that is non-immunogenic or possesses low
inherent immunogenicity. Alternatively, the hydrophilic polymer can
be weakly antigenic if used with appropriate adjuvants. Suitable
non-immunogenic polymers include, but are not limited to, PEG,
polyamides, polylactic acid, polyglycolic acid, polylactic
acid/polyglycolic acid copolymers and combinations thereof. In a
preferred embodiment, the polymer has a molecular weight of about
250 to about 7000 daltons.
[0111] "Y" is a polycationic moiety. The term polycationic moiety
refers to a compound, derivative, or functional group having a
positive charge, preferably at least 2 positive charges at a
selected pH, preferably physiological pH. Suitable polycationic
moieties include basic amino acids and their derivatives such as
arginine, asparagine, glutamine, lysine and histidine; spermine;
spermidine; cationic dendrimers; polyamines; polyamine sugars; and
amino polysaccharides. The polycationic moieties can be linear,
such as linear tetralysine, branched or dendrimeric in structure.
Polycationic moieties have between about 2 to about 15 positive
charges, preferably between about 2 to about 12 positive charges,
and more preferably between about 2 to about 8 positive charges at
selected pH values. The selection of which polycationic moiety to
employ may be determined by the type of liposome application which
is desired.
[0112] The charges on the polycationic moieties can be either
distributed around the entire liposome moiety, or alternatively,
they can be a discrete concentration of charge density in one
particular area of the liposome moiety e.g., a charge spike. If the
charge density is distributed on the liposome, the charge density
can be equally distributed or unequally distributed. All variations
of charge distribution of the polycationic moiety are encompassed
by the present invention.
[0113] The lipid "A", and the non-immunogenic polymer "W", can be
attached by various methods and preferably, by covalent attachment.
Methods known to those of skill in the art can be used for the
covalent attachment of "A" and "W". Suitable linkages include, but
are not limited to, amide, amine, carboxyl, carbonate, carbamate,
ester and hydrazone linkages. It will be apparent to those skilled
in the art that "A" and "W" must have complementary functional
groups to effectuate the linkage. The reaction of these two groups,
one on the lipid and the other on the polymer, will provide the
desired linkage. For example, when the lipid is a diacylglycerol
and the terminal hydroxyl is activated, for instance with NHS and
DCC, to form an active ester, and is then reacted with a polymer
which contains an amino group, such as with a polyamide (see, U.S.
patent application Ser. No. 09/218,988, filed Dec. 22, 1998), an
amide bond will form between the two groups.
[0114] In certain embodiments, "W" is bound, preferably covalently
bound, to "Y". As with "A" and "W", a covalent attachment of "W" to
"Y" can be generated by complementary reactivity of functional
groups, one on the polymer and the other on the polycationic
moiety. For example, an amine functional group on "W" can be
reacted with an activated carboxyl group, such as an acyl chloride
or NHS ester, to form an amide. By suitable choice of reactive
groups, the desired coupling can be obtained. Other activated
carboxyl groups include, but are not limited to, a carboxylic acid,
a carboxylate ester, a carboxylic acid halide and other activated
forms of carboxylic acids, such as a reactive anhydride. Reactive
acid halides include for example, acid chlorides, acid bromides,
and acid fluorides.
[0115] In certain instances, the polycationic moiety can have a
ligand attached, such as a targeting ligand. Preferably, after the
ligand is attached, the cationic moiety maintains a positive
charge. In certain instances, the ligand that is attached has a
positive charge. Suitable ligands include, but are not limited to,
a compound or device with a reactive functional group and includes
lipids, amphipathic lipids, carrier compounds, bioaffinity
compounds, biomaterials, biopolymers, biomedical devices,
analytically detectable compounds, therapeutically active
compounds, enzymes, peptides, proteins, antibodies, immune
stimulators, radiolabels, fluorogens, biotin, drugs, haptens, DNA,
RNA, polysaccharides, liposomes, virosomes, micelles,
immunoglobulins, functional groups, other targeting moieties, or
toxins.
[0116] In certain preferred embodiments, other moieties are
incorporated into the compounds of Formula I to form the compounds
of Formula II: 8
[0117] In Formula II, "A" is a lipid moiety such as, an amphipathic
lipid, a neutral lipid or a hydrophobic lipid moiety. Suitable
lipid examples include, but are not limited to, diacylglycerolyl,
dialkylglycerolyl, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane
and 1,2-dialkyl-3-aminopropane.
[0118] In Formula II, "X" is a single bond or a functional group
that covalently attaches the lipid to at least one ethylene oxide
unit. Suitable functional groups include, but are not limited to,
phosphatidylethanolamino, phosphatidylethanolamido, phosphoro,
phospho, phosphoethanolamino, phosphoethanolamido, carbonyl,
carbamate, carboxyl, carbonate, amido, thioamido, oxygen, NR
wherein R is a hydrogen or alkyl group and sulfur. In certain
instances, the lipid "A" is directly attached to the ethylene oxide
unit by a single bond. The number of ethylene oxide units can range
from about 1 to about 160 and preferably from about 6 to about
50.
[0119] In Formula II, "Z" is a single bound or a functional group
that covalently attaches the ethylene oxide unit to the
polycationic moiety. Suitable functional groups include, but are
not limited to, phospho, phosphoethanolamino, phosphoethanolamido,
carbonyl, carbamate, carboxyl, amido, thioamido, NR wherein R is a
member selected from the group consisting of hydrogen atom or alkyl
group. In certain embodiments, the terminal ethylene oxide unit is
directly attached to the polycationic moiety.
[0120] In Formula II, "Y" is a polycationic moiety as described
above in connection with Formula I. In Formula II, the index "n" is
an integer ranging in value from about 6 to about 160.
[0121] In an illustrative embodiment, compounds of Formula II can
be synthesized using a generalized procedure as outlined in FIG. 2.
FIG. 2 illustrates one particular embodiment of the present
invention and thus, is merely an example that should not limit the
scope of the claims herein. Clearly, one of ordinary skill in the
art will recognize many other variations, alternatives, and
modifications that can be made to the reaction scheme illustrated
in FIG. 2. With reference to FIG. 2, a solution of a lipid, such as
DSPE, and a base, such as triethylamine in a chloroform solution is
added to (t-Boc-NH-PEG.sub.3400-CO.sub.2NHS), and the solution is
stirred at ambient temperature. The solution is then concentrated
under a nitrogen stream to dryness. The residue is then purified by
repeated precipitation of the chloroform mixture solution with
diethyl ether until disappearance of the lipid using
chromatography. The purified CPL conjugate is dissolved in a
solvent, followed by addition of TFA, and the solution is stirred
at room temperature. The solution can again be concentrated under a
nitrogen stream. The residue is then purified by repeated
precipitation of the mixture with diethyl ether to offer a
lipid-PEG-NH.sub.2, such as a DSPE-PEG-NH.sub.2 or, alternatively,
DSPE-CPL-1 with one protonizable cationic head group. The ratio of
the phosphoryl-lipid anchor and the distal primary amine can then
be measured by phosphate and flourescamine assays as described
herein.
[0122] In this illustrative embodiment, the number of protonizable
amino groups can be increased to create a polycationic moiety. By
incrementally adding stoichiometric amounts of, for example, a
N.alpha.,N.epsilon.-di-t- -Boc-L-Lysine N-hydroxysuccinide ester,
the polycationic moiety can be increase from about 2 to about 16
positive charges. As describe previously, the positive charges can
be incorporated using any number of suitable polycationic moieties
such as lysine, arginine, asparagine, glutamine, histidine,
polyamines and derivatives or combinations thereof. Using the
synthesis methods of the present invention, the number of cationic
groups, such as amino groups, can be readily controlled during the
CPL synthesis.
[0123] B. Lipid-Based Drug Formulations
[0124] In certain aspects, the present invention provides a
lipid-based drug formulation comprising:
[0125] (a) a compound having the general structure of Formula
I:
A-W-Y I
[0126] wherein A, W and Y are as previously defined; (b) a
bioactive agent; and optionally, (c) a second lipid. In preferred
embodiments, the lipid-based drug formulation of the present
invention comprises the second lipid, such as a PEG-lipid
derivative.
[0127] In certain preferred embodiments, the lipid-based drug
formulations of the present invention comprise
[0128] (a) a compound of Formula II: 9
[0129] wherein A, X, Z, Y and n have been previously defined; (b) a
bioactive agent; and optionally, (c) a second lipid. In preferred
embodiments, the lipid-based drug formulation of the present
invention comprises the second lipid, such as a PEG-lipid
derivative.
[0130] After the CPLs have been prepared, they can be utilized in a
variety of ways including, for example, in lipid-based drug
formulations. In this aspect, the lipid-based formulations can be
in the form of a liposome, a micelle, a virosome, a lipid-nucleic
acid particle, a nucleic acid aggregate and other forms which can
incorporate or entrap one or more bioactive agents. In certain
aspects, the lipid-based drug formulations of the present invention
comprise a second lipid.
[0131] The compounds of Formulae I and II can be used in
lipid-based formulations such as those described in for example,
the following copending U.S. patent application Ser. Nos.
08/454,641, 08/485,458, 08/660,025, 08/484,282, 60/055,094,
08/856,374, 60/053,813 and 60/063,473, entitled "Methods for
Encapsulating Nucleic Acids in Lipid Bilayers," filed on Oct. 10,
1997 and bearing Attorney Docket No. 016303-004800, U.S. Pat. No.
5,703,055, U.S. patent application Ser. No. 09/218,988, filed Dec.
22, 1998, the teachings all of which are incorporated herein by
reference in their entirety for all purposes. This specification
sets out a variety of liposome types and a variety of methods for
incorporating CPLs into liposomes, all of which are examples of the
broad methods and compositions claimed herein.
[0132] The lipid components and CPLs used in forming the various
lipid-based drug formulations will depend, in part, on the type of
delivery system employed. For instance, if a liposome is employed,
the lipids used in the CPL will generally be selected from a
variety of vesicle-forming or vesicle-adopting lipids, typically
including phospholipids and sterols, such as
phosphatidylenthanolamine (PE), phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic
acid (PA), which have been suitably functionalized, and the like.
In contrast, if a micelle is employed, the lipids used in the CPL
will generally be selected from sterylamines, alkylamines,
C.sub.8-C.sub.22 alkanoic acids, lysophospholipids, detergents and
the like. It will be readily apparent to those of skill in the art
that the acyl chains can be varied in length and can be saturated
or possess varying degrees of unsaturation. The more saturated the
acyl chains the more rigid the membrane. Higher degrees of
unsaturation impart more fluidity into the vesicle's membrane.
Similarly, the other lipid components (e.g., lipids, cationic
lipids, neutral lipids, non-cationic lipids, etc.) making up the
drug delivery systems of the present invention will vary depending
on the drug delivery system employed. Suitable lipids for the
various drug delivery systems will be readily apparent to those of
skill in the art.
[0133] When the lipid-based drug formulations are used to deliver
therapeutic genes or oligonucleotides intended to induce or to
block production of some protein within the cell, cationic lipids
can be included in the formulation, e.g., liposome, micelle,
lipid-nucleic acid particle, etc. Nucleic acid is negatively
charged and can be combined with a positively charged entity to
form a lipid complex suitable for formulation and cellular
delivery.
[0134] As used in this specification, "cationic lipid" generally
refers to a lipid with a cationic head group situated at or near
the liposome membrane (when incorporated in a liposome). CPLs are
distinguished from cationic lipids by the polymer "W" which in
certain instances, has the effect of placing the cationic charge at
a significant distance from the membrane.
[0135] Examples of suitable cationic lipids include, but are not
limited to, the following: DC-Chol, (see, Gao, et al., Biochem.
Biophys. Res. Comm., 179:280-285 (1991); DDAB; DMRIE; DODAC (see,
U.S. patent application Ser. No. 08/316,399, filed Sep. 30, 1994,
which is incorporated herein by reference); DOGS; DOSPA; DOTAP; and
DOTMA. In a presently preferred embodiment,
N,N-dioleoyl-N,N-dimethylammonium chloride is used in combination
with a phosphatidylethanolamine.
[0136] In addition, other cationic lipids useful in producing
lipid-based carriers for gene and oligonucleotide delivery are
LIPOFECTIN (U.S. Pat. Nos. 4,897,355; 4,946,787; and 5,208,036
issued to Eppstein, et al.) and LIPOFECTACE (U.S. Pat. No.
5,279,883 issued to Rose). Both agents, as well as other
transfecting cationic lipids, are available from Life Technologies,
Inc. in Gaithersburg, Md.
[0137] In one preferred embodiment, the CPL-liposomes of the
present invention are optimized for systemic delivery applications.
In certain applications, the polymer length in the CPL is shorter
than the normal neutral PEG chains (M.W. 2000-5000 Daltons) used
for stealth liposomes. In this instance, the shorter polymer in the
CPL is about 250 to about 3000 Daltons and more preferably, about
1000 to about 2000 Daltons. In this embodiment, the second lipid is
for example, a PEG.sub.3400-lipid and the compound of Formula I is,
for example, A-PEG.sub.1000-Y. (see, FIG. 17C).
[0138] Without being bound by any particular theory, when the
shorter polymer is used, it is believed that the distal charge(s)
of the CPL is hidden within the normal PEG exclusion barrier, thus
allowing retention of long circulation lifetimes while at the same
time, extending the positive charges away from the liposomal
surface. This embodiment enhances interactions between liposomes
and a target cell. The use of different sized polymers, such as PEG
chains, in the CPLs and the neutral PEG-lipids used to modulate
vesicle circulation and cellular uptake, allows for a new
generation of stealth liposomes as drug carriers. It is believed
that the optimized polymer length can vary with the specific
conditions such as in vitro or in vivo applications, local or
systemic administration, and different lipid formulations.
[0139] In another embodiment, the polymer length in the CPL has a
larger MW than the normal neutral PEG chains used for stealth
liposomes. In this instance, the second lipid is for example, a
PEG.sub.1000-lipid and the compound of Formula I has a formula of,
for example, A-PEG.sub.3400-Y. (see, FIG. 17B).
[0140] In certain formulations and applications, the type of CPL
i.e. the length of the polymer chain, and the amount of cationic
charge per molecule, and the amount of such CPL in a formulation
e.g., SPLP, can be optimized to obtain the best balancing of
clearance properties. In certain instances, long chain CPLs and
higher levels of such CPLs are to be preferred to increase
transfection. In other instances, short chain CPLs incorporated in
the formulations are optimized for longer circulation lifetimes in
animals.
[0141] In one embodiment of the present invention, a fusogenic
liposome or virosome is provided. It will be readily apparent to
those of skill in the art that the CPLs of the present invention
can advantageously be incorporated into various types of fusogenic
liposomes and virosomes. Such fusogenic liposomes and virosomes can
be designed to become fusogenic at the disease or target site.
Those of skill in the art will readily appreciate that a number of
variables can be used to control when the liposome or virosome
becomes fusogenic. Such variables include, for example, the
composition of the liposome or virosome, pH, temperature, enzymes,
cofactors, ions, etc.
[0142] In one embodiment, the fusogenic liposome comprises: a lipid
capable of adopting a non-lamellar phase, yet capable of assuming a
bilayer structure in the presence of a bilayer-stabilizing
component (such as a PEG-lipid derivative); and a
bilayer-stabilizing component reversibly associated with the lipid
to stabilize the lipid in a bilayer structure. Such fusogenic
liposomes are advantageous because the rate at which they become
fusogenic can be not only predetermined, but varied as required
over a time scale of a few minutes to several tens of hours. It has
been found, for example, that by controlling the composition and
concentration of the bilayer-stabilizing component, one can control
the rate at which the BSC exchanges out of the liposome in vivo
and, in turn, the rate at which the liposome becomes fusogenic
(see, U.S. Pat. No. 5,885,613). For instance, it has been found
that by controlling the length of the lipid acyl chain(s), one can
control the rate at which the BSC exchanges out of the liposome in
vivo and, in turn, the rate at which the liposome becomes
fusogenic. In particular, it has been discovered that shorter acyl
chains (e.g., C-8) exchange out of the liposome more rapidly than
longer acyl chains (e.g., C-20). Alternatively, by controlling the
composition and concentration of the BSC, one can control the rate
at which the BSC is degraded, i.e., broken down, by endogenous
systems, e.g., endogenous enzymes in the serum, and, in turn, the
rate at which the liposome becomes fusogenic.
[0143] The polymorphic behavior of lipids in organized assemblies
can be explained qualitatively in terms of the dynamic molecular
shape concept (see, Cullis, et al., in "Membrane Fusion" (Wilschut,
J. and D. Hoekstra (eds.), Marcel Dekker, Inc., New York, (1991)).
When the effective cross-sectional areas of the polar head group
and the hydrophobic region buried within the membrane are similar
then the lipids have a cylindrical shape and tend to adopt a
bilayer conformation. Cone-shaped lipids which have polar head
groups that are small relative to the hydrophobic component, such
as unsaturated phosphatidylethanolamines, prefer non-bilayer phases
such as inverted micelles or inverse hexagonal phase (H ). Lipids
with head groups that are large relative to their hydrophobic
domain, such as lysophospholipids, have an inverted cone shape and
tend to form micelles in aqueous solution. The phase preference of
a mixed lipid system depends, therefore, on the contributions of
all the components to the net dynamic molecular shape. As such, a
combination of cone-shaped and inverted cone-shaped lipids can
adopt a bilayer conformation under conditions where either lipid in
isolation cannot (see, Madden and Cullis, Biochim. Biophys. Acta,
684:149-153 (1982)).
[0144] A more formalized model is based on the intrinsic curvature
hypothesis (see, e.g., Kirk, et al., Biochemistry, 23:1093-1102
(1984)). This model explains phospholipid polymorphism in terms of
two opposing forces. The natural tendency of a lipid monolayer to
curl and adopt its intrinsic or equilibrium radius of curvature
(R.sub.O) which results in an elastically relaxed monolayer is
opposed by the hydrocarbon packing constraints that result. Factors
that decrease the intrinsic radius of curvature, such as increased
volume occupied by the hydrocarbon chains when double bonds are
introduced, tend to promote H phase formation. Conversely, an
increase in the size of the headgroup increases R.sub.O and
promotes bilayer formation or stabilization. Introduction of apolar
lipids that can fill the voids between inverted lipid cylinders
also promotes H phase formation (see, Gruner, et al., Proc. Natl.
Acad. Sci. USA, 82:3665-3669 (1989); Sjoland, et al., Biochemistry,
28:1323-1329 (1989)).
[0145] As such, in one embodiment, the lipids which can be used to
form the fusogenic liposomes of the present invention are those
which adopt a non-lamellar phase under physiological conditions or
under specific physiological conditions, e.g., in the presence of
calcium ions, but which are capable of assuming a bilayer structure
in the presence of a BSC. Such lipids include, but are not limited
to, phosphatidylenthanolami- nes, ceramides, glycolipids, or
mixtures thereof. Other lipids known to those of skill in the art
to adopt a non-lamellar phase under physiological conditions can
also be used. Moreover, it will be readily apparent to those of
skill in the art that other lipids can be induced to adopt a
non-lamellar phase by various non-physiological changes including,
for example, changes in pH or ion concentration (e.g., in the
presence of calcium ions) and, thus, they can also be used to form
the fusogenic liposomes of the present invention. In a presently
preferred embodiment, the fusogenic liposome is prepared from a
phosphatidylethanolamine. The phosphatidylethanolamine can be
saturated or unsaturated. In a presently preferred embodiment, the
phosphatidylyethanolamine is unsaturated. In an equally preferred
embodiment, the fusogenic liposome is prepared from a mixture of a
phosphatidylethanolamine (saturated or unsaturated) and a
phosphatidylserine. In another equally preferred embodiment, the
fusogenic liposome is prepared from a mixture of a
phosphatidylethanolamine (saturated or unsaturated) and a cationic
lipid.
[0146] In one embodiment, the lipid-based drug formulations of the
present invention comprise a bilayer stabilizing component (BSC).
Suitable BSCs include, but are not limited to, polyamide oligomers,
peptides, proteins, detergents, lipid-derivatives, PEG-lipids such
as PEG coupled to phosphatidylethanolamine, and PEG conjugated to
ceramides (see, U.S. Pat. No. 5,885,613, which is incorporated
herein by reference). Preferably, the bilayer stabilizing component
is a PEG-lipid, or an ATTA-lipid. As discussed herein, in certain
preferred instances, the PEG or the ATTA of the BSC has a greater
molecular weight compared to the polymer "W" of the CPL. In other
instances, the BSC has a smaller molecular weight compared to the
"W" of the polymer. The present invention encompasses all such
variations.
[0147] In accordance with the present invention, lipids adopting a
non-lamellar phase under physiological conditions can be stabilized
in a bilayer structure by BSCs which are either bilayer forming
themselves, or which are of a complementary dynamic shape. The
non-bilayer forming lipid is stabilized in the bilayer structure
only when it is associated with, i.e., in the presence of, the BSC.
In selecting an appropriate BSC, it is preferable that the BSC be
capable of transferring out of the liposome, or of being chemically
modified by endogenous systems such that, with time, it loses its
ability to stabilize the lipid in a bilayer structure. Only when
liposomal stability is lost or decreased can fusion of the liposome
with the plasma membrane of the target cell occur. The BSC-lipid,
therefore, is "reversibly associated" with the lipid and only when
it is associated with the lipid is the lipid constrained to adopt
the bilayer structure under conditions where it would otherwise
adopt a non-lamellar phase. As such, the BSC-lipids of the present
invention are capable of stabilizing the lipid in a bilayer
structure, yet they are capable of exchanging out of the liposome,
or of being chemically modified by endogenous systems so that, with
time, they lose their ability to stabilize the lipid in a bilayer
structure, thereby allowing the liposome to become fusogenic.
[0148] Typically, the CPL is present in the lipid-based formulation
of the present invention at a concentration ranging from about 0.05
mole percent to about 50 mole percent. In a presently preferred
embodiment, the CPL is present at a concentration ranging from 0.05
mole percent to about 25 mole percent. In an even more preferred
embodiment, the CPL is present at a concentration ranging from 0.05
mole percent to about 15 mole percent. One of ordinary skill in the
art will appreciate that the concentration of the CPL can be varied
depending on the CPL employed and the rate at which the liposome is
to become fusogenic.
[0149] In one embodiment of the present invention, the liposomes
contain cholesterol. It has been determined that when
cholesterol-free liposomes are used in vivo, they have a tendency
to absorb cholesterol from the plasma lipoproteins and cell
membranes. Cholesterol, if included, is generally present at a
concentration ranging from 0.2 mole percent to about 50 mole
percent and, more preferably, at a concentration ranging from about
35 mole percent to about 45 mole percent.
[0150] C. Preparation of CPL-Liposomes
[0151] A variety of general methods for making CPL-containing
liposomes (or "CPL-liposomes") are discussed herein.
[0152] Two general techniques include "post-insertion," that is,
insertion of a CPL into for example, a pre-formed liposome vesicle,
and "standard" techniques, wherein the CPL is included in the lipid
mixture during for example, the liposome formation steps. The
post-insertion technique results in liposomes having CPLs mainly in
the external face of the liposome bilayer membrane, whereas
standard techniques provide liposomes having CPLs on both internal
and external faces.
[0153] In particular, "post-insertion" involves forming vesicles
(by any method), and incubating the pre-formed vesicles in the
presence of CPL under appropriate conditions (usually 2-3 hours at
60.degree. C.). Between 60-80% of the CPL can be inserted into the
external leaflet of the recipient vesicle, giving final
concentrations up to 7 mol % (relative to total lipid). The method
is especially useful for vesicles made from phospholipids (which
can contain cholesterol) and also for vesicles containing
PEG-lipids (such as PEG-Ceramide).
[0154] In an example of a "standard" technique, the CPL-LUVs of the
present invention can be formed by extrusion. In this embodiment,
all of the lipids including CPL, are co-dissolved in chloroform,
which is then removed under nitrogen followed by high vacuum. The
lipid mixture is hydrated in an appropriate buffer, and extruded
through two polycarbonate filters with a pore size of 100 nm. The
resulting vesicles contain CPL on both internal and external faces.
In yet another standard technique, the formation of CPL-LUVs can be
accomplished using a detergent dialysis or ethanol dialysis method,
for example, as discussed in U.S. Pat. Nos. 5,976,567 and
5,981,501, both of which are incorporated herein by reference. The
extrusion method and the detergent dialysis method are explained in
detail in the Example section.
[0155] D. Liposome Preparation and Sizing
[0156] A variety of methods are available for preparing and sizing
liposomes as described in, e.g., Szoka, et al., Ann. Rev. Biophys.
Bioeng., 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344,
4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028,
4,946,787, PCT Publication No. WO 91/17424, Deamer and Bangham,
Biochim. Biophys. Acta, 443:629-634 (1976); Fraley, et al., Proc.
Natl. Acad. Sci. USA, 76:3348-3352 (1979); Hope, et al., Biochim.
Biophys. Acta, 812:55-65 (1985); Mayer, et al., Biochim. Biophys.
Acta, 858:161-168 (1986); Williams, et al., Proc. Natl. Acad. Sci.,
85:242-246 (1988), the text Liposomes, Marc J. Ostro, ed., Marcel
Dekker, Inc., New York, 1983, Chapter 1, and Hope, et al., Chem.
Phys. Lip., 40:89 (1986), all of which are incorporated herein by
reference. Suitable methods include, but are not limited to,
sonication, extrusion, high pressure/homogenization,
microfluidization, detergent dialysis, calcium-induced fusion of
small liposome vesicles, and ether-infusion methods, all of which
are well known in the art. One method produces multilamellar
vesicles of heterogeneous sizes. In this method, the
vesicle-forming lipids are dissolved in a suitable organic solvent
or solvent system and dried under vacuum or an inert gas to form a
thin lipid film. If desired, the film may be redissolved in a
suitable solvent, such as tertiary butanol, and then lyophilized to
form a more homogeneous lipid mixture which is in a more easily
hydrated powder-like form. This film is covered with an aqueous
buffered solution and allowed to hydrate, typically over a 15-60
minute period with agitation. The size distribution of the
resulting multilamellar vesicles can be shifted toward smaller
sizes by hydrating the lipids under more vigorous agitation
conditions or by adding solubilizing detergents, such as
deoxycholate.
[0157] Extrusion of liposome through a small-pore polycarbonate
membrane or an asymmetric ceramic membrane is an effective method
for reducing 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. For use in the present invention, liposomes
having a size ranging from about 0.05 microns to about 0.40 microns
are preferred.
[0158] E. Use of Liposomes as Drug Delivery Vehicles
[0159] The lipid-based drug formulations and compositions of the
present invention (e.g., liposomes, micelles, lipid-nucleic acid
particles, virosomes, etc.) are useful for the systemic or local
delivery of bioactive agents such as therapeutic agents,
prophylactic agents and diagnostic agents. Such delivery systems
are described in greater detail in, for example, the following
copending U.S. patent application Ser. Nos. 08/454,641, 08/485,458,
08/660,025, 08/484,282, 60/055,094, 08/856,374, 60/053,813 and
60/063,473, the teachings of all of which are incorporated herein
by reference.
[0160] The following discussion refers generally to liposomes;
however, it will be readily apparent to those of skill in the art
that this same discussion is fully applicable to the other drug
delivery systems of the present invention (e.g., micelles,
virosomes, lipid-nucleic acid particles, etc.).
[0161] For the delivery of therapeutic agents, the compositions can
be loaded with a therapeutic agent and administered to the subject
requiring treatment. The therapeutic agents which are administered
using the present invention can be any of a variety of drugs which
are selected to be an appropriate treatment for the disease to be
treated or prevented. Often the drug will be an antineoplastic
agent, such as vincristine, doxorubicin, mitoxantrone,
camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate,
streptozotocin, and the like. Especially preferred antitumor agents
include, for example, actinomycin D, vincristine, vinblastine,
cystine arabinoside, anthracyclines, alkylative agents, platinum
compounds, antimetabolites, and nucleoside analogs, such as
methotrexate and purine and pyrimidine analogs. It may also be
desirable to deliver anti-infective agents to specific tissues by
the present methods. The compositions of the present invention can
also be used for the selective delivery of other drugs including,
but not limited to, local anesthetics, e.g., dibucaine and
chlorpromazine; beta-adrenergic blockers, e.g., propranolol,
timolol and labetolol; antihypertensive agents, e.g., clonidine and
hydralazine; anti-depressants, e.g., imipramine, amitriptyline and
doxepim; anti-conversants, e.g., phenyloin; antihistamines, e.g.,
diphenhydramine, chlorphenirimine and promethazine;
antibiotic/antibacterial agents, e.g., gentamycin, ciprofloxacin,
and cefoxitin; antifungal agents, e.g., miconazole, terconazole,
econazole, isoconazole, butaconazole, clotrimazole, itraconazole,
nystatin, naftifine and amphotericin B; antiparasitic agents,
hormones, hormone antagonists, immunomodulators, neurotransmitter
antagonists, antiglaucoma agents, vitamins, narcotics, and imaging
agents.
[0162] As mentioned above, cationic lipids can be used in the
delivery of therapeutic genes or oligonucleotides intended to
induce or to block production of some protein within the cell.
Nucleic acid is negatively charged and may be combined with a
positively charged entity to form a lipid complex or a fully
encapsulated stable plasmid-lipid particle.
[0163] Particularly useful antisense oligonucleotides are directed
to targets such as c-myc, bcr-abl, c-myb, ICAM-1, C-erb B-2 and
BCL-2.
[0164] The CPLs of the present invention are also useful in the
delivery of peptides, nucleic acids, plasmid DNA, minichromosomes
and ribozymes.
[0165] Another clinical application of CPLs of this invention is as
an adjuvant for immunization of both animals and humans. Protein
antigens, such as diphtheria toxoid, cholera toxin, parasitic
antigens, viral antigens, immunoglobulins, enzymes and
histocompatibility antigens, can be incorporated into or attached
onto the liposomes containing the CPLs of the present invention for
immunization purposes.
[0166] Liposomes containing the CPLs of the present invention are
also particularly useful as carriers for vaccines that will be
targeted to the appropriate lymphoid organs to stimulate an immune
response.
[0167] Liposomes containing the CPLs of the present invention can
also be used as a vector to deliver immunosuppressive or
immunostimulatory agents selectively to macrophages. In particular,
glucocorticoids useful to suppress macrophage activity and
lymphokines that activate macrophages can be delivered using the
liposomes of the present invention.
[0168] Liposomes containing the CPLs of the present invention and
containing targeting molecules can be used to stimulate or suppress
a cell. For example, liposomes incorporating a particular antigen
can be employed to stimulate the B cell population displaying
surface antibody that specifically binds that antigen. Liposomes
incorporating growth factors or lymphokines on the liposome surface
can be directed to stimulate cells expressing the appropriate
receptors for these factors. Using this approach, bone marrow cells
can be stimulated to proliferate as part of the treatment of cancer
patients.
[0169] Liposome-encapsulated antibodies can be used to treat drug
overdoses. The tendency of liposomes having encapsulated antibodies
to be delivered to the liver has a therapeutic advantage in
clearing substances, such as toxic agents, from the blood
circulation. It has been demonstrated that whereas unencapsulated
antibodies to digoxin caused intravascular retention of the drug,
encapsulated antibodies caused increased splenic and hepatic uptake
and an increased excretion rate of digoxin.
[0170] Liposomes containing the CPLs of this invention also find
utility as carriers for introducing lipid or protein antigens into
the plasma membrane of cells that lack the antigens. For example,
histocompatibility antigens or viral antigens can be introduced
into the surface of viral infected or tumor cells to promote
recognition and killing of these cells by the immune system.
[0171] In addition, liposomes containing the CPLs of the present
invention can be used to deliver any product (e.g., therapeutic
agents, diagnostic agents, labels or other compounds) including
those currently formulated in PEG-derivatized liposomes.
[0172] In certain embodiments, it is desirable to target the
liposomes of this invention using targeting moieties that are
specific to a cell type or tissue. Targeting of liposomes using a
variety of targeting moieties, such as ligands, cell surface
receptors, glycoproteins, vitamins (e.g., riboflavin) and
monoclonal antibodies, has been previously described (see, e.g.,
U.S. Pat. Nos. 4,957,773 and 4,603,044, the teachings of which are
incorporated herein by reference). The targeting moieties can
comprise the entire protein or fragments thereof.
[0173] In some cases, the diagnostic targeting of the liposome can
subsequently be used to treat the targeted cell or tissue. For
example, when a toxin is coupled to a targeted liposome, the toxin
can then be effective in destroying the targeted cell, such as a
neoplasmic cell.
[0174] In another aspect, the present invention provides a method
for increasing intracellular delivery of a lipid-based drug
formulation, comprising: incorporating into the lipid-based drug
formulation, a compound of Formulae I or II, thereby increasing the
intracellular delivery of the lipid based drug formulation compared
to a formulation without a compound of Formulae I or II. The
compounds of Formulae I or II increase intracellular delivery about
10 fold to about 1000 fold and preferably, about 10 fold to about
100000 fold.
[0175] In another aspect, the present invention provides a method
of increasing the blood-circulation time of a parenterally
administered lipid-based drug formulation, the method comprising:
incorporating into the lipid-based drug formulation about 0.1 to 20
mole percent of a compound of Formulae I or II.
[0176] In other aspects, the present invention provides a method
for transfection of a cell with a lipid-based drug formulation,
comprising: contacting the cell with a lipid-based drug formulation
having about 0.1 to 20 mole percent of a compound of Formulae I or
II. Moreover, a method for increasing the transfection of a cell
with a lipid-based drug formulation, comprising: contacting the
cell with a lipid-based drug formulation having about 0.1 to 20
mole percent of a compound of Formulae I or II, whereby the
transfection efficiency of the lipid-based drug formulation is
increased compared to the transfection efficiency of a lipid-based
drug formulation without the compound of Formulae I or II.
[0177] G. Use of the Liposomes as Diagnostic Agents
[0178] The lipid-based drug formulations or compositions, e.g.,
liposomes, prepared using the CPLs of this invention can be labeled
with markers that will facilitate diagnostic imaging of various
disease states including tumors, inflamed joints, lesions, etc.
Typically, these labels will be radioactive markers, although
fluorescent labels can also be used. The use of gamma-emitting
radioisotopes is particularly advantageous as they can easily be
counted in a scintillation well counter, do not require tissue
homogenization prior to counting and can be imaged with gamma
cameras.
[0179] Gamma- or positron-emitting radioisotopes are typically
used, such as .sup.99Tc, .sup.24Na, .sup.51Cr, .sup.59Fe,
.sup.67Ga, .sup.86Rb, .sup.111In, .sup.125I, and .sup.195Pt as
gamma-emitting; and such as .sup.68Ga, .sup.82Rb, .sup.22Na
.sup.75Br, .sup.122I and .sup.18F as positron-emitting.
[0180] The liposomes can also be labelled with a paramagnetic
isotope for purposes of in vivo diagnosis, as through the use of
magnetic resonance imaging (MRI) or electron spin resonance (ESR).
See, for example, U.S. Pat. No. 4,728,575, the teachings of which
are incorporated herein by reference.
[0181] H. Loading and Administering the Liposomes
[0182] The following discussion refers generally to liposomes;
however, it will be readily apparent to those of skill in the art
that this same discussion is fully applicable to the other drug
delivery systems of the present invention (e.g., micelles,
virosomes, lipid-nucleic acid particles, etc.). Methods of loading
conventional drugs into liposomes include, for example, an
encapsulation technique, loading into the bilayer and a
transmembrane potential loading method.
[0183] In one encapsulation technique, the drug and liposome
components are dissolved in an organic solvent in which all species
are miscible and concentrated to a dry film. A buffer is then added
to the dried film and liposomes are formed having the drug
incorporated into the vesicle walls. Alternatively, the drug can be
placed into a buffer and added to a dried film of only lipid
components. In this manner, the drug will become encapsulated in
the aqueous interior of the liposome. The buffer which is used in
the formation of the liposomes can be any biologically compatible
buffer solution of, for example, isotonic saline, phosphate
buffered saline, or other low ionic strength buffers. Generally,
the drug will be present in an amount of from about 0.01 ng/mL to
about 50 mg/mL. The resulting liposomes with the drug incorporated
in the aqueous interior or in the membrane are then optionally
sized as described above.
[0184] Transmembrane potential loading has been described in detail
in U.S. Pat. No. 4,885,172, U.S. Pat. No. 5,059,421, and U.S. Pat.
No. 5,171,578, the contents of which are incorporated herein by
reference. Briefly, the transmembrane potential loading method can
be used with essentially any conventional drug which can exist in a
charged state when dissolved in an appropriate aqueous medium.
Preferably, the drug will be relatively lipophilic so that it will
partition into the liposome membranes. A transmembrane potential is
created across the bilayers of the liposomes or protein-liposome
complexes and the drug is loaded into the liposome by means of the
transmembrane potential. The transmembrane potential is generated
by creating a concentration gradient for one or more charged
species (e.g., Na.sup.+, K.sup.+ and/or H.sup.+) across the
membranes. This concentration gradient is generated by producing
liposomes having different internal and external media and has an
associated proton gradient. Drug accumulation can than occur in a
manner predicted by the Henderson-Hasselbach equation.
[0185] The liposome compositions of the present invention can by
administered to a subject according to standard techniques.
Preferably, pharmaceutical compositions of the liposome
compositions are administered parenterally, i.e.,
intraperitoneally, intravenously, subcutaneously or
intramuscularly. More preferably, the pharmaceutical compositions
are administered intravenously by steady infusion. Suitable
formulations for use in the present invention are found in
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Philadelphia, Pa., 17th ed. (1985). The pharmaceutical compositions
can be used, for example, to diagnose a variety of conditions, or
treat a diseased state. The diseases include, but are not limited
to, inflammation associated with rheumatoid arthritis,
post-ischemic leukocyte-mediated tissue damage (reperfusion
injury), acute leukocyte-mediated lung injury (e.g., adult
respiratory distress syndrome), septic shock, and acute and chronic
inflammation, including atopic dermatitis and psoriasis. In
addition, various neoplasms and tumor metastases can be
treated.
[0186] Preferably, the pharmaceutical compositions are administered
intravenously. Thus, this invention provides compositions for
intravenous administration which comprise a solution of the
liposomes suspended in an acceptable carrier, preferably an aqueous
carrier. A variety of aqueous carriers can be used, e.g., water,
buffered water, 0.9% isotonic saline, and the like. These
compositions can be sterilized by conventional, well known
sterilization techniques, or may be sterile filtered. The resulting
aqueous solutions may be packaged for use as is or lyophilized, the
lyophilized preparation being combined with a sterile aqueous
solution prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc.
[0187] The concentration of active ingredient in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.05%,
usually at or at least about 2-5% to as much as 10 to 30% by weight
and will be selected primarily by fluid volumes, viscosities, etc.,
in accordance with the particular mode of administration selected.
For diagnosis, the amount of composition administered will depend
upon the particular label used (i.e., radiolabel, fluorescence
label, and the like), the disease state being diagnosed and the
judgment of the clinician.
[0188] The following examples serve to illustrate, but not to limit
the invention.
EXAMPLES
I. Example I
[0189] A. General Overview
[0190] Distal cationic-poly(ethylene glycol)-lipid conjugates (CPL)
were designed, synthesized and incorporated into conventional and
stealth liposomes for enhancing cellular uptake. The present
approach uses either inert, nontoxic or naturally occurred
compounds as components for the CPL synthesis. CPLs were
synthesized with the following architectural features: 1) a
hydrophobic lipid anchor of DSPE for incorporating CPLs into
liposomal bilayer; 2) a hydrophilic spacer of polyethylene glycol
for linking the lipid anchor to the cationic head group; and 3) a
naturally occurring amino acid (L-lysine) was used to produce a
protonizable cationic head group. The number of charged amino
groups can be controlled during the CPL synthesis. It has been
demonstrated that DSPE-CPLs were almost quantitatively incorporated
into liposomal bilayer by a hydration-extrusion method. Quite
surprisingly, in an in vitro model, it was confirmed for the first
time that liposomes possessing distal positively charged polymer
conjugates with preferably four or more charges efficiently bind to
host cell surfaces and enhance cellular uptake in mammalian
cells.
[0191] B. Materials and Methods
[0192] 1. Abbreviations: DSPE,
Distearoyl-sn-glycero-3-phosphoethanolamine- ; DSPC,
1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE-PEG.sub.2000,
1,2-distearoyl-3-phosphatidylethanolamine-PEG.sub.2000; TFA,
trifluoroacetic acid; CPL, cationic-poly (ethylene glycol)-lipid
conjugate; DSPE-CPL, (cationic-polyethylene glycol)-DSPE conjugate;
DSPE-CPL-1, DSPE-CPL with one positive charge; DSPE-CPL-2, DSPE-CPL
with two positive charges; DSPE-CPL-4, DSPE-CPL with four positive
charges; DSPE-CPL-8, DSPE-CPL with eight positive charges; Rh-PE,
(or Rho-PE),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
Rhodamine B sulfonyl).
[0193] 2. Chemical: t-Boc-NH-PEG.sub.3400-CO.sub.2NHS was obtained
from Shearwater polymers, Inc (Huntsville, Ala.). N.alpha.,
N.epsilon.-di-t-Boc-L-Lysine N-Hydroxysuccinide Ester,
triethylamine and cholesterol were obtained from Sigma-Aldrich
Canada Ltd (Oakville, ON). Trifluoroacetic acid, ethyl ether and
chloroform were obtained from Fisher Scientific (Fair Lawn, N.J.).
1,2-Distearoyl-sn-glycero-3-phosphoe- thanolamine and
1,2-distearoyl-sn-glycero-3-phosphocholine were obtained from
Avanti Polar Lipids, Inc (Alabaster, Ala.).
1,2-distearoyl-3-phospha- tidylethanolamine-PEG.sub.2000 was
obtained from Genzyme (Cambridge, Mass.).
[0194] 3. Synthesis of DSPE-CPL-1: To a solution of DSPE (121 mg,
161 mmol) and Et.sub.3N (200 .mu.L) in CHCl.sub.3 (2 mL) at
45.degree. C. was added t-Boc-NH-PEG.sub.3400-CO.sub.2NHS (500 mg,
147 .mu.mol in 2 mL dry CHCl.sub.3), and the solution was stirred
for 3 hr at ambient temperature. The solution was concentrated
under a nitrogen stream to dryness. The residue was purified by
repeat precipitation of the chloroform mixture solution with
diethyl ether until disappearance of DSPE spot on TLC. The purified
DSPE-PEG conjugate was dissolved in 2 mL CHCl.sub.3 followed by
addition of 2 mL TFA, and the reaction solution was stirred at room
temperature for 4 hr. The solution was again concentrated under a
nitrogen stream to dryness. The residue was purified by repeat
precipitation of the chloroform mixture solution with diethyl ether
to offer DSPE-PEG-NH.sub.2 as DSPE-CPL-1 with one protonable
cationic head group: yield 500 mg (120 .mu.mol, 80%); R.sub.f 0.4
(CHCl.sub.3/MeOH, 9/1, v/v); The ratio of phosphoryl-lipid anchor
and the distal primary amine was measured by phosphate and
flourescamine assays and .sup.1H NMR.
[0195] 4. General procedure for the synthesis of DSPE-CPL-2.
DSPE-CPL-4 and DSPE-CPL-8 (see FIG. 2 for schematic): To a solution
of DSPE-CPL-1 (250 mg, 60 .mu.mol) and Et.sub.3N (200 .mu.L) in
CHCl.sub.3 (2 mL) was added N.alpha., N.epsilon.-di-t-Boc-L-Lysine
N-Hydroxysuccinide Ester (50 mg, 113 .mu.mol in 2 mL dry
CHCl.sub.3), and the solution was stirred for 3 hr at ambient
temperature. Disappearance of positive amine-active spot on TLC by
nihydrin visualization indicated that the reaction was completed.
The solution was concentrated under a nitrogen stream to dryness.
The residue was purified by repeat precipitation of the chloroform
mixture solution with diethyl ether until disappearance of
t-Boc-Lysine spot on TLC. The purified DSPE-PEG-conjugates were
dissolved in 2 mL CHCl.sub.3 followed by addition of 2 mL TFA, and
the reaction solution was stirred at room temperature for 4 hr. The
solution was again concentrated under a nitrogen stream to dryness.
The residue was purified by repeat precipitation of the chloroform
mixture solution with diethyl ether to offer DSPE-CPL2: yield 250
mg (57 .mu.mol, 95%); R.sub.f 0.4 (CHCl.sub.3/MeOH, 9/1, v/v); The
ratio of phosphoryl-lipid anchor and the distal primary amine was 1
measured by phosphate and flourescamine assays. DSPE-CPL4 and
DSPE-CPL8 were synthesized in a similar manner.
[0196] 5. Preparation of large unilamellar vesicles: Large
unilamellar vesicles (LUV) were prepared by extrusion as described
by Hope et al. (see Hope, M. J., et al., (1985) Production of large
unilamellar vesicles by a rapid extrusion procedure.
Characterization of size distribution, trapped volume and ability
to maintain a membrane potential. Biochim. Biophys. Acta. 812,
55-65). Appropriate amounts of lipid mixtures (DSPC/Chol, 60:40
mol/mol) with or without DSPE-CPLs (as set out in Table 3)
containing trace amounts of Rh-PE in chloroform, were dried under a
stream of nitrogen gas to form a homogeneous lipid film. The trace
amount of solvent was then removed under a vacuum overnight. The
lipid film was hydrated in HBS buffer (pH 7.5) with or without HPTS
(50 mM) by vortex mixing. The resulting multilamellar vesicles
(MLVs) were extruded 10 times through two stacked 100 nm
polycarbonate filters (Nuclepore) employing an extrusion device
(Lipex Biomembranes, Inc., Vancouver, BC, Canada) at 65.degree. C.
Unincorporated DSPE-CPLs and in some cases untrapped free HPTS were
removed by chromatography using a 1.1.times.20 cm Sepharose CL-6B
column (Sigma Chemical Co., St. Louis, Mo., USA) equilibrated with
HBS buffer.
[0197] 6. Determination of liposome size: Liposome size was
determined by quasi-elastic light scattering (QELS) using a Nicomp
370 submicron particle sizer (Santa Barbara, Calif.).
[0198] C. Results and Discussion
[0199] This example was carried out to synthesize and assess the
efficacy of the distal positively charged cationic polymer lipid
conjugates (CPL) to enhance the cellular uptake of CPL-incorporated
liposomes. The present approach uses inert, nontoxic and naturally
occurring compounds, e.g., amino acids, as components for the CPL
synthesis. Several CPLs were designed with the following
architectural features: 1) a hydrophobic lipid anchor for
incorporating the CPLs into the liposomal bilayer; 2) a hydrophilic
spacer for linking the lipid anchor to the cationic head group; and
3) a cationic head group. Moreover, the amount and nature of the
cationic group can be changed according to the final application.
In this example, a naturally occurring amino acid, L-lysine, was
used to produce a protonizable amino group. The number of amino
group can be controlled during the CPL synthesis.
[0200] In analyzing these compounds, structure-function
relationships in these cellular uptake enhancers may be identified.
As an initial step, a variety of these CPLs with differing amounts
of charge were screened for their ability to enhance uptake (see,
FIGS. 5-7). In addition, the physico-chemical properties of the
synthesized CPLs and the ability of these CPLs to incorporate into
the liposome bilayers were also studied (see, Tables 1-3 and FIGS.
5-7). In an in vitro model, it was confirmed that these distal
charged polymer conjugates significantly enhance liposome uptake in
mammalian cells.
1TABLE 1 Physicochemical properties of cationic CPL Sample
NH.sub.2/P ratio DSPE-CPL-1 0.98 DSPE-CPL-2 2.05 DSPE-CPL-4 3.96
DSPE-CPL-8 7.88
[0201]
2TABLE 2 pH gradients for CPL-liposomes Sample Lipid composition
.DELTA. pH 1 DSPC/Ch (60:40) 1.84 2 DSPC/Ch/CPL-4 (57.5:40:2.5)
1.11 3 DSPC/Ch/CPL-8 (57.5:40:2.5) 0.85 4 DSPC/Ch/PEG-PE (54:40:6)
1.59 5 DSPC/Ch/PEG-PE/CPL-4 (54:40:2:4) 1.01 6 DSPC/Ch/PEG-PE/CPL-8
(54:40:2:4) 1.13
[0202]
3TABLE 3 CPL incorporated liposomes and their properties. Lipid
composition Size (nm) CPL incorp. (%) 1, DSPC/Ch(60:40) 110 -- 2,
DSPC/Ch/CPL-1(57.5:40:2.5) 120 98.5 3, DSPC/Ch/CPL-2(57.5:40:2.5)
122 94.5 4, DSPC/Ch/CPL-4(57.5:40:2.5) 122 98.1 5,
DSPC/Ch/CPL-8(57.5:40:2.5) 122 97.6 6, DSPC/Ch/CPL-1(55:40:5) 120
98.5 7, DSPC/Ch/CPL-2(55:40:5) 122 94.5 8, DSPC/Ch/CPL-4(55:40:5)
122 98.1 9, DSPC/Ch/CPL-8(55:40:5) 122 97.6 10,
DSPC/Ch/PEG-PE(54:40:6) 128 -- 11, DSPC/Ch/PEG-PE/CPL-1(54:40:2:4)
130 96.7 12, DSPC/Ch/PEG-PE/CPL-2(54:40:2:4) 130 101 13,
DSPC/Ch/PEG-PE/CPL-4(54:40:2:4) 130 104 14,
DSPC/Ch/PEG-PE/CPL-8(54:40:2:4) 130 110 15, DSPC/Ch(60:40) 110 --
16, DSPC/Ch/CPL-4(57.5:40:2.5) 120 98.5 17,
DSPC/Ch/CPL-8(57.5:40:2.5) 122 94.5 18, DSPC/Ch/PEG-PE(54:40:6) 128
-- 19, DSPC/Ch/PEG-PE/CPL-4(54:40:2:4) 130 96.7 20,
DSPC/Ch/PEG-PE/CPL-8(54:40:2:4) 130 101
II. Example II
[0203] This example illustrates that LUVs containing CPL.sub.4 can
be formed by a detergent dialysis method.
[0204] The LUVs contain DOPE, DODAC, PEG-Cer-C20, and
CPL.sub.4[3.4K] (or CPL.sub.4[1K]). Two preparations were made with
the CPL comprising 4 mol % of the original lipids.
4 TABLE 4 Lipid mol-% DODAC 6 DOPE 79.5 CPL.sub.4 4 PC-C20 10
Rho-PE 0.5
[0205] The lipids indicated above were co-dissolved in chloroform,
which was then removed under nitrogen followed by 2 hours under
high vacuum. The dry lipid mixture (10 .mu.mol total) was then
hydrated in 83 .mu.L of 1 M OGP and 1 mL Hepes-buffered saline (20
mM Hepes 150 mM NaCl pH 7.5) at 60.degree. C. with vortexing until
all the lipid was dissolved in the detergent solution.
[0206] The lipid-detergent mixture was transferred to Slide-A-Lyzer
dialysis cassettes, and dialysed against at least 2 L HBS for 48
hours, with a least two changes of buffer in that time. Removal of
detergent by dialysis results in formation of LUVs. To determine
whether all of the CPL was incorporated into the LUVs following
dialysis, the lipid samples were fractionated on a column of
Sepharose CL-4B (see FIGS. 8A and 8B). The fractionation profiles
show LUVs formed with either CPL.sub.4[3.4K] or CPL.sub.4[1K].
[0207] The final concentration of CPL in the LUV fraction
(fractions 7-10) was estimated from initial and final
dansyl/rhodamine ratios, and from estimating the proportion of
total dansyl and rhodamine fluorescence present in the LUV
peak.
[0208] Essentially identical results were obtained.
[0209] In order to examine the effect of increasing the initial CPL
concentration, a sample was made with the following
proportions:
5 TABLE 5 Lipid mol-% DODAC 6 DOPE 71.5 CPL4 12 PC-C20 10 Rho-PE
0.5
[0210] The column profile for fractionation of this sample is shown
in FIG. 8C.
[0211] The results for all 3 samples are given below:
6 TABLE 6 original final mol Sample mol % CPL %-inserted % CPL 4
64.6 2.6 4 82.8 3.3 12 50.9 6.1
[0212] Conclusion: LUVs containing CPL.sub.4 can be formed by
detergent dialysis. Not all of the CPL.sub.4 is incorporated into
the vesicle, and the proportion that is incorporated falls as the
initial CPL/lipid molar ratio is increased. In the present case,
beginning with 4 mol % CPL, about 3 mol % was incorporated into the
LUV. For an initial CPL content of 12 mol %, a final content of 6
mol % was achieved. It is also worth noting that the behavior of
the CPL.sub.4[1K] is very similar to that of the CPL.sub.4[3.4K].
This is also true in post-insertion studies. In certain instances,
the ideal length of the hydrophilic spacer will allow the cationic
groups to extend out from the liposomal surface at a distance
shorter than the normal neutral PEG that is typically being used to
provide stealth properties for increased liposomal circulation
lifetimes.
III. Example III
[0213] A. Overview
[0214] In this example, a non-specific targeting approach is
described that involves increasing the electrostatic attraction
between liposomes and cells by incorporation of positively-charged
lipid molecules into preformed vesicles. This approach leads to
dramatic increases in cell binding/uptake in vitro in BHK cells.
The methodology is demonstrated to work for neutral vesicles and
for vesicles composed of lipids used in the construct of
lipid-based gene carriers. The approach outlined herein thus has
numerous applications ranging from delivery of conventional drugs
to gene therapy.
[0215] B. Materials and Methods
[0216] 1. Materials: 1,2-dioleoylphosphatidylcholine (DOPC),
1,2-dioleoylphosphatidylethanolamine (DOPE), and
1,2-dioleoyl-sn-glycero-- 3-phosphoethanolamine-N-(Lissamine
Rhodamine B Sulfonyl) (Rhodamine-PE) were obtained from Avanti
Polar Lipids. Cholesterol was obtained from Sigma Chemical Co.
DODAC and PEGCerC20, PEGCerC 14, and PEGCerC8 were generous gifts
from Inex Pharmaceuticals.
[0217] 2. Synthesis of cationic-PEG lipids: The details of the
synthesis of the CPLs is described herein. Two types of CPLs were
synthesized which differed in the lipid anchor portion of the
molecule. In one, the anchor was distearoylglycerol (DSG), while
the other contained distearoylphosphatidylethanolamine (DSPE). The
molecule consists of the anchor portion, to which is attached a
PEG.sub.3400 chain. At the end of the PEG chain, a charged
"headgroup" is attached, often made up of lysine residues linked
together. By modifying the headgroup region, CPLs were synthesized
which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T), and 4
(quad, or Q) positive charges. Several different Quad CPLs were
synthesized, hence these are numbered Q1 through Q5. The
nomenclature chosen to describe these compounds specifies the type
of lipid anchor and the identity of the headgroup (e.g.,
d-DSPE-CPL-Q5). The lower case "d" indicates a dansylated
derivative.
[0218] 3. Preparation of Vesicles by Detergent Dialysis. In
general, vesicles were formed using a detergent dialysis method
(see, Wheeler, J. J., et al. (1999) Stabilized plasmid-lipid
particles: construction and characterization. Gene Therapy 6,
271-281, the teachings of which are incorporated herein by
reference). The lipids, as described in Example II, were
co-dissolved in chloroform in the appropriate ratios, following
which the chloroform was removed under a stream of nitrogen and
placed under high vacuum for 2 hours. An aliquot of the non-ionic
detergent octylglucopyranoside (1 M in water) (OGP) was then added
to the dry lipid film, which was incubated for 10-20 minutes at
60.degree. C. with frequent vortexing. This was followed by
addition of 20 mM HEPES 150 mM NaCl pH 7.5, with further warming
and vortexing until all the lipid was dispersed and a clear
solution was obtained. For 20 mg of lipid, 0.125 mL of OGP and 1 mL
of HBS were used. The lipid-detergent solutions (1-2 mL) were then
transferred to Slide-A-Lyzer dialysis membranes (3 mL volume) and
exhaustively dialysed at room temperature against HBS over a period
of 48 hours. In general, a total volume of 8-10 L of HBS was used
(4-5 changes of 2L) for sample volumes of 1-8 mL.
[0219] Vesicles of DOPC and DOPC/Chol (55:45) were prepared by
extrusion as previously described (Hope, M. J., et al., (1985)
Production of large unilamellar vesicles by a rapid extrusion
procedure. Characterization of size distribution, trapped volume
and ability to maintain a membrane potential. Biochim. Biophys.
Acta 812, 55-65).
[0220] 4. Insertion of Cationic PEG-lipids into preformed vesicles.
The cationic PEG-lipids (CPLs) were stored as micellar solutions in
HBS or, in a few cases, in methanol. The CPL and the vesicles were
combined to give the desired molar ratio (up to 11.6 mol % CPL
relative to vesicle lipid), and incubated for a given time at the
desired temperature. For most insertions, the standard conditions
involved a 3 hour incubation at 60.degree. C. Following insertion,
the samples were cooled on ice, and the CPL-LUV was separated from
free CPL by passage down a column (1.5.times.15 cm) of Sepharose
CL-4B equilibrated in HBS. FIG. 16 illustrates the insertion
protocol for SPLPs (an analogous procedure).
[0221] The insertion levels of CPL were measured by fluorescence.
In all cases, the vesicles contained either 0.25 mol % or 0.5 mol %
rhodamine-PE, and the CPL contained a dansyl group. After combining
the CPL and lipids, a 15 .mu.L aliquot (initial fraction) was set
aside for analysis. The amount of CPL inserted into the vesicles
could then be quantified by measuring the initial dansyl/rhodamine
(D/R) fluorescence ratio, and the D/R ratio of the isolated
CPL-LUVs. Fluorescence parameters: for the rhodamine assay, the
excitation wavelength was 560 nm, and the emission wavelength was
590 nm. For the dansyl assay, the excitation wavelength was 340 nm,
and the emission wavelength was 510 nm. In general, the excitation
and emission slit widths were 10 and 20 nm, respectively. The assay
was performed as follows: to an aliquot of the initial sample (2-3
.mu.L) or the CPL-LUV (20-40 .mu.L) was added 30 .mu.L of 10%
Triton X-100 followed by 2 mL of HBS. The fluorescence levels of
both the dansyl and rhodamine labels were read consecutively using
a wavelength program as per the above parameters with an emission
filter of 410 run. The %-insertion was calculated as follows:
%-insertion=([D/R].sub.CPL-LUV)*100/[D/R].sub.INITIAL
[0222] 5. Measurement of Lipid Concentrations: Following insertion,
it is necessary to know the lipid concentration of each sample for
cell binding studies. This can be done quickly by fluorescence.
Following detergent dialysis, the lipid concentration of each
sample was measured using the standard phosphate assay (Fiske, C.
H., and Subbarow, Y. (1995) The colorimetric determination of
phosphorus. J. Biol. Chem. 66, 375-400). An aliquot was then
diluted to approximately 3 mM. By comparing the rhodamine
fluorescence of this sample, whose lipid concentration is known,
with the CPL-LUVs prepared from that stock, allows determination of
CPL-LUV concentrations. Lipid concentrations of LUVs were measured
using the standard phosphate assay. Following CPL insertion, lipid
concentrations were estimated for cell binding studies from the
rhodamine fluorescence.
[0223] 6. Uptake of CPL-containing LUVs by BHK cells. Approximately
10.sup.5 BHK cells were incubated in PBS/CMG medium with 20 nmol of
DOPE/DODAC/PEGCerC20 (84/6/10) LUVs containing either (1) no CPL,
(2) 8% DSPE-CPL-D, (3) 7% DSPE-CPL-T1, or (4) 4% DSPE-CPL-Q5.
Incubations were performed for 1, 2, 4, and 6 hours at 4.degree. C.
and 37.degree. C., the former giving an estimate of cell binding,
and the latter of binding and uptake. By taking the difference of
the two values, an estimate of lipid uptake at 37.degree. C. was
obtained. For each timepoint, the cells were ruptured and assayed
for lipid and protein. Lipid concentrations were measured from
rhodamine fluorescence, while protein was determined using the BCA
assay. Lipid concentrations were measured using rhodamine
fluorescence, while protein was determined using the BCA assay kit
obtained from Pierce.
[0224] C. Results and Discussion
[0225] 1. Development of Insertion Protocol. The transfer of
pegylated lipids from micellar aggregates to vesicles has been
previously described (see, Uster, P. S., et al., (1996) Insertion
of poly(ethylene glycol) derivatized phospholipid into pre-formed
liposomes results in prolonged in vivo circulation time. FEBS
Letters 386, 243-246; Zalipsky, S., et al., (1997) Poly(ethylene
glycol)-grafted liposomes with oligopeptide or oligosaccharide
ligands appended to the termini of the polymer chains. Bioconjugate
Chem. 8, 111-118). This idea was tested with DSPE-CPL's. This is
demonstrated in FIG. 9(A) for DOPC LUVs. The co-elution of the
dansyl and rhodamine labels demonstrates incorporation of the CPL
in the LUVs. In this case, 84% of the CPL was incorporated into the
LUVs, and thus only a trace of free CPL is observed trailing the
CPL-LUV fractions. This is more clearly seen in FIG. 8(B), where
the DSPE-CPL-Q5 has been inserted into a more complex positively
charged vesicles composed of DOPE/DODAC/PEGCerC20 (84/6/10). Here,
the co-elution of the two fluorescent labels at approx. 9 mLs
demonstrates 70% insertion of the CPL into the vesicles. The free
CPL elutes in a broad peak centered at 16 mLs, which is separate
from the vesicle peak, allowing for easy isolation of the CPL-LUV.
Once inserted, the DSPE-CPL-Q5 is retained and does not exchange
out of the vesicles. The CPL-LUV fraction from FIG. 9(B) was
re-eluted on the column of Sepharose CL-4B. As shown in FIG. 9(C),
all of the CPL remains with the LUVs.
[0226] The effects of incubation temperature and time on the
insertion process are shown in FIG. 10. DSPE-CPL-Q1 was incubated
in the presence of DOPE/DODAC/PEGCerC20 (84/6/10) at room
temperature, 40.degree. C., and 60.degree. C., with aliquots
withdrawn at 1, 3, and 6 hours. The highest insertion levels were
achieved at 60.degree. C., which was therefore used in subsequent
insertions. Although slightly higher insertion was obtained at 6
hr, we chose 3 hr to minimize sample degradation.
[0227] Aside from time and temperature, the parameter that will
have the greatest influence on final CPL insertion levels is the
initial CPL/lipid ratio. Assuming about 70% insertion, a series of
incubations were performed with CPL/lipid molar ratios varying
between 0.011 to 0.14, with the aim to achieve CPL-LUVs containing
1, 2, 4, 6, 8, and 12 mol % CPL. These results are shown in FIG.
11, where it is seen that the insertion level remains close to 70%
up to an initial CPL/lipid ratio of 0.095, above which it drops to
50% for CPL/lipid=0.14.
[0228] Similar results were obtained for other vesicle systems,
including DOPE/DODAC/PEGCerC14 and DOPE/DODAC/PEGCerC8. In general,
the insertion levels obtained with DODAC-containing samples fell in
the range of 70-80% for initial CPL/lipid <0.1. In order to see
whether the insertion levels were effected by the presence of
cationic lipid, several experiments were performed on neutral
vesicles containing DOPC. The compositions examined were: (1) DOPC,
(2) DOPC/Chol, (3) DOPC/PEGCerC20, and (4) DOPC/Chol/PEGCerC20. The
results, shown in FIG. 12, reveal that for the DSPE-CPL-Q1,
somewhat less insertion was achieved in the neutral systems:
between 45-65%. This may be due to reduced attraction between the
negatively-charged DSPE anchor and the membrane surface.
Regardless, the results demonstrate that significant insertion can
be achieved for both neutral and positive vesicles.
[0229] It should be noted that the insertion levels for the
DSPE-CPL-Q5 also shown in FIG. 13 are much higher than for the Q1
(70-84%). There is a reason for the differential behavior of the Q1
and Q5 CPLs in these systems, when in prior experiments they
behaved very similar. This particular batch of Q5 was prepared in
methanol, a solvent in which the lipids may exhibit greater storage
stability. As explained herein, it has been found that the presence
of methanol in the incubation mixture leads to higher
insertion.
[0230] A large number of insertions have been performed using other
CPLs in addition to the Q5 and Q1. These results are summarized in
Tables 7 and 8, (FIGS. 23 and 24) where some composition-dependent
trends can be ascertained. First, the same trend seen above in FIG.
11 with the Q5 hold for several CPLs with differing charge. As the
initial ratio of CPL/lipid is increased, the percentage of CPL
inserted decreases. If we look at the T1, Q1, and Q5 incubations
where CPL/lipid=0.022-0.024, the %-insertion ranges from 76-80%.
However, for CPL/lipid=0.086-0.095, the %-insertion range decreases
to 62-68%.
[0231] Another trend is illustrated in FIG. 13. For initial
CPL/lipid ratios >0.04, slightly less CPL-Q1 is inserted into
LUVs containing PEG-Cer-C20 than into those containing either of
the shorter chain PEGs. In addition to the type of PEG anchor
present, the quantity of PEG-Cer also has an effect on insertion,
as seen in FIG. 14. As the PEG-Cer-C20 content is increased from 4
to 10 mol %, the insertion levels of CPL-Q5 fall from 71 to
62%.
[0232] As those of skill in the art will readily appreciate, the
lipid anchor can be varied and the insertion levels may vary
depending on the lipid used as the lipid anchor. For instance, some
experiments were performed with CPL containing a DSG
(distearoylglycerol) anchor: in all cases the insertion levels were
much lower, from 17-40%, than in CPL containing a DSPE anchor.
Using the methods and assays of the present invention, those of
skill in the art can readily identify suitable lipid anchors.
[0233] In order to check for possible aggregation following CPL
insertion, quasi-elastic light scattering (QELS) was used to
examine the effect of insertion on particle diameter.
DOPE/DODAC/PEG-Cer-C20 vesicles were found to have a diameter of
119.+-.39 nm. Following insertion of 1.8 mol % CPL4b, a slight
increase in diameter to approx. 135.+-.42 nm was observed, but both
the mean diameter and standard deviation remained constant up to 7
mol % CPL. The increase from 120 nm to 135 nm could reflect a
slightly larger diameter resulting from the presence of the longer
CPL PEG chains or it could indicate a small amount of vesicle
aggregation. To differentiate between these two possibilities,
CPL-LUVs were examined by fluorescence microscopy, using a
rhodamine filter. While control LUVs exhibited no signs of
aggregation, significant levels were observed for CPL-LUVs.
However, it was found that addition of 40 mM CaCl.sub.2 completely
prevented this effect.
[0234] As described in Materials & Methods, estimates were
obtained for the uptake of various CPL-LUVs on BHK cells incubated
on PBS/CMG. The data, shown in FIG. 15, reveals that the presence
of positive charge on the CPLs can lead to significant enhancement
in uptake by BHK cells. LUVs composed of DOPE/DODAC/PEGCerC20 (and
thus exhibiting a net positive charge) showed little uptake on the
BHK cells. LUVs containing 8 mol % of DSPE-CPL-D showed similar low
uptake values. Uptake was only slightly increased by the presence
of 7 mol % of DSPE-CPL-T1. However, a significant increase in
uptake was realized for DSPE-CPL-Q5 present at only 4.1 mol %
Several points can be surmised from this data. While it is clear
that an increase in the positive charge present at some distance
from the LUV surface leads to an increase in uptake, it is not
total charge alone that plays a role in enhanced cell binding. The
quantity of positive charge present for the DSPE-CPL-D and
DSPE-CPL-Q5 samples is approximately equal, and yet the former
shows little binding compared to the latter. The DSPE-CPL-T1 sample
has a greater positive charge than the DSPE-CPL-Q5 sample, and yet
exhibits only 1/3 the uptake. It would appear that localization of
a sufficient positive charge density at the distal end of the CPL
molecule is an important parameter in ensuring interaction with
cells. In a preferred embodiment, at least four charges are used to
achieve efficient cell binding.
[0235] The dramatic effect of CPL insertion on LUV binding to BHK
cells is most clearly visualized using fluorescence microscopy. In
the absence of CPL, vesicles composed of DOPE/DODAC/PEG-Cer-C20 and
containing a trace of rhodamine-PE exhibit little binding to cells.
Incorporation of 3 mol % CPL4b leads to high levels of vesicle
binding and uptake. Although much of the lipid appears to be
binding to the cell surface, some small punctate structures can be
seen, indicating that uptake of vesicles is also occurring. An
important point to note is that the cells appear healthy following
incubation in the presence of the CPL-LUVs. In contrast,
DNA-cationic lipid complexes are known to display significant
toxicity.
[0236] One of the major remaining hurdles in liposomal drug
delivery is the problem of how to ensure that the contents of a
carrier system are taken up and utilized by a specific target cell.
It is now believed that the cellular uptake of liposomes involves
adsorption or binding at the cell surface, followed by endocytosis.
Thus factors which interfere with cellular binding will lead to low
levels of intracellular delivery. This is of particular importance
for `stealth` or long-circulating liposomes that are coated with a
surface layer of a hydrophilic polymer such as PEG. The very
characteristic of the PEG coating which imparts long-circulation
lifetimes--the formation of a steric barrier that prevents
interaction with serum proteins, will also minimize interactions
with cells. On the other hand, factors that enhance surface binding
may be expected to lead to increased cellular uptake. One approach
involves attaching molecules specific for membrane receptors to
liposomal surfaces. Possible candidates include oligopeptides (see,
Zalipsky et al., Bioconjugate Chemistry 6, 705-708 (1995); Zalipsky
et al., Bioconjugate Chemistry 8, 111-118 (1997)) oligosaccharides
(see, Zalipsky et al., Bioconjugate Chemistry 8, 111-118 (1997)),
folate (see, Gabizon et al., Bioconjugate Chemistry 10, 289-298
(1999); Lee et al., Journal of Biological Chemistry 269, 3198-3204
(1994); Reddy et al., Critical Reviews in Therapeutic Drug Carrier
Systems 15, 587-627 (1998); Wang et al., Journal of Controlled
Release, 53, 39-48 (1998)), riboflavin (see, Holladay et al.,
Biochimica et Biophysica Acta 1426, 195-204 (1999)), or antibodies
(see, Meyer et al., Journal of Biological Chemistry 273,
15621-15627 (1998); Kao et al., Cancer Gene Therapy 3 250-256
(1996); Hansen et al., Biochimica et Biophysica Acta 1239, 133-144
(1995)]. An alternate approach is to modify the charge
characteristics of the liposome. It is well known that inclusion of
either negative (see, Miller et al., Biochemistry 37, 12875-12883
(1998); Allen et al., Biochimica et Biophysica Acta 1061, 56-64
(1991); Lee et al., Biochemistry 32, 889-899 (1993); Lee et al.,
Biochimica et Biophysica Acta 1103, 185-197 (1992)) or positive
(see, Miller et al., Biochemistry 37, 12875-12883 (1998)) charges
in liposomes can lead to enhanced cellular uptake. Cationic
DNA-lipid complexes, which are efficient in vitro transfection
agents (see, Felgner et al. Nature 337, 387-388 (1989); Felgner et
al., Proceedings of the National Academy of Sciences of the United
States of America 84, 7413-7417 (1987); Kao et al., Cancer Gene
Therapy 3 250-256 (1996); Felgner et al., Annals of the New York
Academy of Sciences 772, 126-139 (1995); Jarnagin et al., Nucleic
Acids Research 20, 4205-4211 (1992)), are taken up via
endocytosis.
[0237] This example describes a new approach for enhancing the
interaction of liposomes with cells, a necessary step in the
development of non-viral systems capable of intracellular delivery.
The approach involves the insertion of novel cationic-PEG-lipids
into pre-formed liposomes, leading to a cationic vesicle in which
the positive charge involved in cell interaction is located some
distance away from the vesicle surface. The process is illustrated
in FIG. 16 for the insertion of a CPL.sub.4 into
sterically-stabilized LUVs composed of DOPE, the cationic lipid
DODAC, and PEG-Cer-C20. This lipid composition was chosen for study
for two reasons: first, it allows for efficient entrapment of
plasmid DNA within small vesicular structures by virtue of the
presence of positively charged DODAC (see, Wheeler et al. Gene
Therapy 6, 271-281 (1999)), and thus has potential as a gene
delivery system (see below). Secondly, this composition is
representative of the many sterically-stabilized drug delivery
systems which contain PEG-lipids. Insertion of CPLs leads to
localization of positive charge above the surface PEG layer,
thereby allowing electrostatic interactions between the CPLs and
cell surfaces. This should lead to increased cellular interactions
for both conventional- and PEG-containing liposomes.
[0238] The CPLs are conjugates of DSPE, a dansyl-lysine moiety, the
hydrophilic polymer PEG.sub.3400, and a mono- or multivalent
cationic headgroup. The PEG functions as a spacer, separating the
charged headgroup from the lipid anchor and vesicle surface.
Incubation of a wide variety of neutral and cationic LUVs with
micellar CPLs resulted in the incorporation of up to 6-7 mol %
(relative to total vesicle lipid) of CPL in the outer vesicle
monolayer (see tables in FIGS. 23 and 24). The insertion efficiency
was quite high, with approximately 70-80% of added CPL
incorporating into the LUVs (see tables in FIGS. 23 and 24). The
most important factors influencing the CPL insertion levels were
the incubation temperature (FIG. 10) and initial CPL/lipid ratio
(FIG. 11). The composition of the liposome was found to affect the
final CPL levels to a lesser degree (see tables in FIGS. 23 and
24). Following insertion, the CPL-LUV could be efficiently
separated from free CPL by gel exclusion chromatography. Similar
insertion levels were obtained for all CPLs, with headgroup charges
ranging from one to four charges per molecule. With this knowledge,
vesicles could be prepared containing a desired level of CPL with
reasonable accuracy.
[0239] High insertion levels (up to 7 mol %) could be achieved for
vesicles containing as much as 10 mol % PEG-Cer-C20. It is possible
that a portion of the PEG-Cer's are lost during the insertion
process, as PEG-Cer's will exchange from vesicles during
circulation. This may explain why the highest insertion levels are
achieved with PEG-Cer-C8, which has the greatest propensity to
exchange. However, analysis of LUVs and SPLPs containing
PEG-Cer-C20 by HPLC before and after insertion of CPL4 reveal only
a slight loss of PEG-Cer-C20 (from about 10 mol % to 8 mol %).
[0240] As shown in FIG. 15, cationic LUVs composed of
DOPE/DODAC/PEG-Cer-C20 exhibit little uptake when incubated on BHK
cells. Although positively charged vesicles exhibit enhanced
binding to some cell lines, this can be attenuated by the presence
of PEG on the liposome surface (see, Miller et al., Biochemistry
37, 12875-12883 (1998)). Clearly, for these systems, the presence
of 6 mol % of positively charged DODAC leads to only low uptake
levels after 6 hours. Incorporation of approximately 7 mol % of
dicationic-CPL has little effect on uptake, which was only slightly
improved in the presence of approx. 7 mol % tricationic-CPL. The
best results were obtained with the CPL4b (at 4 mol %), which
possessed 4 positive charges. At 6 hours incubation, a ten-fold
increase in uptake was observed relative to the starting vesicles.
Several points can be surmised from this data. The first is that
the presence of positively charged groups at some distance from the
LUV surface can lead to significant increases in cellular uptake.
In this case, the positive charges of the CPL (PEG MW=3400) are
located above the surface coating of PEG (MW=2000), and thus are
available for interactions with cells. However, it is not total
charge alone that plays a role in enhanced cell binding. The
quantity of positive charge present for the CPL.sub.2 and
CPL.sub.4b samples is approximately equal, and yet the former shows
little uptake compared to the latter. The CPL.sub.3 sample has a
greater positive charge than the CPL.sub.4b sample, and yet
exhibits only 1/3 the uptake. It would appear that localization of
a sufficient positive charge density at the distal end of the CPL
molecule is an important parameter in ensuring interaction with
cells. At least four charges seem to be required for efficient cell
binding to occur.
[0241] The protocol described for insertion of CPL into
conventional and sterically-stabilized CPL is ideal for
demonstrating the methodology using in vitro applications. In both
cases, the added positive charge is physically distant from the
surface, and is available for interactions with cells. This is
particularly important for polymer-coated vesicles that are
designed for minimal interactions with serum proteins and cells
such as macrophages. However, this system may not be ideal for in
vivo applications, where it may be desirable to initially hide or
screen the CPL charge to reduce clearance and allow accumulation of
the vesicles at the tissue of choice. Thus, alternative embodiments
employ shorter PEG spacer chains in the CPL, or longer PEG chains
in the PEG-Cer molecules. The PEG-Cer molecules are known to
exchange out of the particle during circulation see, Webb et al.,
Biochimica et Biophysica Acta 1372, 272-282 (1998)], which would
leave the CPL exposed for cellular interactions.
[0242] As mentioned above, the cationic liposomes employed in the
present study are composed of a fusogenic lipid (DOPE), a cationic
lipid (DODAC), and a stabilizing lipid (PEG-Cer-C20), the latter of
which imparts long-circulating properties to the vesicles. This
lipid composition was modeled after a new class of lipid-based DNA
carrier systems known as stabilized plasmid-lipid particles (SPLPs)
see, Wheeler et al. Gene Therapy 6, 271-281 (1999)). SPLPs are
small (70 nm) particles that encapsulate a single plasmid molecule.
The presence of a PEG coating on the liposome surface imparts
long-circulation properties as well as protecting the plasmid from
degradation by serum nucleases. SPLPs thus represent the first
carrier systems with real potential for systemic in vivo gene
therapy applications. The approach described here greatly enhances
the tranfection potency of these particles by increasing cellular
binding and uptake, which leads to increased intracellular delivery
of plasmids. The inclusion of CPL in conventional formulations
(e.g., anticancer drugs) also leads to increased efficacy.
IV. Example IV
[0243] A. Overview
[0244] This example employs CPLs incorporated into stable
plasmid-lipid articles (SPLPs) for in vitro transfection of
cells.
[0245] Incubation of these particles on BHK cells for up to 8 hours
resulted in an increase in uptake as the amount of inserted CPL
increased from 2-4 mol %. Transfection of the SPLP system increased
with the addition of CPL with 15 mM CaCl.sub.2 in the transfection
media. The SPLP alone showed very low transfection at both a 4 and
a 9 hour transfection followed by 24 hour complete incubation in
fresh media. The addition of 15 mM CaCl.sub.2 final concentration
in the media to the SPLPs, increased transfection on BHK cells by
10-fold at both time points. In the presence of 15 mM CaCl.sub.2,
SPLP+2%, 3% and 4% CPL transfect 2000- to 5000-times higher than
that of the SPLP alone at both time points. The 4 mol % CPL shows
the greatest increase in transfection: approximately 4500 times
higher, followed by the 3% and then the 2% CPL samples. Therefore,
the presence of the CPL, DSPE-Quad5 in the SPLP increased in both
uptake and transfection to levels comparable to or above those
achieved with the complexes.
[0246] B. Materials and Methods
[0247] 1. Synthesis of the DSPE-Quad5: The dansylated DSPE-Quad5
(CPL) was prepared in our laboratory as described by Chen et al
(2000).
[0248] 2. Incorporation of DSPE-Quad5 Into SPLP: Inex
Pharmaceuticals, Inc. supplied the SPLP. The incorporation of the
CPL into the SPLP was performed by incubation of the CPL with the
SPLP at 60.degree. C. for 2-3 hours in HBS. The resulting mixture
was then passed down a Sepharose CL-4B column equilibrated with
HBS, 75 mM CaCl.sub.2, pH 7.5 to remove the unincorporated CPL from
the SPLP with the incorporated CPL. Fractions (1 mL) were collected
and assayed for CPL (dansyl assay), phospholipid, and DNA
(PicoGreen assay). The final samples were prepared to contain 2, 3,
or 4 mol % of the CPL. The dansyl assay involved preparing a
standard curve of 0.5 to 2.5 mol % of dansylated CPL in BBS and
determining the concentration of the CPL in the sample. The
phospholipid was extracted from the SPLP by extracting the lipid
using the Bligh-Dyer extraction technique (Bligh & Dyer, 1952)
and then performing a Fiske-Subarrow assay on the organic phase of
the extraction. The PicoGreen assay was performed by comparing the
sample in the presence of PicoGreen and Triton X-100 using a DNA
standard curve. The final % insertion of the CPL was determined by
dividing the CPL concentration by the lipid concentration.
[0249] The optimal time for insertion of the CPL into the SPLP was
determined using SPLP prepared with 0.5 mol % Rh-DSPE. 15 nmol of
the dansylated CPL (DSPE-Quad5) was mixed with 200 nmol of the
labeled SPLP and the sample was incubated at 60.degree. C. for
various time points (0.5, 1, 2, 3, and 4 hours). At these time
points the sample was removed from the water bath and was passed
down a Sepharose CL-4B column. The major fraction was collected
from the column and the dansyl to rhodamine fluorescence ratios
were measured. The parameters used for the rhodamine fluorescence
were a .lambda..sub.ex of 560 nm and a .lambda..sub.em of 600 nm
and for the dansyl fluorescence were a .lambda..sub.x of 340 m and
a .lambda..sub.em of 510 nm. The excitation and emission slit
widths for both of these were 10 nm and 20 nm, respectively. By
comparison of the dansyl/rhodamine ratio for the sample before the
column to that after the column, the % insertion was determined at
each time point.
[0250] 3. QELS of CPL-SPLP: The diameter of these particles was
determined using a Nicomp Particle Sizer.
[0251] 4. Freeze-Fracture EM Freeze-fracture EM was performed on
the 2%, 3%, and 4% CPL samples by methods which will be described
by K. Wong
[0252] 5. Serum Stability of Particles: The stability of the DNA
within these CPL-SPLP was determined by incubating the samples (25
.mu.L), containing 6 .mu.g of plasmid DNA (pLuc) for various time
periods (0, 1, 2, and 4 hours) in 50% mouse serum (25 .mu.L) at
37.degree. C. At each time point, other than the zero time point,
11 .mu.L of the mixture was removed, the volume was made up to 45
.mu.L using water and the samples were placed on ice. The DNA was
then extracted from the lipid using one volume of phenol:chloroform
(1:1). Following a 20 min centrifugation in a microfuge, the top
aqueous phase was removed. The zero time point was obtained by
removing 5.5 .mu.L of the sample prior to serum addition and
performing the extraction. Twenty microliters of the aqueous phase
was then mixed with 2 .mu.L of loading buffer and the sample was
run on a 1% agarose gel in TAE buffer. Following one hour, the gel
was placed on a transilluminator and a photograph was taken.
[0253] 6. Lipid Uptake Studies: For the uptake studies,
1.times.10.sup.5 BHK cells were grown on 12 well plates overnight
in 2 mL of complete media (DMEM+10% FBS) at 37.degree. C. in 5%
CO.sub.2. Then 20 nmol of the 2, 3, and 4 mol % CPL-SPLP samples
containing 0.5% rhodamine-DSPE were mixed with HBS+75 mM CaCl.sub.2
to a final volume of 200 .mu.L and this was added to the top of the
cells followed by the addition of 800 .mu.L of complete media. This
was allowed to incubate on top of the cells for 2, 4, 6, and 8
hours at which time the cells were washed three times with PBS and
were lysed with 600 .mu.L of 0.1% Triton X-100 in PBS, pH 8.0. The
rhodamine fluorescence of the lysate was then measured on a
fluorometer using a .lambda..sub.ex of 560 nm and a .lambda..sub.em
of 600 nm using slit widths of 10 and 20 nm, respectively. An
emission filter of 430 nm was also used. A 1.0 mL microcuvette was
used. The lipid uptake was determined by comparison of the
fluorescence to that of a lipid standard (5 nmol). This value was
then normalized to the amount of cells present by measuring the
protein in 50 .mu.L of the lysate using the BCA assay.
[0254] Fluorescence micrographs were taken on a Zeiss fluorescence
microscope.
[0255] 7. Transfection Studies: For the in vitro transfection
studies, 5.times.10.sup.4 BHK cells were plated in 24-well plates
in complete media. These were incubated overnight at 37.degree. C.
in 5% CO.sub.2. SPLP, SPLP+75 mM CaCl.sub.2, DOPE:DODAC (1:1)/DNA
complexes, and CPL-SPLP systems (2, 3, and 4 mol % CPL) containing
2.5 .mu.g of DNA were made up to 100 .mu.L using HBS or HBS+75 mM
CaCl.sub.2 and were placed on the cells. Then 400 .mu.L of complete
media was added to this. At 4 and 9 hours, the transfection media
was removed and replaced with complete media containing penicillin
and streptomycin for a complete 24 hour transfection. At the end of
the transfection period, the cells were lysed with lysis buffer
containing Triton X-100. Following this lysis, 10-20 .mu.L of the
lysated was transferred to a 96-well luminescence plate. The
luminescence of the samples on the plate were measured using a
Luciferase reaction kit and a plate luminometer. The luciferase
activity was determined by using a luciferase standard curve and
was normalized for the number of cells by measuring the protein
with the BCA assay on 10-20 .mu.L of the lysated.
[0256] C. Results and Discussion
[0257] FIGS. 18A and B show that the uptake and transfection of the
SPLP system is on the order of 105 times lower than complexes.
[0258] The CPL, DSPE-Quad5, will be used in the following studies.
Its structure is shown in FIG. 16A. This molecule possesses four
positive charges at the end of a PEG.sub.3400 molecule, which has
been covalently attached to the lipid DSPE. The incorporation of
this CPL into empty liposomes of the same composition as the SPLP
has been described previously in the above examples.
[0259] The incorporation of the CPL into the SPLP involves only a
few steps. These steps are shown in FIG. 16B.
[0260] The DSPE-Quad5 was incorporated into SPLPs containing
DOPE:PEG-CerC20:DODAC (84:10:6) at various concentrations of the
CPL (from 2-4 mol %). The incorporation efficiencies for the
various CPL percentages were between 70 and 80% of the initial. In
order to separate the SPLPs possessing the CPL from the
unincorporated CPL, gel filtration chromatography was employed. A
typical column profile for the 3% DSPE-Quad5 is shown in FIG. 19A.
The CPL, lipid, and DNA all eluted from the column at the same time
in a single peak. There was however a small amount of
unincorporated CPL that eluted at a later stage. To show that the
incorporated CPL remains incorporated, the sample is re-eluted from
the column (FIG. 19A). As it can be seen in FIG. 199B, no CPL is
eluted in the later fractions of the column indicating that the CPL
remains associated with the lipid.
[0261] To determine the optimal incubation period for the insertion
of the CPL, a time course at 60.degree. C. was performed (FIG. 20).
From this figure, it can be determined that the optimal insertion
occurs between 2 and 3 hours.
[0262] The diameter of these particles containing the CPL was
determined by QELS to be 125 nm compared to the SPLP, which had a
diameter of 109 nm. To observe the structure of these particles
compared to the SPLP in the absence of the CPL, freeze-fracture EM
was performed.
[0263] The serum stability of the SPLP in the presence and absence
of various amounts of the CPL was assayed (data not shown).
Incubating free DNA with 50% mouse serum for only 1 hour results in
its complete degradation. The serum stability of the CPL-SPLPs was
similar to that for the SPLP system. This indicates that the DNA in
the CPL-SPLP is as protected as that in the SPLP system without
CPL.
[0264] The major objective of this study is to increase both the
uptake and transfection of the SPLP system using CPLS. FIG. 21
shows the time course for the uptake of rhodamine labeled SPLP in
the presence (2, 3, or 4 mol %) and absence of the DSPE-Quad5 (0%).
The uptake of the 4% system is higher than the 3% system, which is
higher than the 2% system, and all three are much higher than the
system without CPL. FIG. 22 shows 4 h and 9 h time points of the
same formulations.
V. Example V
[0265] This example illustrates the incorporation of a CPL into a
Stabilized Antisense-Lipid Particle ("SALP").
[0266] A. Materials and Results
[0267] Distearoylphosphatidylcholine (DSPC), was purchased from
Northern Lipids (Vancouver, Canada).
1,2-dioleoyloxy-3-dimethylammoniumpropane (DODAP or AL-I) was
synthesized by Dr. Steven Ansell (Inex Pharmaceuticals) or,
alternatively, was purchased from Avanti Polar Lipids. Cholesterol
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. Lipid stock solutions were prepared in 95% ethanol at 20
mg/mL (PEG-Ceramides were prepared at 50 mg/mL).
[0268] SALPs are first prepared according to the methods set out in
PCT Patent Application No. WO 98/51278, published 19 Nov. 1998, and
incorporated herein by reference. See also, J. J. Wheeler et al.,
(1999), Gene Therapy, 6, 271-281. Briefly, a 16mer of
[3H]-phosphorothioate oligodeoxynucleotide Inx-6295 (human c-myc)
having sequence 5' T AAC GTT GAG GGG CAT 3' (SEQ ID. No: 1) (in 300
mM citrate buffer, pH 3.80) was warmed to 65.degree. C. and the
lipids (in ethanol) were slowly added, mixing constantly
(DSPC:CHOL:DODAP:PEG-CerC14; 25:45:20:10, molar ratio). The
resulting volume of the mixture was 1.0 mL and contained 13 mmol
total lipid, 2 mg of antisense oligodeoxynucleotide, and 38%
ethanol, vol/vol. The antisense-lipid mixture was subjected to 5
cycles of freezing (liquid nitrogen) and thawing (65.degree. C.),
and subsequently was passed 10.times. through three stacked 100 nm
filters (Poretics) using a pressurized extruder apparatus with a
thermobarrel attachment (Lipex Biomembranes). The temperature and
pressure during extrusion were 65.degree. C. and 300-400 psi
(nitrogen), respectively. The extruded preparation was diluted with
1.0 mL of 300 mM citric acid, pH 3.8, reducing the ethanol content
to 20%. The extruded sample was dialyzed (12000-14000 MW cutoff;
SpectraPor) against several liters of 300 mM citrate buffer, pH 3.8
for 3-4 hours to remove the excess ethanol. The sample was
subsequently dialyzed against HEPES-buffered saline (HBS), pH 7.5,
for 12-18 hours to neutralize the DODAP and release any antisense
that was associated with the surface of the vesicles. Encapsulation
was assessed either by analyzing the pre-column and post-column
ratios of [.sup.3H]-antisense and [.sup.14C]-lipid or by
determining the total pre-column and post-column
[.sup.3H]-antisense and [.sup.14C]-lipid radioactivity.
[0269] CPL is incorporated after the SALPs are prepared.
Approximately 5 .mu.mol SALP were mixed with 3-10 mol % CPL (i.e.,
0.15-0.5 .mu.mol CPL). CPL were stored as micellar solutions in
HBS, or in methanol. When CPL was added in methanol, the final
methanol concentration of 3-4%. The mixtures were incubated
overnight at room temperature or at 40.degree. C. Unincorporated
CPL was removed from the SALP preparation by column separation
(Sepharose CL-4B equilibrated with HBS, 75 mM CaCL.sub.2 at pH
7.5). Incorporation efficiency was between 34 and 60%. It is
anticipated that other organic solvents may improve incorporation
efficiency.
VI. Example VI
[0270] A. General Overview
[0271] In the present example, distal positively charged cationic
poly(ethylene glycol) lipid conjugates (CPL) were synthesized and
assessed for their efficacy at enhancing the cellular uptake of
CPL-incorporated liposomes. It was confirmed that distal charged
polymer conjugates bound to a liposome surface enhanced liposome
uptake in mammalian cells in vitro.
[0272] B. Methods
[0273] Determination of the Critical Micelle Concentration
(CMC)
[0274] The CMCs of the CPLs were determined using the NPN assay as
previously reported by Brito and Vaz (see, Brito, R. M. M., and
Vaz, W. L. C. (1986) Determination of the critical micelle
concentration of surfactants using the fluorescent probe
N-phenyl-1-naphthylamine. Anal. Biochem. 152, 250-255.). A series
of different concentrations of CPLs were prepared in HBS buffer (25
mM Hepes, 150 mM NaCl, pH 7.4). 5 .mu.M of NPN (from a stock NPN
solution in 95% ethanol) was added into the above CPL solutions.
After incubation of the mixtures at room temperature for 30 min,
the fluorescence intensities at .lambda..sub.em=410 nm using
.lambda..sub.ex=356 nm on a Perkin Elmer LS 50 Luminescence
Spectrometer.
[0275] C. Results
[0276] Uptake Enhancement of CPL-L UVs in Vitro. Cellular Uptake of
Conventional CPL-Liposomes.
[0277] The in vitro cellular uptake of CPL-containing liposomes was
studied on baby hamster kidney (BHK) cells. The liposome-associated
fluorescent lipid marker (Rh-PE) was used as a marker for lipid
uptake. As shown in FIGS. 26 and 27, CPL.sub.4 significantly
enhances the cellular uptake compared to control samples (no CPL)
using both PBS-CMG and serum containing medium. The time dependent
uptake of CPL-LUVs reaches a maximum after 3 hr. FIG. 6 summarized
the cell uptake of the different CPL-containing vesicles after a
four hour incubation. Compared to a control, reduced cell uptake
was observed for CPL.sub.1, a moderate increase for CPL.sub.2 (2
fold), and a large increase for both CPL.sub.4 and CPL.sub.8. The
similar degree of increase resulting from CPL.sub.4 and CPL.sub.8
indicates a charge density of four in the CPLs satisfies the
requirement for maximum enhanced cellular uptake.
VII. Example VII
[0278] A. General Overview
[0279] This experiment describes the synthesis of a new class of
cationic lipids designed to enhance non-specific targeting by
increasing the electrostatic attraction between liposomes and
cells.
[0280] B. Materials and Reagents
[0281] tBoc-NH-PEG.sub.3400-CO.sub.2--NHS was obtained from
Shearwater Polymers (Huntsville, Ala.).
N.sub..alpha.,N.sub..epsilon.-di-tBoc-L-lysi-
ne-N-hydroxysuccinimide ester, N.sub..epsilon.-dansyl-L-lysine,
N-hydroxysuccinimide (NHS), and N,N'-dicyclohexyl-carbodiimide
(DCC) were purchased from Sigma-Aldrich Canada (Oakville, ON).
1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were
obtained from Northern Lipids (Vancouver, BC). Fluorescamine and
Rhodamine-DSPE (Rh-PE) were obtained from Molecular Probes (Eugene,
Oreg.). Cholesterol (Chol) was obtained from Sigma Aldrich Canada
(Oakville, ON). Trifluoroacetic acid, diethyl ether, methanol,
triethylamine, and chloroform were obtained from Fisher Scientific
(Vancouver, BC). All reagents were used without further
purification.
[0282] 1. General Methods
[0283] All reactions were performed in 16.times.100 mm glass test
tubes. .sup.1H NMR spectra were obtained employing a Bruker MSL 200
spectrometer operating at 200 MHz. Deuterated chloroform
(CDCl.sub.3) was used as the solvent in the NMR experiments. Proton
chemical shifts (.delta.) were referenced to CHCl.sub.3 set at 7.24
ppm. When signals were reasonably resolved, their intensities were
integrated to allow an estimation of the number of protons. The
chemical shifts of exchangeable amino group protons, observed
between 7-8 ppm, are not given. These peaks were assigned on the
basis of their removal by a D.sub.2O exchange.
[0284] Phosphorus and fluorescamine assays were performed to
confirm the ratio of primary amine per phosphate in each CPL as
follows.
[0285] The phosphate concentration of the CPL was determined using
the Fiske-Subarrow phosphorus assay (see, Fiske, C. H., and
Subbarow, Y. (1995) The colorimetric determination of phosphorous.
J. Biol. Chem. 66, 375-400.). The primary amine concentration in
the CPL was determined using the fluorophore, fluorescamine. A
fluorescamine solution (0.6 mg/mL) in acetone was prepared. An
aliquot of CPL solution in HBS (2-4 .mu.L) was made up to 250 .mu.L
with 200 mM sodium borate, pH 8.0. To this mixture, 50 L of the
fluorescamine solution was added dropwise with vortexing, followed
by 1700 .mu.L of water. The fluorescence of this solution was
measured using a Perkin-Elmer LS50 Luminescence Spectrometer with
.lambda..sub.ex, of 397 nm and .lambda..sub.em of 475 nm, and
excitation and emission slit widths of 10 nm. The primary amine
concentration of the CPL was determined from a lysine standard
curve.
[0286]
tBoc-NH-PEG.sub.3400-CO.sub.2--(N.sub..epsilon.-dansyl)lysine (1).
tBoc-NH-PEG.sub.3400-CO.sub.2--NHS (500 mg, 147 .mu.mol) in 3 mL of
dry chloroform was added slowly to a solution of
N.sub..epsilon.-dansyl-L-lys- ine (65 mg, 171 .mu.mol) in 1 mL of
methanol and 200 .mu.L of triethylamine. After the reaction mixture
was stirred at room temperature for 3 h, the solvent was removed
under a N.sub.2 stream and further dried under vacuum. The crude
product was washed by first dissolving it in a minimum amount of
chloroform with warming and then precipitating it out with the
addition of 10 mL of diethyl ether. The ether was added while
vortexing. Precipitation of 1 was accelerated by cooling. The
precipitate was then pelleted by centrifugation and the ether was
discarded. This chloroform/ether wash and precipitation procedure
was repeated. The dry solid was then dissolved in 4 mL of
chloroform and cooled in an ice bath for 15 min. Methanol (2 mL)
was added if this cooled solution was clear. If a precipitate
(excess dansyl lysine) developed, it was filtered off prior to the
addition of methanol. The chloroform/methanol solution was washed
with 1.2 mL of 0.1 M HCl. The chloroform phase was extracted,
dried, and the solid redissolved in 6 mL of chloroform/methanol
(2:1 v/v) and washed with 1.2 mL of distilled water. The chloroform
phase was extracted, dried to a thick paste and
tBoc-NH-PEG-CO.sub.2--(N.sub..epsil- on.-dansyl)lysine (1) was
precipitated with 10 mL of ether. After centrifugation and the
removal of ether, the dried product is a light yellow solid. Yield:
520 mg (93%). TLC (silica gel) chloroform/methanol (85:15 v/v):
R.sub.f 0.56. .sup.1H NMR (CDCl.sub.3): .delta. 1.08 (t), 1.40 (s,
11H), 2.66 (s, 1H), 2.86 (s, 8H), 3.27 (q), 3.50 (t), 3.60 (s,
309H), 3.96 (t), 4.19 (m[broad]), 5.03 (s[broad], 1H), 5.22 (t,
1H), 5.43 (d, 1H), 7.16 (d, 1H), 7.51 (q, 2H), 8.19 (d, 1H), 8.27
(d), 8.50 (d, 1H) ppm.
[0287] Dansylated CPL.sub.1-tBoc (3). First,
tBoc-NH-PEG.sub.3400-CO.sub.2- --(N.sub..epsilon.-dansyl)lysine-NHS
(2) was prepared as follows. A solution of
tBoc-NH-PEG.sub.3400-CO.sub.2--(N.sub..epsilon.-dansyl)lysine (1)
(500 mg, 132 .mu.mol) and NHS (31.5 mg, 274 .mu.mol) in 2 mL of dry
chloroform was added to DCC (42.8 mg, 207 .mu.mol) dissolved in 1
mL of dry chloroform. The reaction mixture was stirred for 2 h at
room temperature. The by-product, dicyclohexyl urea (DCU), was
filtered using a Pasteur pipette with a cotton plug. The filtrate,
containing
tBoc-NH-PEG.sub.3400-CO.sub.2--(N.sub..epsilon.-dansyl)lysine-NHS
(2), was slowly added to a solution of DSPE (120.6 mg, 161 .mu.mol)
in 2 mL of dry chloroform and 200 .mu.L of triethylamine. The
dissolution of DSPE in dry chloroform and triethylamine required
warming to 65.degree. C. After the reaction mixture was stirred at
room temperature for 3 h, it was dried, and chloroform/ether washed
and precipitated as described earlier until the disappearance of
DSPE on TLC as visualized with ninhydrin. This removal of excess
DSPE required at least three washings. The product, dansylated CPL,
-tBoc (3), was dissolved in chloroform/methanol (2:1), washed with
dilute HCl and water, and precipitated using ether as described for
(1). Yield: 575 mg (96%). TLC (silica gel) chloroform/methanol
(85:15) R.sub.f 0.58. .sup.1H NMR (CDCl.sub.3): .delta. 0.85 (t,
4H), 1.22 (s, 48H), 1.41 (s, 10H), 1.55 (t), 2.27 (m[broad], 6H),
2.90 (m[broad], 6H), 3.04 (s, 8H), 3.27 (q), 3.61 (s, 275H), 4.14
(m[broad]), 4.32 (d), 4.38 (d), 5.05 (s[broad]), 5.23 (s[broad]),
5.58 (m[broad]), 7.37 (d, 1H), 7.49 (s[broad], 1H), 7.59 (t, 2H),
8.24 (d, 1H), 8.50 (d, 1H), 8.59 (d, 1H) ppm.
[0288] Dansylated CPL.sub.1 (4). Trifluoroacetic acid (TFA) (2 mL)
was added to a solution of dansylated CPL.sub.1-tBoc (3) (550 mg,
121 .mu.mmol) in 2 mL of chloroform and stirred for 4 h at room
temperature. The solution was concentrated to a thick paste and
chloroform/ether washed three times. After the removal of ether,
the solid was dissolved in 6 mL of chloroform/methanol (2:1) and
washed with 1.2 mL of 5% sodium bicarbonate. The chloroform phase
was extracted, dried and redissolved in 6 mL chloroform/methanol
(2:1) and washed with 1.2 mL distilled water. The chloroform phase
was concentrated to a thick paste and the purified CPL.sub.1 (4)
was obtained through a chloroform/ether wash and vacuum dried.
Yield: 535 mg (97%). TLC (silica) chloroform/methanol/water
(65:25:4) R.sub.f 0.76. .sup.1H NMR (CDCl.sub.3). .delta. 0.85 (t,
4H), 1.22 (s, 46H), 1.54 (m[broad], 8H), 2.23 (t, 6H), 2.84 (s,
9H), 3.16 (m[broad], 3H), 3.26 (t, 3H), 3.61 (s, 263H), 3.98 (q),
4.17 (t), 4.33 (d), 4.38 (d), 5.19 (s[broad]), 5.93 (d, 1H), 7.13
(d, 1H), 7.46 (t, 1H), 7.52 (t, 1H), 8.15 (d, 1H), 8.43 (t, 2H)
ppm.
[0289] Dansylated CPL.sub.2-tBoc (5). A solution of
N.sub..alpha.N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester (105 mg, 236 .mu.mol) in 2 mL dry chloroform was gradually
added to a solution of dansylated CPL.sub.1 (4) (510 mg, 112
.mu.mol) in 2 mL chloroform containing 200 .mu.L triethylamine and
stirred at room temperature for 3 h. The completion of the reaction
was indicated by the disappearance of primary amine as visualized
by ninhydrin assay on TLC. The reaction mixture was concentrated to
a thick paste and chloroform/ether washed (.about.3 times) until
the disappearance of excess
N.sub..alpha.N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester as checked by TLC. The product was dissolved in 6 mL
chloroform/methanol (2:1) and washed with 1.2 mL 0.1 M HCl. The
chloroform phase was extracted, dried, redissolved in 6 mL
chloroform/methanol (2:1) and washed with 1.2 mL distilled water.
The chloroform phase was concentrated to a thick paste and the
purified compound was obtained through a chloroform/ether wash and
vacuum dried. Yield: 510 mg (96%). TLC (silica gel)
chloroform/methanol (85:15) R.sub.f 0.58. .sup.1H NMR (CDCl.sub.3).
.delta. 0.85 (t, 3H), 1.22 (s, 44H), 1.41 (s, 20H), 1.56
(m[broad]), 1.78 (m[broad]), 2.27 (m, 5H), 2.88 (s), 2.91 (s), 2.97
(s), 3.06 (s, 7H), 3.26 (t), 3.44 (t), 3.62 (s, 252H), 3.97 (t),
4.05 (d), 4.13 (m), 4.33 (d), 4.38 (d), 4.68 (s[broad]), 5.22
(s[broad]), 5.51 (s[broad]), 6.57 (t[broad], 1H), 7.39 (d, 1H),
7.51 (s[broad], 1H), 7.60 (t, 2H), 8.26 (d, 1H), 8.53 (d, 1H), 8.61
(d, 1H) ppm.
[0290] Dansylated CPL.sub.2 (6). The synthesis of CPL.sub.2 (6) was
the same as that of CPL.sub.1 (4) by deprotecting dansylated
CPL.sub.2-tBoc (5) (490 mg, 103 .mu.mol). Yield: 478 mg (97%). TLC
(silica) chloroform/methanol/water (65:25:4) R.sub.f 0.63. .sup.1H
NMR (CDCl.sub.3). .delta. 0.85 (t, 3H), 1.22 (s, 42H), 1.55 (m,
10H), 1.93 (s[broad], 4H), 2.24 (t, 5H), 2.85 (s, 8H), 3.26 (t,
3H), 3.61 (s, 271H), 3.95 (q), 4.17 (s), 4.34 (s), 5.18 (s[broad],
1H), 6.31 (d, 1H), 6.89 (s, 1H), 7.10 (d, 1H), 7.49 (m, 1H), 8.15
(d, 1H), 8.34 (d, 1H), 8.47 (d, 2H) ppm.
[0291] Dansylated CPL.sub.4-tBoc (7). The synthesis of
CPL.sub.4-tBoc (7) was the same as that of CPL.sub.2-tBoc (5) by
reacting
N.sub..alpha.,N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester (170 mg, 383 .mu.mol) with dansylated CPL.sub.2 (6) (455 mg,
95 .mu.mol). Yield: 475 mg (96%). TLC (silica gel)
chloroform/methanol (85:15) R.sub.f 0.58. .sup.1H NMR (CDCl.sub.3).
.delta. 0.85 (t, 3H), 1.22 (s, 43H), 1.40 (s, 39H), 1.71 (m[broad],
6H), 2.27 (m, 5H), 2.88 (s), 2.90 (s), 2.95 (s), 3.05 (s, 10H),
3.25 (t, 3H), 3.43 (s), 3.61 (s, 262H), 3.97 (t), 4.05 (d), 4.15
(m), 4.32 (d), 4.37 (d), 4.51 (s[broad]), 4.75 (s[broad]), 4.90
(s[broad]), 5.23 (t[broad], 1H), 5.52 (s[broad]), 5.80 (s[broad],
1H), 7.15 (m[broad], 1H), 7.38 (d, 1H), 7.50 (s, 1H), 7.59 (t, 2H),
8.25 (d, 1H), 8.51 (d, 1H), 8.60 (d, 1H) ppm.
[0292] Dansylated CPL.sub.4 (8). The synthesis of CPL.sub.4 (8) was
the same as that of CPL.sub.1 (4) by deprotecting dansylated
CPL.sub.4-tBoc (7) (450 mg, 86 .mu.mol). Yield: 440 mg (97%). TLC
(silica) chloroform/methanol/water (65:25:4) R.sub.f 0.19. .sup.1H
NMR (CDCl.sub.3). .delta. 0.85 (t), 1.22 (s), 1.53 (m[broad]), 2.34
(m[broad]), 2.86 (s), 3.26 (t), 3.62 (s), 3.87 (s[broad]), 3.97
(t), 4.17 (s[broad]), 4.33 (d), 5.18 (s[broad]), 7.15 (d), 7.43
(s), 7.51 (t), 8.15 (d), 8.32 (d), 8.48 (d), 9.05 (s[broad])
ppm.
[0293] Dansylated CPL.sub.8-tBoc (9). The synthesis of
CPL.sub.8-tBoc (9) was the same as that of CPL.sub.2-tBoc (5) by
reacting
N.sub..alpha.N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester (70 mg, 158 .mu.mol) with dansylated CPL.sub.4 (8) (100 mg,
19 .mu.mol). Yield: 112 mg (96%). TLC (silica gel)
chloroform/methanol (85:15) R.sub.f 0.58. .sup.1H NMR (CDCl.sub.3).
.delta. 0.84 (t, 3H), 1.08 (s), 1.21 (s, 39H), 1.39 (s, 75H), 1.66
(m [broad]), 2.26 (m, 4H), 2.89 (s, 4H), 3.06 (s, 11H), 3.25 (t,
3H), 3.43 (s), 3.49 (s), 3.60 (s, 248H), 3.96 (t), 4.04 (d), 4.12
(t), 4.31 (d), 4.36 (m), 5.19 (m [broad]), 6.77 (m [broad], 1H),
6.91 (s [broad], 1H), 7.24 (CHCl.sub.3), 7.41 (d), 7.50 (s
[broad]), 7.60 (t), 8.25 (d, 1H), 8.53 (d, 1H), 8.63 (d, 1H)
ppm.
[0294] Dansylated CPL.sub.8 (10). The synthesis of CPL.sub.8 (8)
was the same as that of CPL.sub.1 (4) by deprotecting dansylated
CPL.sub.8-tBoc (9) (50 mg, 8 .mu.mol). Yield: 48 mg (96%). TLC
(silica) chloroform/methanol/water (65:25:4) R.sub.f 0.13. .sup.1H
NMR (CDCl.sub.3). .delta.0.85 (t, 3H), 1.22 (s, 34H), 1.52 (s
[broad]), 2.23 (s [broad]), 2.86 (d), 3.27 (d), 3.61 (s, 274H),
3.96 (t), 4.18 (m [broad]), 7.14 (s [broad]), 7.24 (CHCl.sub.3),
7.50 (m [broad]), 8.12-8.27 (s [broad]), 8.47 (m [broad]) ppm.
[0295] C. Results and Discussion
[0296] The CPL were synthesized by repeated coupling reaction steps
involving amines and NHS-activated carbonate groups as outlined in
FIG. 29. This consists of (a) incorporating the dansyl fluorescent
label to the hydrophilic PEG spacer, (b) coupling of the DSPE
anchor, and (c) attachment of the cationic headgroup to the lipid.
The heterobifunctional PEG polymer
tBoc-NH-PEG.sub.3400-CO.sub.2--NHS (MW 3400), was chosen for two
reasons. Firstly, it was commercially available. Secondly, it is
insoluble in ether that provided a very convenient means of
purifying its derivatives, 1-10. Other reagents were used in excess
to ensure the complete conversion of the PEG polymer to its
derivatives. The excess reagents were soluble in ether and
therefore could be removed by washing in ether during
purification.
[0297] Incorporation of the fluorescent label,
N.sub..epsilon.-dansyl lysine, to the PEG polymer by coupling the
.alpha.-amino group of dansyl lysine with the NHS activated
carbonate of PEG gave the lysine derivative 1. The DSPE anchor was
coupled via intermediate 2 that was formed by the esterification of
1 using NHS and DCC. The resulting PEG lipid, 3, was deprotected by
removing the tBoc to form CPL.sub.1, 4, with one positive charge.
The positive charges in the other CPL are carried by the amino
groups of lysine. Here, the NHS activated and di-tBoc protected
lysine was attached to the free amino function of CPL.sub.1 to form
intermediate 5 which, upon deprotection, yielded CPL.sub.2, 6, with
two positive charges. The attachment of two lysine residues to the
amino groups of CPL.sub.2 via intermediate 7 gave CPL.sub.4, 8,
with four positive charges. Thus, CPL.sub.8, 10, with eight
positive charges was synthesized with the attachment of four lysine
residues as the headgroup. As can be seen, this provides a very
convenient means of synthesizing multivalent CPL that are of
particular interest for non-viral drug delivery applications.
[0298] The structures of the purified intermediates and CPL in FIG.
29 were verified by .sup.1H NMR spectroscopy and chemical analysis.
The .sup.1H NMR spectra showed well-resolved resonances for the
PEG, tBoc and acyl chains of DSPE at approximately 3.61, 1.41 and
1.21 ppm, respectively, and for the resonances of the dansyl moiety
(aromatic protons at 7.1-8.5 ppm; methyl protons at 2.8-3.0 ppm).
From the integrated signal intensities of the former three peaks,
it was found that the ratio of tBoc/PEG or tBoc/DSPE was 1.0, 2.1,
4.0, and 8.1 for CPL.sub.1-tBoc, CPL.sub.2-tBoc, CPL.sub.4-tBoc,
and CPL.sub.8-tBoc, respectively. As each tBoc is attached to an
amino group, this gives the number of amino groups in the headgroup
of each CPL relative to the CPL.sub.1. That essentially identical
results were obtained using the ratios of tBoc relative to both PEG
and DSPE demonstrates the presence of lipid and polymer in correct
proportion to the headgroup. The complete cleavage of the tBoc
protecting groups was verified by the loss of tBoc NMR peaks and
chemical analysis which determined the ratio of primary amine to
phosphate in each of the CPL by using the fluorescamine and
phosphorus assays. The amine/phosphate ratios for CPL.sub.1,
CPL.sub.2, CPL.sub.4, and CPL.sub.8 were found to be 1.0, 2.2, 3.7,
and 8.0, respectively. These corresponded well with the expected
number of positive charge bearing amino groups of the respective
CPL.
[0299] The CPL described here possess several attributes which may
increase their usefulness relative to other cationic lipids.
Firstly, the phospholipid anchor will readily allow efficient
incorporation of CPL into liposomal systems. Secondly, the dansyl
label will permit accurate and convenient quantification of the CPL
in the bilayer using fluorescence techniques. Finally, the valency
of the cationic headgroup in the CPL can easily be modified using
lysine residues.
VIII. Example VIII
[0300] A. General Overview
[0301] The synthesis of a fluorescent cationic poly(ethylene
glycol) (MW 1000) lipid conjugates (CPL).sup.1 is described. The
procedure is very similar to that of PEG 3400 described in detail
previously. However the lower molecular weight PEG derivatives may
not be insoluble in ether, and therefore could not be readily
purified by ether wash as before. The synthetic procedure is
similar to the one outlined in FIG. 29.
[0302] B. Abbreviations
[0303] tBoc, tert-butyloxycarbonyl;
tBoc-NH-PEG.sub.1000-CO.sub.2--NHS, tBoc protected and NHS
activated PEG.sub.1000; CPL, cationic poly(ethylene glycol) lipid
conjugate; CPL.sub.1, CPL with one positive charge; CPL.sub.2, CPL
with two positive charges; CPL.sub.4, CPL with four positive
charges; DCC, N,N'-dicyclohexyl-carbodiimide; DCU, dicyclohexyl
urea; NHS, N-hydroxysuccinimide; di-tBoc-lysine-NHS,
N.sub..alpha.,N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine;
PEG.sub.1000, poly(ethylene glycol) with an average MW of 1000;
TFA, trifluoroacetic acid.
[0304] C. Materials and Reagents
[0305] tBoc-NH-PEG.sub.1000-CO.sub.2--NHS was obtained from
Shearwater Polymers (Huntsville, Ala.).
N.sub..alpha.,N.sub..epsilon.-di-tBoc-L-lysi-
ne-N-hydroxysuccinimide ester, N.sub..epsilon.-dansyl-L-lysine,
N-hydroxysuccinimide (NHS), and N,N'-dicyclohexyl-carbodiimide
(DCC) were purchased from Sigma-Aldrich Canada (Oakville, ON).
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) was obtained
from Northern Lipids (Vancouver, BC). Fluorescamine was obtained
from Molecular Probes (Eugene, Oreg.). Trifluoroacetic acid,
diethyl ether, methanol, triethylamine, and chloroform were
obtained from Fisher Scientific (Vancouver, BC). All other reagents
were used without further purification.
[0306]
tBoc-NH-PEG.sub.1000-CO.sub.2--(N.sub..epsilon.-dansyl)lysine (1).
tBoc-NH-PEG.sub.1000-CO.sub.2--NHS (500 mg, 500 .mu.mol) in 3 mL of
dry chloroform was added slowly to a solution of
N.sub..epsilon.-dansyl-L-lys- ine (200 mg, 536 .mu.mol) in 1.5 mL
of methanol and 300 .mu.L of triethylamine. After the reaction
mixture was stirred at room temperature for 3 h, the solvent was
removed under a N.sub.2 stream and further dried under vacuum. The
crude product was dissolved in 6 mL of chloroform/methanol (2:1
v/v), washed once with 1.2 mL of 0.5 M HCl and twice with 1.2 mL of
distilled water. The chloroform phase was extracted, dried to a
thick paste and tBoc-NH-PEG-CO.sub.2--(N.sub..epsilon.-dansyl)-
lysine (I) was obtained as a light yellow solid. Yield: 600 mg
(95%). TLC (silica gel) chloroform/methanol (85:15 v/v): R.sub.f
0.50.
[0307] Dansylated CPL.sub.1-tBoc (3). First,
tBoc-NH-PEG.sub.1000-CO.sub.2- --(N.sub..epsilon.-dansyl)lysine-NHS
(2) was prepared as follows. A solution of
tBoc-NH-PEG.sub.1000-CO.sub.2--(N.sub..epsilon.-dansyl)lysine (1)
(600 mg, 474 .mu.mol) and NHS (113 mg, 982 mmol) in 2 mL of dry
chloroform was added to DCC (150 mg, 728 .mu.mol) dissolved in 1 mL
of dry chloroform. The reaction mixture was stirred for 5 h at room
temperature. The by-product, dicyclohexyl urea (DCU), was filtered
using a Pasteur pipette with a cotton plug. The filtrate,
containing
tBoc-NH-PEG.sub.1000-CO.sub.2--(N.sub..epsilon.-dansyl)lysine-NHS
(2), was slowly added to a solution of DSPE (365 mg, 488 .mu.mol)
in 3 mL of dry chloroform and 300 .mu.L of triethylamine. The
dissolution of DSPE in dry chloroform and triethylamine required
warming to 65.degree. C. After the reaction mixture was stirred
overnight at room temperature, it was filtered to remove some
precipitate (unreacted DSPE) and dried to a viscous paste. The
paste was dissolved in chloroform/methanol (2:1), washed with
dilute HCl and water as before. The product, dansylated
CPL.sub.1-tBoc (3), was obtained after the removal of solvent and
precipitated using 10 mL of ether. Yield: 900 mg (96%). TLC (silica
gel) chloroform/methanol (85:15) R.sub.f 0.58.
[0308] Dansylated CPL.sub.1 (4). Trifluoroacetic acid (TFA), 3 mL,
was added to a solution of dansylated CPL.sub.1-tBoc (3) (900 mg,
456 .mu.mol) in 3 mL of chloroform and stirred for 4 h at room
temperature. The solution was concentrated to a thick paste and
chloroform/ether washed three times. After the removal of ether,
the solid was dissolved in 6 mL of chloroform/methanol (2:1) and
washed twice with 1.2 mL of 5% sodium bicarbonate and twice with
1.2 mL distilled water. The chloroform phase was concentrated to a
thick paste and the purified CPL.sub.1 (4) was obtained through a
chloroform/ether wash and vacuum dried. Yield: 750 mg (88%). TLC
(silica) chloroform/methanol/water (65:25:4) R.sub.f 0.72.
[0309] Dansylated CPL.sub.2-tBoc (5). A solution of
N.sub..alpha.N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester (350 mg, 789 [mol) in 3 mL dry chloroform was gradually added
to a solution of dansylated CPL.sub.1 (4) (750 mg, 400 .mu.mol) in
3 mL chloroform containing 300 .mu.L triethylamine and stirred at
room temperature for 3 h. The completion of the reaction was
indicated by the disappearance of primary amine as visualized by
ninhydrin assay on TLC. The reaction mixture was concentrated to a
thick paste, redissolved in 6 mL chloroform/methanol (2:1) and
washed once with 1.2 mL 0.5 M HCl four times with 1.2 mL distilled
water. The chloroform phase was extracted and dried. No further
purification was performed. Yield: 700 mg (81%). TLC (silica gel)
chloroform/methanol (85:15) R.sub.f 0.58.
[0310] Dansylated CPL.sub.2 (6). The synthesis of CPL.sub.2 (6) was
the same as that of CPL.sub.1 (4) by deprotecting dansylated
CPL.sub.2-tBoc (5) (700 mg, 318 .mu.mol). Yield: 650 mg (92%). TLC
(silica) chloroform/methanol/water (65:25:4) R.sub.f 0.63.
[0311] Dansylated CPL.sub.4-tBoc (7). The synthesis of
CPL.sub.4-tBoc (7) was the same as that of CPL.sub.2-tBoc (5) by
reacting
N.sub..alpha.,N.sub..epsilon.-di-tBoc-L-lysine-N-hydroxysuccinimide
ester (500 mg, 1127 .mu.mol) with dansylated CPL.sub.2 (6) (650 mg,
292 .mu.mol). Besides washing with dilute HCL and water no further
attempts were made to purify CPL.sub.4-tBoc before deblocking to
generate CPL.sub.4. Yield: 800 mg (Crude). TLC (silica gel)
chloroform/methanol (85:15) R.sub.f 0.58 (dansyl peak only).
[0312] Dansylated CPL.sub.4 (8). The synthesis of CPL.sub.4 (8) was
the same as that of CPL.sub.1 (4) by deprotecting dansylated
CPL.sub.4-tBoc (7) (800 mg). The final product was purified by
column chromatography using silica gel 60, 70-230 mesh, and
chloroform/methanol/ammonia solution (65:25:4 v/v). Yield: 300 mg
(38%). TLC (silica) chloroform/methanol/water (65:25:4) R.sub.f
0.15.
IX. Example IX
[0313] A. General Overview
[0314] We show here that CPL.sub.4 can be inserted into preformed
SPLP and that the resulting SPLP-CPL.sub.4 exhibit improved uptake
and markedly improved in vitro transfection potency in BHK cells.
These results establish that the SPLP system is intrinsically a
highly potent transfection vector.
[0315] B. Materials and Methods
[0316] 1. Preparation of SPLP, SPLP-CPL.sub.4, and Complexes
[0317] (i). SPLP: SPLP composed of DOPE:DODAC:PEG-CerC.sub.20
(84:6:10) and containing the plasmid pLuc, a modified marker gene
expressing luciferase, was supplied by INEX Pharmaceuticals
Inc.
[0318] (ii) SPLP-CPL.sub.4: Dansylated CPL.sub.4 was prepared in
our laboratory and incorporated into SPLP as follows: SPLP at a
dose of 500 nmol lipid was incubated with different amounts of
CPL.sub.4 (12.5, 19, and 30 nmol) at 60.degree. C. for 2 to 3 hours
in Hepes Buffered Saline, pH 7.5 (HBS) to achieve a final
incorporation of 2, 3, and 4 mol %, respectively. SPLP-CPL was
separated from unincorporated CPL by gel filtration chromatography
on a Sepharose CL-4B column equilibrated in HBS. Fractions (1 mL)
were collected and assayed for CPL, phospholipid and DNA contents.
Fractions containing all three components were pooled and
concentrated for use in transfection and uptake studies. The
samples from the column were greatly aggregated. To deaggregate the
systems, addition of CaCl.sub.2 or MgCl.sub.2 was required.
Experiments to determine the optimal amount of cation for
deaggregation will be described later in the Methods.
[0319] CPL Assay: The presence of CPL was determined by measuring
the fluorescence of the dansyl group in CPL on a Perkin Elmer LS52
Luminescence spectrophotometer using .lambda..sub.ex=340 nm and
.lambda..sub.em=510 nm with excitation and emission slit widths of
10 and 20 nm, respectively. Fluorescence of the dansyl was
quantified using a standard curve of dansylated CPL in HBS.
[0320] Phospholipid Assay: Phospholipid was determined by first
extracting the lipids from SPLP using the Bligh-Dyer technique. and
then measuring phosphate in the organic phase according to the
Fiske-Subbarow method (see, Bligh E G, Dyer W J A rapid method of
total lipid extraction and purification. Can J Biochem Physiol
1959; 37: 911-917; and Fiske C H, Subbarow Y. The calorimetric
determination of phosphorous. J Biol Chem 1925; 66: 375-400.).
[0321] DNA Assay: DNA content was measured using the PicoGreen
Assay kit (Molecular Probes, Eugene, Oreg.) as previously
described. (see, Mok KWC, Lam AMI, Cullis PR. Stabilized
plasmid-lipid particles: factors influencing plasmid entrapment and
transfection properties. Biochim Biophys Acta 1999; 1419:
137-150).
[0322] (iii) Complexes: The complexes were prepared, at a charge
ratio of 1.5:1+ve/-ve, by mixing 25 .mu.L of DOPE:DODAC (0.8 mM),
kindly supplied by Inex, with 25 .mu.L of 88 .mu.g/mL pLuc, also
supplied by Inex, followed by incubation for 30 min before addition
to cells.
[0323] 2. Preparation of SPLP Containing 0.5 mol % of Rh-PE for
Optimal Insertion Time Determination and Lipid Uptake
Experiments
[0324] SPLP were prepared as described by Wheeler et al. (see,
Wheeler et al., Gene Therapy; 6:271-281 (1999)) with a few
modifications. The lipids DOPE, PEG-CerC.sub.20, DODAC, and
rhodamine-DOPE (Rh-PE), all stocks in CHCl.sub.3, were mixed
together in a molar ratio of (83.5:10:6:0.5) and the CHCl.sub.3 was
completely evaporated. The resulting lipid film was dissolved in 20
mM octyl glucopyranoside (OGP) and 200 .mu.g/mL of plasmid DNA was
added to a total volume of 1 mL. The OGP was dialysed from the
sample in a dialysis bag with two changes of buffer (HBS) over 48
hours. The resulting sample was passed down a DEAE Sepharose column
and the effluent was run on a discontinuous sucrose gradient as
described previously. (see, Gabizon A, Papahadjopoulos D. Liposome
formulations with prolonged circulation time in blood and enhanced
uptake by tumors. Proc Natl Acad Sci USA 1988; 85: 6949-6953.). The
resulting rhodamine-labeled SPLP possessed a DNA/Lipid ratio of
.about.60 .mu.g/.mu.mol.
[0325] 3. Determination of Optimal Incubation Time for Insertion of
CPL4 into SPLP
[0326] To determine the time required for optimal insertion of
CPL.sub.4 into SPLP, 5 mol % of CPL.sub.4 (0.3 nmol) was mixed with
6 nmol of SPLP (containing 0.5 mol % Rh-PE) in a total volume of
1.5 mL and incubated in a 60.degree. C. water bath. At time points
(30 min, 1 h, 2 h, 3 h, and 4 h), 250 .mu.L of the mixture was run
down a Sepharose CL-4B column equilibrated with HBS. The fractions
possessing fluorescent dansyl were combined and the dansyl
fluorescence was measured using the parameters described above
while the rhodamine fluorescence was measured using
.lambda..sub.ex=560 nm, .lambda.em=590 nm, and excitation and
emission slit widths of 10 and 20 nm, respectively. These
measurements were also made on a small fraction of the original
solution before the column. The dansyl/rhodamine ratios are
calculated for both the initial and final samples to determine the
percentage of the initial 5 mol % that was inserted.
[0327] 4. Deaggregation of SPLP-CPL4 Using CaCl2 and MgCl2
[0328] As stated above, the preparation of SPLP-CPL.sub.4 results
in aggregation of the particles. To deaggregate the system an
increase in ionic strength is required. This was achieved by the
addition of increasing amounts of CaCl.sub.2 or MgCl.sub.2 (500 mM
stock solution) to a solution of SPLP-CPL.sub.4. To 60 .mu.L of
SPLP-CPL.sub.4 (3 mM lipid) was added 360 .mu.L of HBS in a Nicomp
tube. The mean diameter.+-.standard deviation of the SPLP-CPL.sub.4
(0 mM Cation) was then determined by QELS using a Nicomp Model 270
Submicron Particle Sizer. Then the salt (CaCl.sub.2 or MgCl.sub.2)
was added to concentrations from 20 mM to 70 mM. At each interval
the mean diameter.+-.standard deviation was determined by QELS. The
mean diameter of the particles hardly changes with increasing
[Cation], however, the QELS Gaussian distribution gets broader.
Therefore, the standard deviations were used as a measure of
deaggregation.
[0329] 5. Size Determination of SPLP-CPL4 and SPLP
[0330] Freeze-fracture EM was performed on the SPLP-CPL.sub.4 (no
CaCl.sub.2), SPLP-CPL.sub.4+40 mM CaCl.sub.2, and SPLP, according
to Wheeler et al. (see, Wheeler J J et al. Stabilized plasmid-lipid
particles: construction and characterization. Gene Therapy 1999; 6:
271-281.). The SPLP-CPL.sub.4 contained 4 mol % CPL.sub.4. The
micrographs of SPLP-CPL.sub.4, in the presence and absence of
CaCl.sub.2, were compared to show the visual effect of Ca.sup.2+ on
the aggregation. Vesicle diameters of the SPLP-CPL.sub.4+40 mM
CaCl.sub.2 and SPLP were analyzed by QELS using a Nicomp Model 270
Submicron Particle Sizer.
[0331] 6. Serum Stability of SPLP-CPL4 Particles
[0332] The serum stability of the SPLP-CPL containing various % of
CPL were determined by mixing the particles with mouse serum to a
final serum concentration of 50% v. These mixtures were then
incubated for 0, 1, 2, or 4 hours at 37.degree. C. At these time
points, a volume of the mixture containing about 1 .mu.g of plasmid
DNA was removed and the DNA was extracted from the lipid and
protein using a phenol:chloroform extraction. The resulting DNA
solutions were then run on a 1% agarose gel following which the DNA
was transferred to nitrocellulose and a Southern blot was
performed.
[0333] 7. Lipid Analysis of SPLP-CPL.sub.4
[0334] To determine the loss of PEG-CerC.sub.20 from the SPLP
during the insertion of CPL.sub.4, lipid was extracted for the SPLP
sample and SPLP-CPL.sub.4 sample by the Bligh-Dyer extraction. The
mixtures were then passed through an HPLC and were assayed for DOPE
and PEG-CerC.sub.20 by Northern Lipids, Inc (Vancouver, BC). The
DOPE:PEG-CerC.sub.20 ratios for the SPLP-CPL was compared to that
for the SPLP and the amount of PEGylated lipid in the outer
monolayer of the SPLP was determined.
[0335] 8. Uptake Studies
[0336] For all in vitro experiments, the cells used were a
transformed BHK cell line (tk-). For the uptake studies,
1.times.10.sup.5 BHK cells were grown on 12-well plates overnight
in 2 mL of complete media (DMEM+10% FBS) at 37.degree. C. in 5%
CO.sub.2. SPLP, SPLP-CPL.sub.4+40 mM CaCl.sub.2, or DOPE:DODAC
complexes (200 .mu.L), each containing 0.5 mol % Rh-PE as lipid
marker were mixed with 800 .mu.L of complete media and this mixture
was added to the top of the cells at a lipid dose of 20 .mu.M.
After incubation at 37.degree. C. for 2, 4, 6, or 8 hours, the
cells were washed with PBS and lysed with 600 .mu.L of lysis buffer
(0.1% Triton X-100 in PBS). The rhodamine fluorescence of the
lysate was measured in a 1.0 mL microcuvette on a Perkin-Elmer LS52
Luminescence Spectrophotometer using a .lambda..sub.ex of 560 nm
and a .lambda..sub.em of 600 nm with slit widths of 10 and 20 nm,
respectively. An emission filter of 430 nm was also used. Lipid
uptake was determined by comparison of the fluorescence in the
lysate to that of a lipid standard and normalized to the amount of
cells as determined by the BCA protein assay (Pierce, Rockford,
Ill.). Where indicated, fluorescence micrographs were taken on an
Axiovert 100 Zeiss Fluorescent microscope (Carl Zeiss Jena GmbH)
using a rhodamine filter from Omega Opticals (Brattleboro, Vt.)
with the following specifications, .lambda..sub.ex=560.+-.20 nm,
600 nm LP, and DC 590 nm.
[0337] 9. Effect of Type and Concentration of Cation on Lipid
Binding and Uptake
[0338] This uptake experiment was performed with the same
SPLP-CPL.sub.4 (containing 0.5 mol % Rh-PE) as above.
5.times.10.sup.4 BHK cells were plated overnight in 1 mL of
complete media in 24-well plates. The SPLP-CPL.sub.4 (40 nmol) was
mixed with CaCl.sub.2 or MgCl.sub.2 at various initial
concentrations of 20 mM to 70 mM in a total volume of 100 .mu.L. To
this was added 400 .mu.L of complete media resulting in final
[Cation] of 4 mM to 14 mM. This mixture was then added to the top
of the cells and the cells incubated for 4 hours. After incubation
the cells were washed twice with PBS and 600 .mu.L of lysis buffer
(0.1% Triton X-100 in PBS) was added. As above, the rhodamine
fluorescence was measure and the lipid uptake was determined
comparing the resulting fluorescence to that of a standard sample
containing a known amount of lipid. The resulting values were then
normalized to the number of cells by measuring the protein content
using the BCA protein assay kit.
[0339] 10. Transfection Studies
[0340] 1.times.10.sup.4 BHK cells were plated in 96-well plates in
150 .mu.L complete media and incubated overnight at 37.degree. C.
in 5% CO.sub.2. SPLP and SPLP-CPL, containing between 2 and 4 mol %
CPL, were prepared to deliver 0.5 .mu.g of DNA in a total volume of
20 .mu.L using HBS (SPLP), or HBS+40 mM CaCl.sub.2 (SPLP-CPL.sub.4)
and were added to 90 .mu.L of complete media. Samples were
incubated with the cells for 4 hours. The transfection media was
then replaced with complete media for a complete 24 hour
incubation. Cells were then lysed with 100 .mu.L of lysis buffer,
and 40 .mu.L of the lysate was transferred to a 96-well
luminescence plate. Luciferase activity was determined using a
Lucifcrase reaction kit (Promega, Madison, Wis.), a luciferase
standard (Boehringer-Manheim), and a ML3200 microtiter plate
luminometer from Molecular Dynamics (Chantilly, Va.). Activity was
normalized to the number of cells as measured by the BCA protein
assay (Pierce, Rockford, Ill.). From the uptake and transfection
experiments above, it was determined that 4 mol % CPL.sub.4 in
SPLP-CPL.sub.4 gave optimal results. Thus, the rest of the
experiments were performed with SPLP-CPL.sub.4 containing 4 mol %
CPL.sub.4.
[0341] 11. Time Course for the transfection of SPLP-CPL versus SPLP
and Complexes
[0342] Samples and cells were prepared as described for the above
transfection study, and incubated together at 37.degree. C. As
well, Lipofectin (Gibco BRL, ) complexes containing pLuc were
prepared at a charge ratio of 1.5:1. At 4, 9, and 24 hours, the
transfection media was removed and in the case of the 4 and 9 hour
transfections, replaced with complete media for a complete 24-hour
incubation. At 24 h, all cells were lysed and assayed for
luciferase activity and protein content (BCA assay), as above.
[0343] 12. Transfection Potency and Toxicity of SPLP-CPL4
[0344] BHK cells were incubated with SPLP, SPLP-CPL.sub.4+40 mM
CaCl.sub.2, and Lipofectin complexes for 24 or 48 hours. After the
incubation period the cells were immediately lysed and the
luciferase activity was measured and was normalized to the amount
of protein present, as above.
[0345] As a rough measure of cell survival at the above time
points, the protein concentration after cell lysis at 24 and 48
hours was measured and compared for the SPLP-CPL.sub.4+40 mM
CaCl.sub.2 and the Lipofectin complexes.
[0346] 13. Comparison of Effect of Ca2+ and Mg2+ on Transfection of
BHK Cells
[0347] Cells were plated and used as above. SPLP-CPL.sub.4 (5.0
.mu.g/mL) with either CaCl.sub.2 or MgCl.sub.2 at concentrations of
20 mM to 70 mM were combined in a volume of 20 .mu.L and mixed with
complete media, resulting in final [Cation] of 4 mM to 14 mM.
Following incubation on the cells for 48 hours, the cells were
washed and lysed, and the luciferase activity and protein content
were measured as above.
[0348] 14. Measurement of Transfection Efficiency of SPLP-CPL4
[0349] The transfection efficiency of the SPLP-CPL was measured by
preparing SPLP-CPL.sub.4 containing encapsulated pEGFP (kindly
supplied by Inex), that expresses GFP (green fluorescence protein),
using the detergent dialysis procedure. (see, Wheeler et al.
supra). 400 .mu.g/mL of pEGFP was encapsulated within 10 mM
DOPE:PEG-CerC20:DODAC (84:10:6), followed by the insertion of 4 mol
% of CPL.sub.4. DOPE:DODAC complexes and Lipofectin complexes
containing pEGFP were also prepared at a charge ratio of 1.5:1. The
transfections were performed as described earlier at a DNA dose of
5.0 .mu.g/mL. Following incubation of the samples for 24 and 48
hours, the transfection media was removed, the cells were washed,
and fresh media was added to the cells. The cells were then viewed
under the Zeiss fluorescence microscope. The total number of cells
within the frame were counted; then the number of cells expressing
the GFP were counted using a fluorescein filter (Omega Opticals)
with the following specifications, .lambda..sub.ex=470.+-.20 nm,
.lambda..sub.em=535.+-.22.5 nm, and DC .about.500 nm. The
efficiency of transfection is the number of cells expressing the
GFP divided by the total number of cells.
[0350] C. Results and Discussion
[0351] 1. SPLP-CPL4 Aggregate Following Insertion of CPL4 and
De-aggregate Following Addition of Divalent Cations.
[0352] LUV containing CPL tend to aggregate, and that this
aggregation can be inhibited by increasing the ionic strength of
the medium. It was found that SPLP-CPL.sub.4 were also susceptible
to aggregation, and that this aggregation could be reversed by
adding NaCl, CaCl.sub.2 or MgCl.sub.2 to the SPLP-CPL.sub.4
formulation. This effect is illustrated in FIG. 31 which shows the
effect of the addition of CaCl.sub.2 and MgCl.sub.2 on aggregation
of SPLP-CPL.sub.4 as monitored by the change in the standard
deviation of the mean diameter of the particles measured by
quasi-elastic light scattering (QELS). For both cations the
standard deviation decreases with increasing cation concentration
with optimal de-aggregation occurring above 30 to 40 mM. This
behavior could also be visualized by freeze-fracture electron
microscopy. Freeze-fracture micrographs of SPLP reveal small
monodisperse particles, whereas SPLP-CPL.sub.4 prepared in the
absence of CaCl.sub.2 are highly aggregated. The addition of 40 mM
CaCl.sub.2 reverses this aggregation to produce monodisperse
particles similar to the SPLP preparation. For details of sample
preparation and electron microscopy, (see, Wheeler et al., Gene
Therapy; 6:271-281 (1999)).
[0353] The sizes of SPLP and SPLP-CPL.sub.4 in the presence of
CaCl.sub.2 were compared using QELS and freeze-fracture electron
microscopy. QELS studies revealed the mean diameter of SPLP and
SPLP-CPL.sub.4 to be 80.+-.19 nm and 76.+-.15 nm, respectively,
whereas the freeze-fracture studies indicated to diameters of
68.+-.11 nm and 64.+-.14 nm. These values for SPLP are in close
agreement with previous studies.
[0354] 2. Chemical Composition and Stability of SPLP-CPL4.
[0355] The lipid composition of SPLP-CPL.sub.4 and SPLP are given
in Table 9 below:
7TABLE 9 Loss of PEG-CerC.sub.20 from SPLP following CPL.sub.4
insertion. [DOPE] [PEG-CerC.sub.20] % PEG-CerC.sub.20 after (mM)
(mM) DOPE:PEG-C.sub.20 insertion 0.786 0.0714 .+-. 0.0004 11.0 .+-.
0.1 79.7 .+-. 0.9% (81.6:7.4; mol) (x = 5.9 .+-. 0.1 mol %)
SPLP-CPL.sub.4 0.790 .+-. 0.007 0.0572 .+-. 0.0003 13.8 .+-. 0.1
(81.6:x; mol)
[0356] By analysis of the SPLP itself, the molar ratio of
DOPE:PEG-CerC.sub.20 was 11.0(.+-.0.1):1. This corresponds to a
system of DOPE:PEG-CerC.sub.20:DODAC of (81.6:10.9:7.4). From the
results, 79.7.+-.0.9% of the PEG-CerC.sub.20 remains following
CPL.sub.4 insertion. This corresponds to a final mol % of
PEG-CerC.sub.20 of 5.9.+-.0.1 mol %. This means that about
1.5.+-.0.1 mol % of PEG-CerC.sub.20 was replaced during the
insertion of CPL.sub.4. If we assume that on the inner leaflet and
outer leaflet the same amount of PEG-CerC.sub.20 is initially
present at 7.4 mol %, the outer leaflet will possess 4.4.+-.0.1 mol
% of PEG-CerC.sub.20 after insertion. Since we inserted .about.4.5
mol % CPL.sub.4 into SPLP (9.0 mol % in the outer leaflet),
resulting in a total of 13.4.+-.0.1 mol % of total PEG in the outer
leaflet.
[0357] The stability of SPLP and SPLP-CPL.sub.4 in 50% mouse serum
for up to 4 hours. In all cases, the DNA was completely protected
from serum degradation.
[0358] 3. SPLP-CPL.sub.4 Exhibit Enhanced Uptake into BHK Cells and
Dramatically Enhanced Transfection Potency.
[0359] The next set of experiments was aimed at determining the
influence of incorporated CPL.sub.4 on the uptake of SPLP into BHK
cells and the resulting transfection potency of the SPLP-CPL.sub.4
system. SPLP containing up to 4 mol % CPL.sub.4 were prepared in
the presence of 40 mM CaCl.sub.2 and were added to BHK cells (final
CaCl.sub.2 concentration 8 mM) and incubated for varying times. The
cells were then assayed for associated SPLP-CPL.sub.4 as indicated
in Methods. As shown in FIG. 32, uptake of SPLP that contain no
CPL.sub.4 is minimal even after 8 h of incubation, however uptake
is dramatically improved for SPLP containing 3 mol % or higher
levels of CPL.sub.4. For example, SPLP containing 4 mol % CPL.sub.4
exhibit accumulation levels at 8 h that are approximately 50-fold
higher than achieved for SPLP. This enhanced uptake can be visually
detected using fluorescence micrographs of BHK cells following
incubation with rhodamine-labeled SPLP and SPLP-CPL.sub.4 for 4 h.
The presence of 4 mol % CPL.sub.4 clearly results in improved
levels of cell-associated SPLP.
[0360] The transfection properties of SPLP, SPLP-CPL.sub.4 and
plasmid DNA-cationic lipid complexes (DODAC/DOPE; 1:1; 1.5:1+ve/-ve
c.r.) were examined using the incubation protocol usually employed
for complexes. This consisted of incubation of 10.sup.4 BHK cells
with SPLP, SPLP-CPL.sub.4 and complexes containing 0.5 .mu.g
pCMVLuc for 4 h, followed by removal of SPLP, SPLP-CPL.sub.4 or
complexes that are not associated with the cells, replacement of
the media, incubation for a further 20 h and then assaying for
luciferase activity. The SPLP-CPL.sub.4 preparations contained 7 mM
CaCl.sub.2 in the incubation medium. As shown in FIG. 33, the
presence of the CPL.sub.4 resulted in dramatic increases in the
transfection potencies of the SPLP system. SPLP-CPL.sub.4
containing 4 mol % CPL.sub.4 exhibited luciferase expression levels
some 3.times.10.sup.3 higher than achieved with SPLP. (see, Mok et
al., Biochim Biophys Acta, 1419:137-150 (1999)).
[0361] 4. Ca2+ is Required for Transfection Activity of
SPLP-CPL.sub.4.
[0362] It was of interest to determine the influence of Ca.sup.2+
on the transfection activity of SPLP-CPL.sub.4. SPLP containing 4
mol % CPL.sub.4 were incubated with BHK cells for 48 h in the
presence of 0-14 mM MgCl.sub.2 and CaCl.sub.2 and the luciferase
activities then determined. As shown in FIG. 34, the transfection
activity was influenced by the presence of Ca.sup.2+ in the
transfection medium. At the optimum CaCl.sub.2 concentration of 10
mM, SPLP-CPL.sub.4 exhibited transfection potencies that were more
than 10.sup.4 times higher than if MgCl.sub.2 was present.
[0363] Uptake of SPLP-CPL.sub.4 into BHK cells was monitored
following a 4 h incubation in the presence of 0-14 mM MgCl.sub.2
and CaCl.sub.2. As shown in FIG. 35 the amount of SPLP-CPL.sub.4
taken up by BHK cells is the same for both Mg.sup.2+ and
Ca.sup.2+-containing media. The uptake of the SPLP-CPL.sub.4
decreases as the concentration of divalent cations increases, which
likely arises due to shielding of the negatively charged binding
sites for the CPL.sub.4 on the surface of the BHK cells.
[0364] 5. SPLP-CPL.sub.4 Exhibit Transfection Potencies in Vitro
that are Comparable to or Greater than Achieved Using
Complexes.
[0365] The results shown in FIG. 33 indicating that complexes give
rise to .about.100-fold higher levels of transfection than
SPLP-CPL.sub.4 were obtained for a fixed 4 h incubation time with
the BHK cells, followed by a 20 h hold time to achieve maximum
expression. Given that the SPLP-CPL.sub.4 are stable systems it is
likely that uptake into the BHK cells would continue over extended
time periods. The transfection levels achieved when the incubation
time of the SPLP-CPL.sub.4 and the complexes with the BHK cells was
extended to 8 and 24 h, followed by hold times of 16 and 0 h
respectively were examined. Two types of plasmid DNA-cationic lipid
complexes were used, namely DOPE:DODAC (1:1) complexes (1.5:1,
c.r.) and complexes obtained using the commercial transfection
reagent Lipofectin (DOPE/DOTMA [1:1] complexes, 1.5:1 c.r.). As
shown in FIG. 36, the transfection potency of the SPLP-CPL.sub.4
increases markedly with increased incubation times, suggesting that
a limiting factor for transfection achieved at a 4 h incubation
time was the rate of uptake of the SPLP-CPL.sub.4 system. At the 24
h incubation time transfection levels are achieved that are
comparable to those achieved by Lipofectin or DOPE/DODAC
complexes.
[0366] Further experiments were conducted to determine transfection
levels after 24 and 48 h incubation times with luciferase
activities assayed immediately following the incubation period. As
shown in FIG. 37A the activity of Lipofectin (DOPE/DOTMA; 1:1)
complexes leveled off at .about.2000 ng/mg after 24 h. In contrast,
the activity of SPLP-CPL.sub.4 formulation continued to increase as
the incubation time was increased, achieving luciferase expression
levels corresponding to 4000 ng/mg at 48 h. This activity is
approximately 10.sup.6 times higher than observed for SPLP (in the
absence of Ca.sup.2+) and almost double the levels that can be
achieved by Lipofectin complexes. Similar results were obtained for
the DOPE:DODAC complexes.
[0367] 6. SPLP-CPL4 are Non-Toxic and Efficient Transfection
Agents.
[0368] It is well known that plasmid DNA-cationic lipid complexes
can be toxic to cells. The SPLP-CPL.sub.4 contain low levels of
cationic lipid and are potentially less toxic than complexes. The
toxicity of SPLP-CPL.sub.4 and complexes was assayed by determining
cell viability following a 48 h exposure to levels of
SPLP-CPL.sub.4 and complexes corresponding to 0.5 .mu.g plasmid and
.about.30 nmol total lipid. As shown in FIG. 37B, SPLP-CPL.sub.4
exhibit little if any toxicity. Cell survival was only 30% after a
48 h incubation with Lipofectin complexes whereas .about.95% of the
cells were viable following a 48 hour incubation with
SPLP-CPL.sub.4.
[0369] The efficiency of transfection, indicated by the proportion
of cells transfected by a vector, is also an important parameter.
The proportion of cells transfected were estimated using plasmid
carrying the green fluorescent protein (GFP) gene. Transfection was
detected by expression of the fluorescent protein inside a cell
employing fluorescence microscopy. As shown in FIGS. 37A and 38B,
approximately 35% of the cells at 24 h and 50% at 48 h were
transfected by SPLP-CPL.sub.4, with no apparent cell death. In
contrast, Lipofectin complexes exhibit maximum transfection
efficiencies of less than 35% and only .about.50% cell survival
after the 24 h transfection period. Similar low transfection
efficiencies and high toxicities were also seen with DOPE:DODAC
complexes.
[0370] The results of this study demonstrate that the incorporation
of CPL.sub.4 into SPLP results in improved uptake into BHK cells
and a dramatically enhanced transfection potency of SPLP when Ca is
present. There are three points of interest. The first concerns the
chemical composition and structure of the SPLP-CPL.sub.4 system and
the generality of the post-insertion procedure for modifying the
trophism and transfection potency of SPLP. The second concerns the
relation between enhanced uptake of SPLP, the presence of Ca.sup.2+
and the transfection activities observed. Finally, it is of
interest to compare the properties of the SPLP-CPL.sub.4 system
with plasmid DNA-cationic lipid complexes.
[0371] The second point of discussion concerns the mechanism
whereby CPL.sub.4 increases the transfection potency of the SPLP
system. Clearly the presence of the CPL.sub.4 increases the uptake
of SPLP into the BHK cells, however the increase in transfection
potency is almost entirely dependent on the additional presence of
Ca.sup.2+. It may be noted that, following an 8 h incubation, the
presence of 4 mol % CPL.sub.4 increases the uptake of SPLP into BHK
cells by approximately 50-fold, whereas the transfection potency
(in the presence of Ca.sup.2+) is increased by a factor of
.about.10.sup.4. Previous work conducted on SPLP has shown that the
presence of Ca.sup.2+ results in a maximum increase in transfection
potency of .about.600 and that this increase in potency results
from an ability of Ca.sup.2+ to assist in destabilizing the
endosomal membrane following uptake, rather than an increase in
uptake itself. In turn, this suggests that the improvement in
transfection potency for the SPLP-CPL.sub.4 system over the SPLP
system arises from the CPL.sub.4-dependent increase in uptake
multiplied by the Ca.sup.2+-dependent improvement in intracellular
delivery following uptake.
[0372] The final area of discussion concerns the advantages of the
SPLP-CPL.sub.4 system over other non-viral vectors, which include
the well-defined modular nature of the SPLP-CPL.sub.4 system as
well as toxicity and potency issues. First, the well-characterized
nature of the SPLP-CPL.sub.4 as small, homogeneous, stable systems
containing one plasmid per particle contrast with non-viral systems
such as plasmid DNA-cationic lipid complexes which are large,
inhomogeneous, unstable systems containing ill-defined numbers of
plasmids per complex. An important point is that SPLP are basic
components of more sophisticated systems, such as SPLP-CPL.sub.4,
which can be constructed in a modular fashion. For example,
post-insertion of PEG-lipids which contain specific targeting
ligands in place of the cationic groups of CPL should result in
SPLP that are specifically targeted to particular cells and
tissues. With regard to toxicity, it is clear that SPLP-CPL.sub.4
are markedly less toxic to BHK cells in tissue culture. This is
presumably related to the low proportions of cationic lipid
contained in SPLP as compared to complexes. The transfection
potency and efficiency of SPLP-CPL.sub.4 is clearly comparable to
the levels that can be achieved with complexes. It should be noted
that this finding suggests that models of transfection by complexes
that involve.
[0373] In the present example, the superiority of SPLP-CPL.sub.4
compared to commercially available complex systems (e.g.
Lipofectin) has been demonstrated. Thus, a synthetic virus has been
developed that will have high transfection potency but none of the
problems associated with viruses. Many points can be made to
corroborate these statements. The first point revolves around the
placement of the charge. Whereas on complexes the charges are
located on the surface of the lipid bilayer, the SPLP-CPL.sub.4
possess charges on the vesicle surface which are localized a good
distance from the liposomal surface, above the protective PEG
coating which surrounds the liposome. In the case of the complexes,
proteins binding to the liposome surface can lead to recognition
and clearance by macrophages of the RES. (see, Chonn et al., J Biol
Chem; 267:18759-18765 (1992)) In the SPLP-CPL.sub.4, the charge on
the surface of the bilayer is protected by the PEG coating, such
that this should not occur. However, the charge on the
SPLP-CPL.sub.4 will allow the association of the liposomes with
cells resulting in eventual uptake and transfection.
[0374] The size and serum stability of the SPLP-CPL.sub.4 compared
to complexes are important parameters for effective gene delivery
systems, especially if one wishes to approach the capabilities of
viral systems. The SPLP-CPL.sub.4 have been shown here to be of
relative small size (.about.100 nm) compared to complexes, which
are frequently on the order of microns in diameter. The small size
should allow for accumulation at sites with larger fenestration
(e.g. tumors, and inflammation sites). (see, Kohn et al., Lab
Invest; 67:596-607 (1992)). As stated earlier, DNA in the
SPLP-CPL.sub.4 was shown to be protected from the external
environment (i.e. inaccessible to degradation by DNase within
serum), whereas DNA in complexes is susceptible to DNase. (see,
Wheeler et al., Gene Therapy; 6:271-281 (1999)).
[0375] Viruses (see, Hermonat et al., Proc. Natl. Acad. Sci. USA;
81:6466-6470 (1984); Lebkowski et al., Molec Cell Biol; 8:3988-3996
(1988); Keir et al., J Neurovirology, 3:322-330 (1997)]and
lipid/DNA complexes (see, Felgner et al., Proc Natl Acad Sci USA,
84:7413-7417 (1987); Felgner et al., J Biol Chem; 269:2550-61
(1994); Hofland et al., Proc Natl Acad Sci USA; 93:7305-7309
(1996); Bebok et al., J Pharm Exp Ther; 279:1462-1469 (1996); Gao
et al., Gene Therapy; 2:710-722 (1995)) have been shown to possess
high in vitro transfection potencies. It therefore reasons that the
SPLP-CPL.sub.4 system, if it is to attain viral qualities, should
be capable of attaining these high transfections. This has actually
been achieved by the SPLP-CPL.sub.4 system on BHK cells, with
transfection levels reaching a factor of two higher than a
commercially available complex system (i.e. Lipofectin). This is a
huge improvement over SPLP, which showed only a small amount of
transfection.
[0376] Efficient systemic delivery and transfection of genetic
drugs are achieved using this SPLP-CPL.sub.4 system due to the
above benefits. Very high transfections in vitro with
SPLP-CPL.sub.4 have been achieved. In addition, a system wherein
the positioning of the positive charges on the CPL, so that the PEG
of the PEG-Cer initially masks it. This is achieved by the
synthesis of DSPE-PEG-CPL.sub.4 with a shorter PEG moiety. This
allows for its accumulation at disease sites followed by the
controlled release of the PEG-Cer, exposing the positive charges to
the surrounding cells.
Example X
[0377] This example shows transfection rates of BHK cells by long-
versus short-chained CPLs.
[0378] Using synthesis methods from above, CPL (PEG 3.4k) and
CPL(PEG 1k) were generated and each inserted into a separate SPLP
system containing PEG-.sub.2000-Cer C20 as described above. FIG. 38
illustrates transfection rates of the CPLs having a PEG 3.4k versus
a CPL having a PEG 1k. The short-chained PEG in the CPL results in
a decrease by a factor of about 4 compared to the transfection by
the long chained CPL. Without being bound by any particular theory,
it is believed that the long chain CPL (PEG.sub.3400) sticks out
above the surface, whereas the short chain CPL (PEG.sub.1000) is
buried (masked) in the surface of the SPLP. The reduced in vitro
transfection of the short chain CPL clearly suggests that it has
improved in vivo circulation.
Example XI
[0379] This example shows that CPL8 behaves similar to CPL4 with
respect to insertion into LUVs, and that transfection can be
achieved with CPL8-LUV systems.
8TABLE 10 Insertion of CPL.sub.8 in SPLP and LUV. Initial mol %
CPL.sub.8 % Insertion Final mol % CPL.sub.8 SPLP 1.11 97% 1.07 1.39
85% 1.19 1.67 95% 1.60 1.94 87% 1.70 LUV 1.05 79% 0.82 1.39 71%
0.99 1.74 76% 1.32 2.11 89% 1.88
[0380] The insertions of the CPL.sub.8 into LUV and SPLP is very
similar to what was observed for the insertions of CPL.sub.4. For
the transfection and uptake of these particles on BHK cells,
variable results are obtained, with the CPL.sub.8 performing better
than the CPL.sub.4 sometimes and vice versa at other times.
Example XII
[0381] In this in vitro example using mouse neuroblastoma cell line
Neuro-2a (ATCC--CCL-131), the SPLP-CPL4[1k] is used to determine
gene expression with respect to varying Ca.sup.2+ concentrations
and to compare to gene expression using a standard SPLP (PEG-CerC20
10%; CPL.sub.4[1k] 4%; and other components; DNA:lipid
ratio=0.05).
[0382] 5.times.10.sup.4 cells/well are plated in 24-well plates in
1 mL of complete media (MEM(Eagle) with non-essential amino acids
and Hanks' buffered salt solution with 10% FBS). Plates are
incubated overnight at 37.degree. C. with 5.0% CO.sub.2. To each
group set out below is added 500 .mu.L transfection media in
triplicate.
9TABLE 11 SPLP or SPLP-CPL [Ca2+] Complete GROUP (.mu.g) (250 mM)
(.mu.L) Media (.mu.L) A (0 mM Ca2+) 2.5 0 1980 B (2 mM Ca2+) 2.5 48
1932 C (4 mM Ca2+) 2.5 96 1884 D (6 mM Ca2+) 2.5 144 1836 E (8 mM
Ca2+) 2.5 192 1788 F (10 mM Ca2+) 2.5 240 1740 G (12 mM Ca2+) 2.5
288 1692 H (14 mM Ca2+) 2.5 336 1644
[0383] 2.5 .mu.g DNA is added per well in fully encapsulated SPLPs
(0.5 mL total solution). Plates are incubated for 8 hrs.
Transfection media is removed. 1 mL of complete media is added
back. Cells are incubate for another 24 hrs at 37.degree. C., 5.0%
CO.sub.2.
[0384] For analysis, media is removed from cells and they are
washed 2.times. with PBS then frozen at -70.degree. C. Cells are
lysed with 150-200 .mu.L 1.times.CCLR; then shaken 5 minutes on
plate shaker. 20 .mu.L lysate is transferred to a 96-well
luminescence plate. Plates are read to determine luciferase
activity.
[0385] The results are shown in FIG. 39. As shown therein, SPLP+4
mol % CPL4-1k produces 4 orders of magnitude of gene expression
more than SPLP alone in Neuro-2a cells. Effects of calcium are not
considered to be significant in this experiment. The amount of
luciferase produced remains the same from 2-14 mM Ca2+.
Example XIII
[0386] This in vivo example discloses pharmacokinetics and
biodistribution of CPL.sub.4-1-k LUVs (SPLPs containing short chain
CPLs) in C57/b16 mice. Different SPLP formulations containing
increasing amounts of CPL-4-1k are assayed in vivo to determine
optimal clearance characteristics.
[0387] CPL.sub.4-1k SPLPs are prepared according to previous
protocols. Before use, all samples are characterized to determine
actual composition prior to administration. All samples are filter
sterilized prior to dilution to working concentration. All samples
are to provided in sterile crimp top vials. All vials are labeled
with the formulation date, lipid composition, and specific
activity. .sup.3[H]CHE is incorporated at 1 .mu.Ci/mg Lipid. The
following formulations are made and analyzed:
[0388] A: .sup.3[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10
[0389] B: .sup.3[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+1 mol %
CPL-4-1k
[0390] C: .sup.3[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+2 mol %
CPL-4-1k
[0391] D: .sup.3[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+3 mol %
CPL-4-1k
[0392] E: .sup.3[H]CHE-LUV DOPE:DODAC:PEGC20:84:6:10+4 mol %
CPL-4-1k
[0393] Experiments used 100 C57/b16 mice, female, 18-23 g all
ordered from Harlan Sprague Dawley. All animals housed in cages of
4 animals per group in 25 groups.
10TABLE 12 Group Mice Treatment Timepoint Assay A 4
A:DOPE:DODAC:PEGC20:84:6:10 15 min PK B 4
A:DOPE:DODAC:PEGC20::84:6:10 1 hr PK C 4 A:DOPE:DODAC:PEGC20::84:-
6:10 4 hr PK D 4 A:DOPE:DODAC:PEGC20::84:6:10 8 hr PK E 4
A:DOPE:DODAC:PEGC20::84:6:10 24 hr PK F 4 B:DOPE:DODAC:PEGC20::84:-
6:10 + 1 mol % CPL-4-1k 15 min PK G 4 B:DOPE:DODAC:PEGC20::84:6:10
+ 1 mol % CPL-4-1k 1 hr PK H 4 B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol
% CPL-4-1k 4 hr PK I 4 B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol %
CPL-4-1k 8 hr PK J 4 B:DOPE:DODAC:PEGC20::84:6:10 + 1 mol %
CPL-4-1k 24 hr PK K 4 C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol %
CPL-4-1k 15 min PK L 4 C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol %
CPL-4-1k 1 hr PK M 4 C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol %
CPL-4-1k 4 hr PK N 4 C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol %
CPL-4-1k 8 hr PK O 4 C:DOPE:DODAC:PEGC20::84:6:10 + 2 mol %
CPL-4-1k 24 hr PK P 4 D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol %
CPL-4-1k 15 min PK Q 4 D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol %
CPL-4-1k 1 hr PK R 4 D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol %
CPL-4-1k 4 hr PK S 4 D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol %
CPL-4-1k 8 hr PK T 4 D:DOPE:DODAC:PEGC20::84:6:10 + 3 mol %
CPL-4-1k 24 hr PK U 4 E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol %
CPL-4-1k 15 min PK V 4 E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol %
CPL-4-1k 1 hr PK W 4 E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol %
CPL-4-1k 4 hr PK X 4 E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol %
CPL-4-1k 8 hr PK Y 4 E:DOPE:DODAC:PEGC20::84:6:10 + 4 mol %
CPL-4-1k 24 hr PK
[0394] Mice were treated with .sup.3[H]CHE-LUV administered by tail
vein I.V. in a total volume of 200 .mu.l . Mice receive one
treatment only. At the indicated time-points mice are weighed,
sacrificed, and blood will be collected by cardiac puncture then
evaluated for .sup.3[H]CHE. Formulations are expected to be well
tolerated. Mice are treated according to certified animal care
protocols. Any mice exhibiting signs of distress associated with
the treatment are terminated at the discretion of vivarium staff.
All mice are terminated by CO.sub.2 inhalation followed by cervical
dislocation. Measurement of .sup.3[H]CHE from blood is determined
according to standard protocols.
[0395] In vivo pharmacokinetics of SPLP containing short chain
CPL.sub.4 are illustrated in FIG. 40. It is observed that that
increasing amounts of the CPL.sub.4 in the SPLP tends to increase
the rate of clearance from the blood. CPL.sub.4 incorporated a 1
mol % gives clearance results which are similar to SPLPs without
CPL.sub.4. Incorporation of higher amounts of CPL4 tends to
increase the rate of clearance of the SPLP from the blood.
SPLP-CPL.sub.4 [1k] (1%) shows best plasma clearance
characteristics with a t.sub.1/2 of 6-7 hours. Anything greater
than 1 mol % clears more rapidly.
[0396] The results disclosed in this specification indicate a
further refinement of SPLP technology. In particular, from these
results it is clear that the type of CPL (i.e. the length of the
polymer chain; and the amount of cationic charge per molecule) and
the amount of such CPL in an SPLP must be optimized to obtain the
best balancing of clearance properties in vivo with enhanced
transfection ability. In vitro data has shown long chain CPLs and
higher levels of such CPLs are to be preferred to increase
transfection. However, as seen in previous comparisons of SPLPs
versus lipid complexes, lipid formulations that work best in vitro
are not best suited in vivo. In vivo results herein demonstrate
that short chain CPLs incorporated at approximately 1% are
optimized for circulation lifetimes in animals.
[0397] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *