U.S. patent application number 15/011576 was filed with the patent office on 2016-06-02 for amphoteric liposome formulations.
The applicant listed for this patent is Marina Biotech, Inc.. Invention is credited to Silke Lutz, Claudia Muller, Steffen Panzner, Evgenios Siepi.
Application Number | 20160151282 15/011576 |
Document ID | / |
Family ID | 39103329 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151282 |
Kind Code |
A1 |
Panzner; Steffen ; et
al. |
June 2, 2016 |
AMPHOTERIC LIPOSOME FORMULATIONS
Abstract
An amphoteric liposome composed of a mixture of lipids, said
mixture comprising a cationic amphiphile, an anionic amphiphile and
optionally one or more neutral amphiphiles, at least one of said
cationic and anionic amphiphiles being chargeable and the
respective amounts of said cationic and anionic amphiphiles being
selected such there is a stoichiometric excess of positively
charged cationic amphiphile at a first lower pH, a stoichiometric
excess of negatively charged anionic amphiphile at a second higher
pH and said mixture has an isoelectric point intermediate said
first and second pHs; characterised in that said positively charged
cationic and negatively charged anionic amphiphiles are adapted to
form a lipid salt with one another at said isoelectric point. Also
disclosed are methods of predicting the fusogenicity of an
amphoteric liposome at a given pH, formulating an amphoteric
liposome and loading an amphoteric liposome with a cargo
moiety.
Inventors: |
Panzner; Steffen; (Halle,
DE) ; Lutz; Silke; (Halle, DE) ; Siepi;
Evgenios; (Leipzig, DE) ; Muller; Claudia;
(Nerchau, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Marina Biotech, Inc. |
Bothell |
WA |
US |
|
|
Family ID: |
39103329 |
Appl. No.: |
15/011576 |
Filed: |
January 31, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13350137 |
Jan 13, 2012 |
9283186 |
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15011576 |
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11974350 |
Oct 12, 2007 |
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13350137 |
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11581054 |
Oct 13, 2006 |
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11974350 |
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Current U.S.
Class: |
424/450 ;
514/44A; 514/44R |
Current CPC
Class: |
Y02P 20/582 20151101;
A61K 9/127 20130101; A61K 31/713 20130101; A61K 9/1272
20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/713 20060101 A61K031/713 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 13, 2006 |
EP |
06255277.3 |
Claims
1. An amphoteric liposome formulation comprising a cationic
amphiphile, an anionic amphiphile, and one or more neutral
amphiphiles.
2. The amphoteric liposome formulation of claim 1, wherein the
cationic amphiphile is selected from the group consisting of
4-(2-aminoethyl)-morpholino-cholesterol-2,3-dimethylhemisuccinate
(DmC4Mo2),
4-(2-aminoethyl)-morpholino-cholesterol-2,3-dimethylhemimalonate
(DmC3Mo2), 4-(2-aminobutyl)-morpholino-cholesterol-hemisuccinate
(C4Mo4), 4-(2-aminopropyl)-morpholino-cholesterol-hemimalonate
(C3Mo3), 4-(2-aminoethyl)-morpholino-cholesterol-hemimalonate
(C3Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemiglutarate
(C5Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemiadipate
(C6Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemiadipate
(C8Mo2), 4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate
(MoChol), Cholesterol-(3-imidazol-1-yl propyl)carbamate (CHIM), and
Histaminyl-Cholesterolhemisuccinate (HisChol).
3. The amphoteric liposome formulation of claim 1, wherein the
anionic amphiphile is selected from the group consisting of
cholesterol hemisuccinate CHEMS, Dimyristoylglycerolhemisuccinate
DMGS, Dimyristoylglycerolhemimalonate DMGM,
Dimyristoylglycerolhemiglutarate DMGG,
Dimyristoylglycerolhemiadipate DMGA,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxobutanoic acid DMAS,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxopropanoic acid DMAM,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxopentanoic acid DMAG,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxohexanoic acid DMAA,
Dioleoylglycerolhemisuccinate DOGS, Dioleoylglycerolhemimalonate
DOGM, Dioleoylglycerolhemiglutarate DOGG,
Dioleoylglycerolhemiadipate DOGA,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxobutanoic acid DOAS,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxopropanoic acid DOAM,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxopentanoic acid DOAG,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxohexanoic acid DOAA,
5,6-Dimyristoyl-hexanoic acid DMS, 4,5-Dimyristoyl-pentanoic acid
DMM, 6,7-Dimyristoyl-heptanoic acid DMG, 7,8-Dioleoyl-octanoic acid
DMA, 5,6-Dioleoyl-hexanoic acid DOS, 4,5-Dioleoyl-pentanoic acid
DOM, 6,7-Dioleoyl-heptanoic acid DOG, 7,8-Dioleoyl-octanoic acid
DOA, Cholesterolhemimalonate Chol-C3, Cholesterolhemiglutarate
Chol-C5, and Cholesterolhemiadipate Chol-C6.
4. The amphoteric liposome formulation of claim 1, wherein the
neutral amphiphiles are selected from the group consisting of
phosphatidylcholines, sphingomyelins, ceramides,
phosphatidylethanolamines, cholesterol, and mixtures thereof.
5. The amphoteric liposome formulation of claim 1, wherein the
neutral amphiphiles are selected from phosphatidylcholines and
phosphatidylethanolamines.
6. The amphoteric liposome formulation of claim 1, wherein the
neutral amphiphiles are selected from POPC and DOPE.
7. The amphoteric liposome formulation of claim 1, wherein the
formulation has an isoelectric point of from pH 4 to pH 8.
8. The amphoteric liposome formulation of claim 1, wherein the
neutral amphiphiles are less than 50 mol % of the composition and
the composition has an isoelectric point of from pH 4 to pH 8.
9. The amphoteric liposome formulation of claim 1, wherein the
formulation comprises an amphoteric liposome.
10. The amphoteric liposome formulation of claim 1, wherein the
formulation has an isoelectric point of from pH 5 to pH 7.
11. The amphoteric liposome formulation of claim 1, wherein the
formulation has a stable lamellar phase at pH 7 to pH 8.
12. The amphoteric liposome formulation of claim 1, wherein the
molar ratio of the cationic amphiphile to the anionic amphiphile is
greater than or equal to 1.
13. The amphoteric liposome formulation of claim 1, wherein the
neutral amphiphiles are less than 40 mol % of the formulation.
14. The amphoteric liposome formulation of claim 1, wherein the
formulation comprises liposomes that encapsulate at least one
active agent.
15. The amphoteric liposome formulation of claim 14, wherein the at
least one active agent is a nucleic acid.
16. The amphoteric liposome formulation of claim 14, wherein the at
least one active agent is an oligonucleotide.
17. The amphoteric liposome formulation of claim 14, wherein the at
least one active agent is a biologically active agent.
18. An amphoteric liposome-nucleic acid particle, comprising a
cationic amphiphile, an anionic amphiphile, one or more neutral
amphiphiles, and a nucleic acid.
19. The amphoteric liposome-nucleic acid particle of claim 18,
wherein the cationic amphiphile is selected from the group
consisting of
4-(2-aminoethyl)-morpholino-cholesterol-2,3-dimethylhemisuccinate
(DmC4Mo2),
4-(2-aminoethyl)-morpholino-cholesterol-2,3-dimethylhemimalonate
(DmC3Mo2), 4-(2-aminobutyl)-morpholino-cholesterol-hemisuccinate
(C4Mo4), 4-(2-aminopropyl)-morpholino-cholesterol-hemimalonate
(C3Mo3), 4-(2-aminoethyl)-morpholino-cholesterol-hemimalonate
(C3Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemiglutarate
(C5Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemiadipate
(C6Mo2), 4-(2-aminoethyl)-morpholino-cholesterol-hemiadipate
(C8Mo2), 4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate
(MoChol), Cholesterol-(3-imidazol-1-yl propyl)carbamate (CHIM), and
Histaminyl-Cholesterolhemisuccinate (HisChol), the anionic
amphiphile is selected from the group consisting of cholesterol
hemisuccinate CHEMS, Dimyristoylglycerolhemisuccinate DMGS,
Dimyristoylglycerolhemimalonate DMGM,
Dimyristoylglycerolhemiglutarate DMGG,
Dimyristoylglycerolhemiadipate DMGA,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxobutanoic acid DMAS,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxopropanoic acid DMAM,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxopentanoic acid DMAG,
4-((1,2-Dimyristoyl-ethyl)amino)-4-oxohexanoic acid DMAA,
Dioleoylglycerolhemisuccinate DOGS, Dioleoylglycerolhemimalonate
DOGM, Dioleoylglycerolhemiglutarate DOGG,
Dioleoylglycerolhemiadipate DOGA,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxobutanoic acid DOAS,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxopropanoic acid DOAM,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxopentanoic acid DOAG,
4-((1,2-Dioleoyl-ethyl)amino)-4-oxohexanoic acid DOAA,
5,6-Dimyristoyl-hexanoic acid DMS, 4,5-Dimyristoyl-pentanoic acid
DMM, 6,7-Dimyristoyl-heptanoic acid DMG, 7,8-Dioleoyl-octanoic acid
DMA, 5,6-Dioleoyl-hexanoic acid DOS, 4,5-Dioleoyl-pentanoic acid
DOM, 6,7-Dioleoyl-heptanoic acid DOG, 7,8-Dioleoyl-octanoic acid
DOA, Cholesterolhemimalonate Chol-C3, Cholesterolhemiglutarate
Chol-C5, and Cholesterolhemiadipate Chol-C6, and the neutral
amphiphiles are selected from the group consisting of
phosphatidylcholines, sphingomyelins, ceramides,
phosphatidylethanolamines, cholesterol, and mixtures thereof.
20. The amphoteric liposome-nucleic acid particle of claim 19,
wherein the nucleic acid is a plasmid, a linear DNA construct, an
oligonucleotide, a mRNA, a shRNA, a miRNA, a ribozyme, an aptamer,
or an RNA that can specifically regulate a protein expression
level.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/581,054, filed on Oct. 13, 2006 and
European Patent Application No: 06255277.3, also filed on Oct. 13,
2006.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the U.S.
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference. Documents incorporated
by reference into this text may be employed in the practice of the
invention.
FIELD OF THE INVENTION
[0003] The present invention relates to improvements in or relating
to amphoteric liposomes. In particular, the present invention
provides a novel method for formulating such liposomes and a method
for loading them, as well as liposomes produced by such
methods.
BACKGROUND OF THE INVENTION
[0004] Amphoteric liposomes have been found to exhibit excellent
biodistribution and to be well tolerated in animals. They can
encapsulate active agents, including nucleic acid molecules, with
high efficiency.
[0005] In contrast to zwitterionic structures, amphoteric liposomes
advantageously have an isoelectric point and are negatively charged
at higher pH values and positively charged at lower pH values.
Amphoteric liposomes belong to the larger group of pH-sensitive
liposomes that were introduced by Straubinger, et al. (FEBS Lett.,
1985, 179(1), 148-154). Typical pH-responsive elements in
pH-sensitive liposomes are cholesterol hemisuccinate (CHEMS),
palmitoylhomocysteine, dioleoylglycerol hemisuccinate (DOG-Succ)
and the like. CHEMS can stabilise dioleoylphosphatidylethanolamine
(DOPE), a lipid which preferentially adopts the inverted hexagonal
phase at temperatures above 10.degree. C., into the lamellar phase
at pH 7.4. Lamellar CHEMS/DOPE systems can be prepared at neutral
or slightly alkaline pH but these systems become unstable and fuse
at acidic pH (Hafez and Cullis, Biochim. Biophys. Acta, 2000, 1463,
107-114).
[0006] Fusogenic liposomes are very useful in pharmaceutical
applications, especially for the intracellular delivery of drugs,
e.g., nucleic acids, such, for example, as plasmids and
oligonucleotides. After the uptake of a liposome into a cell by
endocytosis the release of the drug from the endosome is a crucial
step for the delivery of a drug into the cytosol of cells. The pH
within an endosome is slightly acidic and therefore pH sensitive
liposomes can fuse with the endosomal membrane and thereby allowing
the release of the drug from the endosome. This means that
destabilisation of the lipid phase, e.g., by enhanced fusogenicity,
facilitates endosome escape and intracellular delivery. Also other
environments of low pH can trigger the fusion of such liposomes,
e.g., the low pH found in tumors or sites of inflammation.
[0007] Hafez, et al. (Biophys. J. 2000, 79(3), 1438-1446) were
unsatisfied with the limited control over the pH at which such
fusion occurs and demonstrated a rational approach to fine-tune the
fusion point by adding cationic lipids. Such mixtures have true
amphoteric properties in that they exist in a cationic state at low
pH and as anionic particles at higher pH, typically at
physiological pH. According to Hafez, et al. fusion starts at pH
values where the net charge of the particles is zero (their
isoelectric point), and once such point is crossed (the pH is lower
to any extent) fusion is a continuous process. This view is shared
by Li and Schick (Biophys. J., 2001, 80, 1703-1711) who analysed
the fusion tendency for amphoteric lipid mixtures using a
mathematical model.
[0008] Israelachvili and Mitchell in 1975 (Biochim. Biophys. Acta,
1975, 389, 13-19) introduced the molecular shape concept which
assumes that the overall form of lipid molecules determines the
structure of the hydrated lipid membrane. This means that the lipid
geometry and more specifically the size ratio between the polar
head-group and the hydrophobic membrane anchor is the key parameter
determining the lipid phase (Israelachvili, et al. Biochim Biophys
Acta. 1977 17; 470(2):185-201). The original theory however did not
consider counterions being a steric part of the polar head-group,
but this was contributed by Li and Schick (Biophys. J., 2001, 80,
1703-1711). In their description of the DODAC/CHEMS system, the
sodium ion enlarges the head-group of CHEMS at neutral pH, but
dissociates as the pH drops, thus minimising the head-group volume
and promoting a hexagonal phase; DODAC as a strong cation is
assumed to be in constant association with its respective
counterion, irrespective of the pH. The model predicts fusion at
some pH and below.
[0009] Lipid phases according to the molecular shape concept
(Israelachvili et al., 1980, Q. Rev. Biophys., 13(2), 121-200):
TABLE-US-00001 Shape Organisation Lipid phase Examples Inverted
cone Micelles Isotropic Detergents Hexagonal I Lysophopholipids
Cylinder Bilayer Lamellar PC, PS, PI, SM (Cubic) Cone Reverse
Hexagonal II PE, PA at low micelles pH or with Ca2+, Cholesterol,
Cardiolipin
[0010] The addition of neutral lipids to amphoteric lipid mixtures
has been found to have little impact on the isoelectric point of
amphoteric liposomes. WO 02/066012 (Panzner, et al.) discloses
certain amphoteric liposomes comprising neutral lipids with a
stable size at both low and neutral pHs. WO 02/066012 also
describes a method of loading such particles with nucleic acids
starting from a low pH.
[0011] Amphoteric liposomes are complex structures and comprise at
least a complementary pair of charged lipids. The inclusion of one
or more such neutral lipids significantly adds to the complexity of
the mixture, especially since the individual amounts of the
components may vary. Hafez, et al. (Biophys. J. 2000, 79(3),
1438-1446) and WO 02/066012 provide some guidance as to how to
select lipid mixtures with truly amphoteric properties and more
specifically how to determine their isoelectric point and onset of
fusion. Nevertheless, the very high number of possible combinations
of lipids represents a practical hurdle towards a more rapid
optimisation of amphoteric liposomes, and there remains a need in
the art for a method of predicting or analysing which mixtures of
lipids form satisfactorily stable lamellar phases at high and low
pH, whilst forming a fusogenic, hexagonal phase at an intermediate
pH.
[0012] It is an object of the present invention therefore to
provide improved methods for formulating such fusogenic amphoteric
liposomes. Amphoteric liposomes that form a stable lipid phase at
neutral pH and a fusogenic phase at low pH represent another object
of the invention. Yet another object of the invention is the
provision of amphoteric liposomes that form stable lipid phases
both at low pH and at neutral pH, but undergo fusion at an
intermediate pH. The inventors have recognised that it would be
desirable to control the pH at which an amphoteric liposome is
fusogenic, so as to enable the liposome to be better targeted in
some applications to the endosomal environment where its cargo is
desired to be released. Yet another object of the invention
therefore is to provide a way of controlling the pH at which fusion
of such amphoteric liposomes occurs.
SUMMARY OF THE INVENTION
[0013] According to one aspect of the present invention therefore
there is provided a method of formulating amphoteric liposomes
comprising: [0014] (i) selecting an anionic amphiphile, a cationic
amphiphile, each of said anionic and cationic amphiphiles having
respective polar head and apolar tail-groups, cationic and anionic
counterions for said anionic and cationic amphiphiles respectively,
and optionally one or more neutral amphiphiles, at least one of
said anionic and cationic amphiphiles being chargeable; [0015] (ii)
calculating the .kappa. values for each of said anionic and
cationic amphiphiles, when uncharged and when charged and
associated respectively with said cationic and anionic counterions,
and said one or more optional neutral amphiphiles and the
.kappa..sub.salt value for a lipid salt comprising said anionic and
cationic amphiphiles in charged form, .kappa. being the ratio of
the molecular volume of the polar head-group V.sub.head to the
molecular volume of the apolar tail-group V.sub.apolar of the
respective species, the molecular volumes of the polar head-groups
of the charged anionic and cationic amphiphiles including the
respective counterions, .kappa..sub.salt being defined as:
[0015] .kappa. salt = V head ( cat ) + V head ( an ) V apolar ( cat
) + V apolar ( an ) ##EQU00001##
wherein V.sub.head(cat) is the molecular volume of the polar
head-group of the cationic amphiphile without the respective
counter-anion, V.sub.head(an) is the molecular volume of the polar
head-group of the anionic amphiphile without the respective
counter-cation, V.sub.apolar (cat) is the molecular volume of the
apolar tail-group of the cationic amphiphile and V.sub.apolar(an)
is the molecular volume of the apolar tail-group of the anionic
amphiphile; [0016] (iii) modelling the function
.kappa..sub.total(pH) for a lipid mixture of said anionic and
cationic amphiphiles and said one or more optional neutral
amphiphiles, assuming said cationic and anionic amphiphiles form
said lipid salt when charged, the respective amounts of said
amphiphiles in said lipid mixture being chosen such that said
mixture of lipids has an isoelectric point between a first lower pH
and a second higher pH and has a stoichiometric excess of
positively charged cationic amphiphile at said first pH and a
stoichiometric excess of negatively charged anionic amphiphile at
said second pH, .kappa..sub.total (pH) being defined as:
[0016] .kappa..sub.total (pH)=.kappa..sub.anc.sub.an
(pH)+.kappa..sub.catc.sub.cat
(pH)+.kappa..sub.an-c.sub.an-(pH)+.kappa..sub.cat++c.sub.cat+(pH)+.kappa.-
.sub.saltc.sub.salt (pH)+.SIGMA..kappa..sub.nc.sub.n
wherein c.sub.an (pH), c.sub.cat (pH), c.sub.an- (pH), c.sub.cat+
(pH) and c.sub.salt (pH) are the respective concentrations in the
lipid mixture of the uncharged anionic, uncharged cationic, charged
anionic and charged cationic amphiphiles and said lipid salt as a
function of pH, c.sub.n is the concentration in the lipid mixture
of the or each optional neutral amphiphile, and .kappa..sub.an,
.kappa..sub.cat, .kappa..sub.an-, .kappa..sub.cat+,
.kappa..sub.salt and .kappa..sub.n are the respective .kappa.
values for the uncharged anionic, uncharged cationic, charged
anionic and charged cationic amphiphiles, said lipid salt and the
or each optional neutral amphiphile; [0017] (iv) determining that
.kappa..sub.total(pH) exhibits a minimum at said isoelectric point;
[0018] (v) making liposomes composed of said lipid mixture and
empirically confirming that said mixture exhibits a stable lamellar
phases at said second pH and, optionally, at said first pH and a
fusogenic, hexagonal phase at or around said isoelectric point; and
thereafter [0019] (vi) manufacturing an amphoteric liposome
composed of said lipid mixture.
[0020] Suitably said molecular volumes may be calculated by
molecular modelling.
[0021] Said mixture may have an isoelectric point in the range pH 4
to pH 8, preferably in the range pH 5 to pH 7.
[0022] Suitably, said first pH may be in the range pH 4 to pH 5 or
in the range pH 2 to pH 4. Said second pH may be in the range pH 7
to pH 8. Advantageously, said second pH is about physiological pH
(about pH 7.4).
[0023] According to another aspect of the present invention there
is provided an amphoteric liposome composed of a mixture of lipids,
said mixture comprising a cationic amphiphile, an anionic
amphiphile and optionally one or more neutral amphiphiles, at least
one of said cationic and anionic amphiphiles being chargeable and
the respective amounts of said cationic and anionic amphiphiles
being selected such there is a stoichiometric excess of positively
charged cationic amphiphile at a first lower pH, a stoichiometric
excess of negatively charged anionic amphiphile at a second higher
pH, and said mixture has an isoelectric point intermediate said
first and second pHs; characterised in that said positively charged
cationic and negatively charged anionic amphiphiles are adapted to
form a lipid salt with one another at said isoelectric point.
[0024] Anionic and cationic amphiphiles have respective polar head
and apolar tail-groups. In accordance with the invention, the polar
head and apolar tail-groups of the anionic and cationic amphiphiles
and said counterions may be selected such that
.kappa..sub.total(pH) for the mixture exhibits a minimum at said
isoelectric point, whereby said mixture exhibits stable lamellar
phases at said second pH and, optionally, at said first pH and a
fusogenic, hexagonal phase at or around said isoelectric point.
[0025] Suitably, therefore, .kappa..sub.total (pH) (as defined
above) for the amphoteric liposome according to the invention
exhibits a minimum at said isoelectric point.
[0026] It has been found therefore that the combination of the
following assumptions allows a proper description of lipid phase
behaviour: [0027] 1) shape theory as a basis for the description;
[0028] 2) for polar head-groups in the charged state, counterions
become part of the head-group volume; and [0029] 3) lipid-lipid
salt formation occurs in the membrane
[0030] The method of the present invention therefore facilitates
the identification of fusogenic amphoteric liposomes, the
description of their fusion behaviour with respect to the
relationship between lipid composition and fusion pH and the impact
of counterions on the stability and fusion behaviour of amphoteric
liposomes.
[0031] The amphoteric liposomes of the present invention comprise a
lipid pair that is capable of forming a lipid-lipid salt within a
bilayer. In some embodiments the capacity of the lipid pair to form
a lipid-lipid salt may render the liposome bistable. Alternatively,
the liposome may be stable only at said second pH and fusogenic at
or around said isoelectric point.
[0032] In some embodiments, said amphoteric liposome of the
invention may comprise a lipid mixture comprising a chargeable
anionic amphiphile and a chargeable cationic amphiphile that are
adapted to form a lipid salt with one another, wherein
.kappa..sub.salt<0.34.
[0033] Said anionic amphiphile may be selected from Chems, DMGS,
DMGM, DMGG, DMGA, DMAS, DMAM, DMAG, DMAA, DOGS, DOGM, DOGG, DOGA,
DOAS, DOAM, DOAG, DOAA, DMS, DMM, DMG, DMA, DOS, DOM, DOG, DOA,
Chol-C3, Chol-C5 and Chol-C6. Said cationic amphiphile may be
cholesterol-based or based on diacylglycerols, and may be selected
from MoChol, Chim, HisChol and Desh4.
[0034] Alternatively, said amphoteric liposome may comprise a
chargeable anionic amphiphile and a chargeable cationic amphiphile
that are adapted to form a lipid salt with one another, wherein
.kappa..sub.salt<0.45; and wherein said chargeable cationic
amphiphile is selected from DmC4Mo2, DmC3Mo2, C4Mo4, C3Mo3, C3Mo2,
C5Mo2, C6Mo2 and C8Mo2; and said chargeable anionic amphiphile is
selected from Chems, DMGS, DMGM, DMGG, DMGA, DMAS, DMAM, DMAG,
DMAA, DOGS, DOGM, DOGG, DOGA, DOAS, DOAM, DOAG, DOAA, DMS, DMM,
DMG, DMA, DOS, DOM, DOG, DOA, Chol-C3, Chol-C5 and Chol-C6.
[0035] In yet another alternative, said amphoteric liposome may
comprise a chargeable anionic amphiphile and a stable cationic
amphiphile that are adapted to form a lipid salt with one another,
said chargeable anionic amphiphile being in excess; wherein
.kappa..sub.salt.gtoreq.0.34 and the difference between
.kappa..sub.total(pH 8) for C/A=0.5 and
.kappa..sub.salt>0.08.
[0036] In such case, said chargeable anionic amphiphile may be
selected from Chems, DMGS, DMGM, DMGG, DMGA, DMAS, DMAM, DMAG,
DMAA, DOGS, DOGM, DOGG, DOGA, DOAS, DOAM, DOAG, DOAA, DMS, DMM,
DMG, DMA, DOS, DOM, DOG, DOA, Chol-C3, Chol-C5 or Chol-C6 and fatty
acids.
[0037] In yet another alternative, said amphoteric liposome may
comprise a chargeable anionic amphiphile and a stable cationic
amphiphile that are adapted to form a lipid salt with one another,
said lipid mixture comprising an excess of said chargeable anionic
amphiphile and .kappa.salt being <0.34; wherein said stable
cationic amphiphile is selected from DDAB, DC-Chol, DAC-Chol,
TC-Chol, DODAP, N-methyl-PipChol, DOTAP, DOEPC and CTAB, and said
chargeable anionic lipid is selected from DMGS, DMGM, DMGG, DMGA,
DMAS, DMAM, DMAG, DMAA, DOGS, DOGM, DOGG, DOGA, DOAS, DOAM, DOAG,
DOAA, DMS, DMM, DMG, DMA, DOS, DOM, DOG, DOA, Chol-C3, Chol-C5 and
Chol-C6.
[0038] In still yet another alternative, said amphoteric liposome
may comprise a lipid mixture comprising an excess of a chargeable
cationic amphiphile and a stable anionic amphiphile, wherein said
cationic lipid is selected from MoChol, Chim, HisChol or Desh4,
DmC4Mo2, DmC3Mo2, C4Mo4, C3Mo3, C3Mo2, C5Mo2, C6Mo2 and C8Mo2, DOIM
and DPIM.
[0039] Suitably, said amphoteric liposome may comprise one or more
neutral or zwitterionic amphiphiles. In some embodiments, said
neutral amphiphile may be cholesterol. The cholesterol can be
present in the amphoteric liposomes as essentially the only neutral
lipid, and can comprise more than 80 mol % of the total neutral
lipids present in the liposomes. In other embodiments, cholesterol
is the only neutral lipid present in the amphoteric liposomes of
the invention. Alternatively, said neutral or zwitterionic lipid
may be selected from phosphatidylcholines, sphingomyelins,
ceramides, phosphatidylethanolamines, cholesterol and mixtures
thereof.
[0040] In some embodiments, said neutral or zwitterionic lipids may
be phosphatidylcholines, sphingomyelins or ceramides and may be
present in the lipid mix in an amount of less than 40 mol %.
[0041] Alternatively, said neutral or zwitterionic lipids may be
DOPE or cholesterol or a mixture thereof and maybe present in the
lipid mix in an amount of less than 65 mol %.
[0042] In a further alternative, said neutral or zwitterionic
lipids may comprise a mix of phosphatidylcholines (PC),
sphingomyelins or ceramides and phosphatidylethanolamines (PE) or a
mix of phosphatidylcholines (PC), sphingomyelins or ceramides and
cholesterol (Choi); wherein said mix of neutral lipids is present
in the lipid mix in an amount of not more than 80 mol %.
[0043] In another aspect of the invention the liposome may comprise
a lipid salt other than those of the following specific
combinations of amphiphiles DODAC/CHEMS; DDAB/CHEMS; DOTAP/DOGS;
DOTAP/DMGS; DOTAP/DPGS; DOTAP/CHEMS; CHIM/CHEMS; CHIM/DMGS;
CHIM/DOGS; HisChol/CHEMS; HisChol/DMGS; HisChol/DPGS; HisChol/DOGS;
HisChol/DPPS; MoChol/CHEMS; MoChol/DMGS; MoChol/DPGS; MoChol/DOGS;
MoChol/Cetyl-P; MoChol/DMPS; MoChol/DPPS; DC-CHOL/DOPA;
DOTAP+CHIM/CHEMS; DC-Chol/Chems; DOIM/DMGS; DOIM/DOGS; DOTAP/oleic
acid.
[0044] There are a number of amphoteric lipid combinations that
show little or no fusion and might not form a lipid salt. Such
lipid combinations are:
MoCHOL/POPG; MoCHOL/DPPG; HisCHOL/POPG; HisChol/DPPG.
[0045] In another aspect of the invention, the liposome may
comprise a lipid mixture other than one having one of the following
specific combinations of amphiphiles:
TABLE-US-00002 Cationic Anionic amphiphile amphiphile Other Ratio
(mol. %) DOTAP Chems 30:40 DOTAP Chems POPC 10:40:50 DOTAP Chems
POPC 25:25:50 DOTAP Chems POPC 20:30:50 DOTAP Chems POPC 20:20:60
DOTAP Chems POPC 10:30:60 DOTAP Chems POPC 15:25:60 DOTAP Chems
POPC:N- 10:30:50:10 glutaryl- DPPE DOTAP Chems DPPC:Chol
10:30:50:10 DOTAP Chems DPPC 10:30:60 DOTAP Chems DPPC 15:35:50
DOTAP Chems POPC:Chol 10:20:30:40 DOTAP Chems DMPC:Chol 10:30:20:40
DOTAP Chems POPC 15:45:40 DOTAP Chems POPC 20:60:20 DOTAP Chems
25:75 DOTAP Chems POPC 40:40:20 DOTAP Chems POPC 30:50:20 DOTAP
Chems POPC 10:70:20 DOTAP Chems POPC 28:47:25 DOTAP Chems DOPE
40:40:20 DOTAP Chems DOPE 30:50:20 DOTAP Chems DOPE 20:60:20 DOTAP
Chems DOPE 10:70:20
[0046] In yet another aspect of the invention, the liposome may
comprise a lipid mixture other than one having one of the following
specific combinations of amphiphiles
TABLE-US-00003 Cationic Anionic amphiphile amphiphile Other Ratio
(mol. %) CHIM Chems DPPC 15:35:50 CHIM Chems POPC 15:35:50 DC-Chol
DOPA 66:34 DC-Chol DOPA Chol 40:20:40 DC-Chol DOPA DMPC 27:13:60
DC-Chol DOPA DMPC:Chol 27:13:20:40 DC-Chol DOPA DMPC:Chol
20:10:30:40 DC-Chol DOPA DMPC:Chol 13:7:40:40 HisChol DG-Succ
DMPC:Chol 10:10:40:40 MoChol DG-Succ DMPC:Chol 10:15:35:40 MoChol
DG-Succ DMPC:Chol 10:10:40:40 MoChol DG-Succ DMPC:Chol 10:30:20:40
MoChol DG-Succ DPPC:Chol 10:30:20:40 MoChol DG-Succ POPC:Chol
10:15:35:40 MoChol DG-Succ POPC:Chol 10:30:20:40 MoChol DG-Succ
POPC:Chol 20:10:30:40
[0047] In yet another aspect of the invention, the liposome may
comprise a lipid mixture other than one having one of the following
specific combinations of amphiphiles:
TABLE-US-00004 Cationic Anionic amphiphile amphiphile Other Ratio
(mol. %) CHIM DMG-Succ POPC:DOPE 17:33:12.5:37.5 CHIM DMG-Succ
POPC:DOPE 33:17:12.5:37.5 CHIM DMG-Succ POPC:DOPE 23:47:7.5:22.5
CHIM DMG-Succ POPC:DOPE 47:23:7.5:22.5 CHIM Chems POPC:DOPE
17:33:12.5:37.5 CHIM Chems POPC:DOPE 33:17:12.5:37.5 CHIM Chems
POPC:DOPE 23:47:7.5:22.5 CHIM Chems POPC:DOPE 47:23:7.5:22.5 MoChol
Cetyl-P POPC:DOPE 20:10:10:60 MoChol Cetyl-P POPC:Chol 20:10:35:35
MoChol DOG-Succ POPC:DOPE 17:33:12.5:37.5 MoChol DOG-Succ POPC:DOPE
33:17:12.5:37.5 MoChol DOG-Succ POPC:DOPE 23:47:7.5:22.5 MoChol
DOG-Succ POPC:DOPE 47:23:7.5:22.5
[0048] According to yet another aspect of the present invention
there is provided a method of loading amphoteric liposomes
according to the present invention with a negatively charged cargo
moiety, said method comprising generating said liposomes in the
presence of said negatively charged cargo moiety at said first pH
using a first solvent comprising anionic counterions, and
thereafter exposing said liposome to said second pH using a second
solvent comprising cationic counterions.
[0049] Preferably such liposomes are bistable, exhibiting a stable
lamellar phase at said first pH as well as at said second pH.
[0050] Suitably, said solvent changes are performed by the one-step
admixture of the respective second solvent, such that said liposome
is rapidly brought to the desired pH. Said first pH may be about pH
2-5, preferably pH 2-4. Said second pH may be about pH 7.4. In
order to stabilise the liposome at said first pH, said
counter-anions may preferably have a molecular volume of at least
50 A.sup.3. Thus, said counter-anions may be selected from citrate,
pyrophosphate, barbituric acid and methyl sulphate.
[0051] In order to stabilise the liposome at said second pH, said
counter-cations may preferably have a molecular volume of at least
50 A.sup.3.
[0052] In accordance with another aspect of the present invention,
the liposome may encapsulate at least one active agent. In some
embodiments, said active agent may comprise a nucleic acid. In
particular, said active agent may comprise an oligonucleotide.
[0053] Without being limited to such use, the amphoteric liposomes
according to the present invention are well suited for use as a
carrier for nucleic acid-based drugs such, for example, as
oligonucleotides and DNA plasmids. These drugs are classified into
nucleic acids that encode one or more specific sequences for
proteins, polypeptides or RNAs and into oligonucleotides that can
specifically regulate protein expression levels or affect the
protein structure through inter alia interference with splicing and
artificial truncation.
[0054] In some embodiments of the present invention, therefore, the
nucleic acid-based therapeutic may comprise a nucleic acid that is
capable of being transcribed in a vertebrate cell into one or more
RNAs, which RNAs may be mRNAs, shRNAs, miRNAs or ribozymes, wherein
such mRNAs code for one or more proteins or polypeptides. Such
nucleic acid therapeutics may be circular DNA plasmids, linear DNA
constructs, like MIDGE vectors (Minimalistic Immunogenically
Defined Gene Expression) as disclosed in WO 98/21322 or DE
19753182, or mRNAs ready for translation (e.g., EP 1392341).
[0055] In another embodiment of the invention, oligonucleotides may
be used that can target existing intracellular nucleic acids or
proteins. Said nucleic acids may code for a specific gene, such
that said oligonucleotide is adapted to attenuate or modulate
transcription, modify the processing of the transcript or otherwise
interfere with the expression of the protein. The term "target
nucleic acid" encompasses DNA encoding a specific gene, as well as
all RNAs derived from such DNA, being pre-mRNA or mRNA. A specific
hybridisation between the target nucleic acid and one or more
oligonucleotides directed against such sequences may result in an
inhibition or modulation of protein expression. To achieve such
specific targeting, the oligonucleotide should suitably comprise a
continuous stretch of nucleotides that is substantially
complementary to the sequence of the target nucleic acid.
[0056] Oligonucleotides fulfilling the abovementioned criteria may
be built with a number of different chemistries and topologies.
Oligonucleotides may be single stranded or double stranded.
[0057] Oligonucleotides are polyanionic structures having 8-60
charges. Oligonucleotides are therefore well adapted for use as
negatively charged cargo in the loading method of the present
invention. In most cases these structures are polymers comprising
nucleotides. The present invention is not limited to a particular
mechanism of action of the oligonucleotides and an understanding of
the mechanism is not necessary to practice the present
invention.
[0058] The mechanisms of action of oligonucleotides may vary and
might comprise effects on inter alia splicing, transcription,
nuclear-cytoplasmic transport and translation.
[0059] In a preferred embodiment of the invention single stranded
oligonucleotides may be used, including, but not limited to,
DNA-based oligonucleotides, locked nucleic acids, 2'-modified
oligonucleotides and others, commonly known as antisense
oligonucleotides. Backbone or base or sugar modifications may
include, but are not limited to, Phosphothioate DNA (PTO),
2'O-methyl RNA (2'Ome), 2'Fluoro RNA (2'F), 2'O-methoxyethyl-RNA
(2'MOE), peptide nucleic acids (PNA), N3'-P5' phosphoamidates (NP),
2'fluoroarabino nucleic acids (FANA), locked nucleic acids (LNA),
Morpholine phosphoamidate (Morpholino), Cyclohexene nucleic acid
(CeNA), tricyclo-DNA (tcDNA) and others. Moreover, mixed
chemistries are known in the art, being constructed from more than
a single nucleotide species as copolymers, block-copolymers or
gapmers or in other arrangements. In addition to the aforementioned
oligonucleotides, protein expression can also be inhibited using
double stranded RNA molecules containing the complementary sequence
motifs. Such RNA molecules are known as siRNA molecules in the art
(e.g., WO 99/32619 or WO 02/055693). Other siRNAs comprise single
stranded siRNAs or double stranded siRNAs having one non-continuous
strand. Again, various chemistries were adapted to this class of
oligonucleotides. Also, DNA/RNA hybrid systems are known in the
art.
[0060] In another embodiment of the present invention, decoy
oligonucleotides can be used. These double stranded DNA molecules
and chemical modifications thereof do not target nucleic acids but
transcription factors. This means that decoy oligonucleotides bind
sequence-specific DNA-binding proteins and interfere with the
transcription (e.g., Cho-Chung, et al. in Curr. Opin. Mol. Ther.,
1999).
[0061] In a further embodiment of the invention, oligonucleotides
that may influence transcription by hybridizing under physiological
conditions to the promoter region of a gene may be used. Again
various chemistries may adapt to this class of
oligonucleotides.
[0062] In a still further alternative of the invention, DNAzymes
may be used. DNAzymes are single-stranded oligonucleotides and
chemical modifications thereof with enzymatic activity. Typical
DNAzymes, known as the "10-23" model, are capable of cleaving
single-stranded RNA at specific sites under physiological
conditions. The 10-23 model of DNAzymes has a catalytic domain of
15 highly conserved deoxyribonucleotides, flanked by 2
substrate-recognition domains complementary to a target sequence on
the RNA. Cleavage of the target mRNAs may result in their
destruction and the DNAzymes recycle and cleave multiple
substrates.
[0063] In yet another embodiment of the invention, ribozymes can be
used. Ribozymes are single-stranded oligoribonucleotides and
chemical modifications thereof with enzymatic activity. They can be
operationally divided into two components, a conserved stem-loop
structure forming the catalytic core and flanking sequences which
are reverse complementary to sequences surrounding the target site
in a given RNA transcript. Flanking sequences may confer
specificity and may generally constitute 14-16 nt in total,
extending on both sides of the target site selected.
[0064] In a still further embodiment of the invention, aptamers may
be used to target proteins. Aptamers are macromolecules composed of
nucleic acids, such as RNA or DNA, and chemical modifications
thereof that bind tightly to a specific molecular target and are
typically 15-60 nt long. The chain of nucleotides may form
intramolecular interactions that fold the molecule into a complex
three-dimensional shape. The shape of the aptamer allows it to bind
tightly against the surface of its target molecule including but
not limited to acidic proteins, basic proteins, membrane proteins,
transcription factors and enzymes. Binding of aptamer molecules may
influence the function of a target molecule.
[0065] All of the above-mentioned oligonucleotides may vary in
length between as little as 10, preferably 15 and even more
preferably 18, and 50, preferably 30 and more preferably 25,
nucleotides. The fit between the oligonucleotide and the target
sequence is preferably perfect with each base of the
oligonucleotide forming a base pair with its complementary base on
the target nucleic acid over a continuous stretch of the
abovementioned number of oligonucleotides. The pair of sequences
may contain one or more mismatches within the said continuous
stretch of base pairs, although this is less preferred. In general,
the type and chemical composition of such nucleic acids is of
little impact for the performance of the inventive liposomes as
vehicles be it in vivo or in vitro, and the skilled artisan may
find other types of oligonucleotides or nucleic acids suitable for
combination with the inventive liposomes.
[0066] According to yet another aspect of the present invention
there is provided a pharmaceutical composition comprising an
active-agent loaded amphoteric liposome according to the invention
and pharmaceutically acceptable vehicle therefor.
[0067] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
Figures, incorporated herein by reference, in which:
[0069] FIGS. 1 and 2 are graphical representations of the
calculation of .kappa. for different ratios between anionic and
cationic model lipids. Left panel: Surface plot for .kappa. in
response to pH and percentage of anionic lipid. Right panel:
Detailed analysis of the pH response for selected amounts of
anionic lipids.
[0070] FIG. 3 is a graphical representation of the calculation of
.kappa. for different ratios between anionic and cationic model
lipids in amphoter III systems. Left panel: Surface plot for
.kappa. in response to pH and percentage of anionic lipid. Right
panel: Detailed analysis of the pH response for selected amounts of
anionic lipids.
[0071] FIG. 4: is a graphical representation of the calculation of
.kappa. for different ratios between anionic and cationic model
lipids in amphoter III systems. Left panel: Salt bridge model;
Right panel: independent ion model.
[0072] FIG. 5 shows the stabilisation of the anionic or cationic
state of an amphoter II mixture through various counterion sizes.
Left panel: Analysis for equal counterion sizes. Right panel:
exclusive stabilisation of the anionic state through larger
cationic counterions. CA--counter-anion; CC--counter-cation; the
numbers in the legend indicate molecular volumes in .ANG..sup.3
[0073] FIG. 6 illustrates the asymmetric stabilisation of a
cationic amphoter II lipid phase through various counter-anions.
During production, the cationic lipid phase is stabilised with
larger anions (CA120). Liposomes are adjusted to a neutral pH and
the buffer composition is changed for a smaller counter-anion
(CA21). Liposomes that now encounter acidic pH are prone to fusion
since the lipid phase has much lower values of .kappa..
CA--counter-anion; CC--counter-cation; the numbers in the legend
indicate molecular volumes in .ANG..sup.3.
[0074] FIG. 7 illustrates the impact of absolute molecular volumes
for given values of .kappa. and counterion size. .kappa. was
adjusted to 0.175 for the cationic and anionic lipid in an amphoter
II system, but the absolute lipid size was varied; the numbers in
the legend indicate molecular head and tail volumes for both the
cationic and anionic lipid in .ANG..sup.3
[0075] FIG. 8 illustrates the impact of absolute molecular volumes
for given values of .kappa. and counterion size. .kappa. was
adjusted to 0.175 for the cationic and anionic lipid in an amphoter
II system, but the anionic lipid had a smaller molecular volume
than the cationic lipid.
[0076] FIG. 9 is a graphical representation of the calculation for
.kappa. in response to external pH in amphoter II systems further
comprising neutral lipids. 50% of neutral lipids were added to the
system with the .kappa. values given in the figure legend.
[0077] FIG. 10 shows the size of DOTAP/CHEMS liposomes after
pH-jump in CiP buffer. DOTAP liposomes containing 66 mol. % CHEMS
(crosses), 75 mol. % CHEMS (asterisks) or 100 mol. % CHEMS (dots)
were produced at pH 8, jumped to the indicated pH and neutralized
after one hour incubation at the lower pH. Size was measured at the
end of the cycle.
[0078] FIG. 11 shows the fusion behaviour of an amphoter II system
comprising a MoCHol and CHEMS. Left--calculation of .kappa. values
for the system. Right--experimental fusion results after pH-jump of
different mixtures of CHEMS and MoChol in CiP buffer. The
percentage in the legend stands for the amount of CHEMS in the
mixture.
[0079] FIG. 12 shows the fusion behaviour of an amphoter III system
comprising a MoCHol and DOPA. Left--calculation of .kappa. values
for the system. Right--experimental fusion results after pH-jump of
different mixtures of DOPA and MoChol in CiP buffer. The percentage
in the legend stands for the amount of DOPA in the mixture.
[0080] FIG. 13 shows the fusion behaviour of an amphoter II system
comprising a monoalkyl lipid. Left--calculation of .kappa. values
for the system. Right--experimental fusion results after pH-jump of
different mixtures of oleic acid and MoChol in CiP buffer. The
percentage in the legend stands for the amount of oleic acid in the
mixture.
[0081] FIG. 14 illustrates fusion behaviour in the presence of
POPC: Liposomes were produced at pH 7.5 and adjusted to acidic
conditions to promote aggregation or fusion. Addition of 20 mol. %
POPC greatly reduced the fusion tendency and liposomes were stable
in size even at the lower pH. The composition in the legend
represents the percentages for DOTAP and CHEMS; remainder is POPC.
pI stands for the calculated isoelectric point of the mixture.
[0082] FIG. 15 illustrates fusion behaviour in the presence of
DOPE: Liposomes were produced at pH 7.5 and adjusted to acidic
conditions to promote aggregation or fusion. Addition of 20 mol. %
DOPE maintains the fusion tendency of the amphoteric membrane. The
composition in the legend represents the percentages for DOTAP and
CHEMS; the remainder is DOPE. pI stands for the calculated
isoelectric point of the mixture.
[0083] FIG. 16 shows the fusion behaviour of an amphoter I system
comprising 20 mol % DOTAP and 80 mol % CHEMS in the presence of
various countercations. The size ratio on the y-axis is used to
normalize the liposome sizes to the value found at pH 8. All
liposomes were within a 170 nm to 220 nm size range under these
conditions.
[0084] FIG. 17 shows the fusion behaviour of an amphoter II system
comprising 20 mol % MoCHOL and 80 mol % CHEMS in the presence of
various countercations. The size ratio on the y-axis indicates
liposome sizes after exposure to the pH values indicated on the
x-axis and the values were normalized to a mock-treatment at pH 8.
All liposomes sizes were between 140 and 300 nm under these
conditions.
[0085] FIG. 18 shows the fusion behaviour of an amphoter III system
comprising 50 mol % MoCHOL and 50 mol % DOPA in the presence of
various countercations. The size ratio on the y-axis indicates
liposome sizes after exposure to the pH values indicated on the
x-axis and the values were normalized to a mock-treatment at pH 8.
All liposomes sizes were between 220 and 260 nm under these
conditions.
[0086] FIG. 19a shows the inverse correlation between fusion
intensity (expressed as .SIGMA.FRET in the matrix C/A=0.33-3 vs.
pH) and k(salt) for amphoter II systems, being formed from
cholesterol based pH sensitive cationic lipids (excepting DmC4Mo2)
and different pH sensitive anionic lipids.
[0087] FIG. 19b shows the inverse correlation between fusion
intensity (expressed as .SIGMA.FRET in the matrix C/A=0.7-1.5 vs.
pH) and k(salt) for amphoter II systems, being formed from
cholesterol based pH sensitive cationic lipids (excepting DmC4Mo2)
and different pH sensitive anionic lipids.
[0088] FIG. 19c illustrates the inverse correlation between fusion
intensity (expressed as .SIGMA.FRET in the matrix C/A=0.33-3 vs.
pH) and k(salt) for amphoter II systems comprising the cationic
lipid DmC4Mo2 in combination with different anionic pH sensitive
lipids.
[0089] FIG. 20 shows inverse correlation between fusion intensity
(expressed as .SIGMA.FRET in the matrix C/A=0.4-0.75 vs. pH) and
k(salt) for amphoter I systems comprising DOTAP or DODAP and
various pH sensitive anions.
[0090] FIGS. 21a and 21b show plots of the intensity of fusion
(expressed as % .SIGMA.FRET in the matrix C/A=0.17-0.75 for
DOTAP/DMGS; C/A=0.33-3 for MoChol/DOGS vs. pH) for liposomes from
DOTAP/DMGS or MoChol/DOGS against k(min) for mixtures with 0%-50%
POPC. The reference k(min) was modelled for C/A=0.66 (DOTAP/DMGS)
or C/A=1 (MoChol/DOGS). The %.SIGMA.FRET for 0% POPC is set to
100.
[0091] FIGS. 22a and 22b show plots of the intensity of fusion
(expressed as % .SIGMA.FRET in the matrix C/A=0.17-0.75 for
DOTAP/DMGS; C/A=0.33-3 for MoChol/DOGS vs. pH) for liposomes from
DOTAP/DMGS or MoChol/DOGS against k(min) for mixtures with 0%-50%
DOPE. The reference k(min) was modelled for C/A=0.66 (DOTAP/DMGS)
or C/A=1 (MoChol/DOGS). The % .SIGMA.FRET for 0% DOPE is set to
100.
[0092] FIGS. 23a and 23b show plots of the intensity of fusion
(expressed as % .SIGMA.FRET in the matrix C/A=0.17-0.75 for
DOTAP/DMGS; C/A=0.33-3 for MoChol/DOGS vs. pH) for liposomes from
DOTAP/DMGS or MoChol/DOGS against k(min) for mixtures with 0%-50%
cholesterol. The reference k(min) was modelled for C/A=0.66
(DOTAP/DMGS) or C/A=1 (MoChol/DOGS). The % .SIGMA.FRET for 0%
cholesterol is set to 100.
[0093] FIGS. 24a and 24b show plots of the intensity of fusion
(expressed as % .SIGMA.FRET in the matrix C/A=0.17-0.75 for
DOTAP/DMGS; C/A=0.33-3 for MoChol/DOGS vs. pH) for liposomes from
DOTAP/DMGS or MoChol/DOGS against k(min) for mixtures with 0%-50%
of a mixture POPC/cholesterol 1:1. The reference k(min) was
modelled for C/A=0.66 (DOTAP/DMGS) or C/A=1 (MoChol/DOGS). The %
.SIGMA.FRET for 0% POPC/cholesterol is set to 100.
[0094] FIG. 25 shows the intensity of fusion (expressed as
.SIGMA.FRET in the matrix C/A=0.33-3 vs. pH) of liposomes
comprising MoChol/DOGS and 10%-50% of different neutral or
zwitterionic lipids. The dotted line indicates the intensity of
fusion of the liposomes with 0% neutral or zwitterionic lipid.
[0095] FIG. 26 shows the correlation between the fusion zone and
the isoelectric point of liposomes comprising DC-Chol/Chems.
d(pH-IP) is the difference between the pH for which FRET was
measured and the isoelectric point for the appropriate C/A
ratio.
DETAILED DESCRIPTION OF THE INVENTION
[0096] By "chargeable" is meant that the amphiphile has a pK in the
range pH 4 to pH 8. A chargeable amphiphile may therefore be a weak
acid or base. A "stable" amphiphile is a strong acid or base,
having a substantially stable charge on the range pH 4 to pH 8.
[0097] By "amphoteric" herein is meant a substance, a mixture of
substances or a supra-molecular complex (e.g., a liposome)
comprising charged groups of both anionic and cationic character
wherein: [0098] 1) at least one, and optionally both, of the cation
and anionic amphiphiles is chargeable, having at least one charged
group with a pK between 4 and 8, [0099] 2) the cationic charge
prevails at pH 4, and [0100] 3) the anionic charge prevails at pH
8.
[0101] As a result the substance or mixture of substances has an
isoelectric point of neutral net charge between pH 4 and pH 8.
Amphoteric character is by this definition different from
zwitterionic character, as zwitterions do not have a pK in the
range mentioned above. In consequence, zwitterions are essentially
neutrally charged over a range of pH values; phosphatidylcholines
and phosphatidylethanolamines are neutral lipids with zwitterionic
character.
[0102] By "C/A" or "C/A ratio" or "C/A molar ratio" herein is meant
the molar ratio of cationic amphiphiles to anionic amphiphiles in a
mixture of amphiphiles.
[0103] By ".kappa.(min)" herein is meant the minimum of the
function .kappa..sub.total (pH)
[0104] The following abbreviations for lipids are used herein, the
majority of which abbreviations are in standard use in the
literature: [0105] PC Phosphatidylcholine (unspecified membrane
anchor) [0106] PE Phosphatidylethanolamine (unspecified membrane
anchor) [0107] SM Sphingomyelin [0108] DMPC
Dimyristoylphosphatidylcholine [0109] DPPC
Dipalmitoylphosphatidylcholine [0110] DSPC
Distearoylphosphatidylcholine [0111] POPC
Palmitoyl-oleoylphosphatidylcholine [0112] DOPC
Dioleoylphosphatidylcholine [0113] DOPE
Dioleoylphosphatidylethanolamine [0114] DMPE
Dimyristoylphosphatidylethanolamine [0115] DPPE
Dipalmitoylphosphatidylethanolamine [0116] CHEMS
Cholesterolhemisuccinate [0117] Chol-C3 Cholesterolhemimalonate
[0118] Chol-C5 Cholesterolhemiglutarate [0119] Chol-C6
Cholesterolhemiadipate [0120] DGS or DG-Succ
Diacylglycerolhemisuccinate (unspecified membrane anchor) [0121]
DOGS or DOG-Succ Dioleoylglycerolhemisuccinate [0122] DMGS or
DMG-Succ Dimyristoylglycerolhemisuccinate [0123] DPGS or DPG-Succ
Dipalmitoylglycerolhemisuccinate [0124] DSGS or DSG-Succ
Distearoylglycerolhemisuccinate [0125] POGS or POG-Succ
Palmitoyloleoylglycerolhemisuccinate [0126] DOGM
Dioleoylglycerolhemimalonate [0127] DOGG
Dioleoylglycerolhemiglutarate [0128] DOGA
Dioleoylglycerolhemiadipate [0129] DMGM
Dimyristoylglycerolhemimalonate [0130] DMGG
Dimyristoylglycerolhemiglutarate [0131] DMGA
Dimyristoylglycerolhemiadipate [0132] DOAS
4-{(1,2-Dioleoyl-ethyl)amino}-4-oxobutanoic acid [0133] DOAM
4-{(1,2-Dioleoyl-ethyl)amino}-4-oxopropanoic acid [0134] DOAG
4-{(1,2-Dioleoyl-ethyl)amino}-4-oxopentanoic acid [0135] DOAA
4-{(1,2-Dioleoyl-ethyl)amino}-4-oxohexanoic acid [0136] DMAS
4-{(1,2-Dimyristoyl-ethyl)amino}-4-oxobutanoic acid [0137] DMAM
4-{(1,2-Dimyristoyl-ethyl)amino}-4-oxopropanoic acid [0138] DMAG
4-{(1,2-Dimyristoyl-ethyl)amino}-4-oxopentanoic acid [0139] DMAA
4-{(1,2-Dimyristoyl-ethyl)amino}-4-oxohexanoic acid [0140] DOS
5,6-Dioleoyl-hexanoic acid [0141] DOM 4,5-Dioleoyl-pentanoic acid
[0142] DOG 6,7-Dioleoyl-heptanoic acid [0143] DOA
7,8-Dioleoyl-octanoic acid [0144] DMS 5,6-Dimyristoyl-hexanoic acid
[0145] DMM 4,5-Dimyristoyl-pentanoic acid [0146] DMG
6,7-Dimyristoyl-heptanoic acid [0147] DMA 7,8-Dioleoyl-octanoic
acid [0148] DOPS Dioleoylphosphatidylserine [0149] DPPS
Dipalmitoylphosphatidylserine [0150] DOPG
Dioleoylphosphatidylglycerol [0151] DPPG
Dipalmitoylphosphatidylglycerol [0152] Chol-SO4 Cholesterol
sulphate [0153] DOPA Dioleoylphosphatidic acid [0154] SDS Sodium
dodecyl sulphate [0155] CHIM Cholesterol-(3-imidazol-1-yl
propyl)carbamate [0156] DDRB Dimethyldioctadecylammonium bromide
[0157] DOTAP, DMTAP, DPTAP, DSTAP:
1,2-Diacyl-3-Trimethylammonium-Propane
[0157] [0158] DODAP, DMDAP, DPDAP, DSDAP:
1,2-Diacyl-3-Dimethylammonium-Propane
[0158] [0159] DOEPC, DMEPC, DPEPC, DSEPC:
1,2-Diacyl-sn-Glycero-3-Ethylphosphocholine
[0159] [0160] DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl
ammonium chloride [0161] DOTIM
1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium
chloride [0162] TMAG
N-(a-trimethylammonioacetyl)-didodecyl-D-glutamate chloride [0163]
BCAT
O-(2R-1,2-di-O-(19Z,99Z-octadecadienyl)-glycerol)-N-(bis-2-aminoethyl)car-
bamate [0164] DODAC Dioleyldimethylammonium chloride [0165] DORIE
1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide [0166] DMRIE
1,2-dimyristoyl-3-dimethyl-hydroxyethyl ammonium bromide [0167]
DOSC 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester [0168] DORI
1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride
[0169] DHMHAC N,N-di-n-hexadecyl-N,Ndihydroxyethylammoniumbromide
[0170] DHDEAB
N,N-di-n-hexadecyl-N-methyl,N-(2-hydroxyethyl)ammonium chloride
[0171] DMHMAC
N,N-myristyl-N-(1-hydroxyprop-2-yl)-N-methylammoniumchloride [0172]
DOTB 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol
[0173] SAINT lipids Synthetic Amphiphiles INTerdisciplinary [0174]
DPIM, DOIM 4,(2,3-bis-acyloxy-propyl)-1-methyl-1H-imidazole
(unspecified membrane anchor) [0175] DPAPy
2,3-bis-palmitoyl-propyl-pyridin-4-yl-amine [0176] DC-Chol
3b-[N--(N9,N9-dimethylaminoethane)carbamoyl]cholesterol [0177]
TC-Chol 3b-[N--(N9,N9-trimethylaminoethane)carbamoyl]cholesterol
[0178] DAC-Chol
3b(N--(N,N'-Dimethylaminoethan)-carbamoyl)cholesterol [0179]
PipC2Chol 4{N-2-ethylamino[(3'-cholesteryl)carbamoyl]}piperazine
[0180] MoC2Chol
{N-2-ethylamino[(3'-cholesteryl)carbamoyl]}morpholine [0181]
MoC3Chol {N-2-propylamino[(3'-cholesteryl)carbamoyl]}morpholine
[0182] N-methyl-PipChol N-methyl
{4-N-amino[(3'-cholesteryl)carbamoyl]}piperazine [0183] PyrroC2Chol
{N-2-ethylamino[(3'-cholesteryl)carbamoyl]}pyrrolidine [0184]
PipeC2Chol {N-2-ethylamino[(3'-cholesteryl)carbamoyl]}piperidine
[0185] ImC3Chol
{N-2-propylamino[(3'-cholesteryl)carbamoyl]}imidazole [0186]
PyC2Chol {N-2-ethylamino[(3'-cholesteryl)carbamoyl]}pyridine [0187]
CTAB Cetyltrimethylammonium bromide [0188] NeoPhectin.TM. cationic
cardiolipins (e.g.
[1,3-Bis-(1,2-bis-tetradecyloxy-propyl-3-dimethylethoxyammoniumbromide)-p-
ropane-2-ol] [0189] HistChol N-Histidinyl-Cholesterol-hemisuccinate
[0190] MoChol
4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate:
[0190] ##STR00001## [0191] HisChol
Histaminyl-Cholesterolhemisuccinate:
[0191] ##STR00002## [0192] DmC4Mo2
4-(2-Aminoethyl)-Morpholino-Cholesterol-2,3-dimethylhemisuccinate
[0192] ##STR00003## [0193] DmC3Mo2
4-(2-Aminoethyl)-Morpholino-Cholesterol-2,2-dimethylhemimalonate
[0193] ##STR00004## [0194] C3Mo2
4-(2-Aminoethyl)-Morpholino-Cholesterol-hemimalonate
[0194] ##STR00005## [0195] C3Mo3
4-(2-Aminopropyl)-Morpholino-Cholesterol-hemimalonate
[0195] ##STR00006## [0196] C4Mo4
4-(2-Aminobutyl)-Morpholino-Cholesterol-hemisuccinate
[0196] ##STR00007## [0197] C5Mo2
4-(2-Aminoethyl)-Morpholino-Cholesterol-hemiglutarate
[0197] ##STR00008## [0198] C6Mo2
4-(2-Aminoethyl)-Morpholino-Cholesterol-hemiadipate
[0198] ##STR00009## [0199] C8Mo2
4-(2-Aminoethyl)-Morpholino-Cholesterol-hemiadipate
##STR00010##
[0199] Molecular Volumes
[0200] Lipid shape theory is built on a shape balance between the
hydrophobic part and the polar head-group of a given amphiphile
rather than on absolute values for the two molecular portions. In
accordance with the present invention, .kappa. is the volume ratio
between the polar and apolar section of a lipid.
.kappa.=molecular volume(head)/molecular volume(tail)
[0201] Various different ways are available to those skilled in the
art to calculate molecular volumes and alternative methods and
sources are discussed for example in Connolly, M. J. Am. Chem. Soc.
(1985) 107, 1118-1124 and the references therein or are given at:
http://www.ccl.net/cca/documents/molecular-modeling/node5.html
[0202] Molecular volume is commonly calculated by assigning a value
called a van der Waals radius, r.sup.i.sub.vdW, to each atom type
in such a way that the sum of these quantities for a given atom
pair, i and j, is equal to their closest possible distance
(dij):
r.sup.i.sub.vdW+r.sup.i.sub.vdWdij
[0203] Many different tables of "best" van der Waals radii exist,
even though the values for corresponding atoms coming from
different authors are similar. In geometric terms, the van der
Waals radius may be imagined as a spherical "shield" surrounding
the atom, and the closest distance between two non-bonded atoms is
when their respective shields touch. However, the shields of
covalently bonded atoms intersect since bond lengths are shorter
than the sum of the van der Waals radii partaking atoms. A
molecular van der Waals surface, also called a van der Waals
envelope, is composed of the spheres for individual atoms with
their intersecting sections removed.
[0204] For a single molecule (i.e., molecule for which there is a
path between any two atoms along covalent bonds), the van der Waals
envelope is a closed surface, and hence, it contains volume.
[0205] This volume is called the molecular volume, or van der Waals
volume and is usually given in .ANG..sup.3. The straightforward way
of calculating molecular volume on a computer is by numerical
integration.
[0206] In some embodiments, molecular volumes for lipid molecules
and the respective head and tail fragments may be calculated using
DS Viewer Pro 5.0 (Accelrys Inc., San Diego, Calif.) and volumes
within the respective van der Waals radii were calculated.
[0207] Typical membrane fragments are 1,2-diacyl-ethyleneglycols
that represent the hydrophobic section for common phospholipids,
leaving the 3' carbon atom of the original glycerol with the
phosphocholine head-group. The same fragment is also found in the
common cationic lipid DOTAP and its derivatives but also in
diacylglycerols with other polar head-groups such as
dimyristoylglycerol hemisuccinate and the like.
[0208] For the cholesterol derivatives, the entire sterol, but not
the 3' oxygene, is defined as the hydrophobic section and the
head-group being complementary to that.
[0209] Likewise, for cationic or anionic alkyl derivatives the
polar head-group is defined as the polar fragment involving the C1
carbon of the alkyl chain. Consequently, the residual chain with
n-1 carbon atoms represents the hydrophobic apolar part.
[0210] Molecular volumes depend on the constants used for the
calculations and may be affected by the conformation of the
molecule. Typical values obtained for the hydrophobic apolar
fragments are and were used for further calculations:
TABLE-US-00005 TABLE 1 Membrane fragment Volume in .ANG..sup.3
di-lauroylethyleneglycol 356 di-myristoylethyleneglycol 407
di-palmitoylethyleneglycol 458 di-stearoylethyleneglycol 509
di-oleoylethyleneglycol 501 Palmitoyl-oleoylethyleneglycol 478
di-phytanoylethylenglycol 566 di-oleylethyleneglycol (e.g., in 495
DOTMA) di-palmitylethylenglycol 452 Didoceyl-D-glutamate (e.g., in
395 TMAG) Cholesteryl 334 C11 hydrophobic part in lauryl 132
derivatives C13 hydrophobic part in myristyl 158 derivatives C15
hydrophobic part in palmityl 184 derivatives C17 hydrophobic part
in stearyl 210 derivatives C17 hydrophobic part in oleyl 208
derivatives Sphingomyelin/Ceramide 467 backbone
[0211] Molecular volumes for most counter-anions were derived the
same way, but for Na+ or K+ the strongly bound hydration sphere is
taken into account. The following values were used for further
calculations:
TABLE-US-00006 TABLE 2 Counterion Volume in .ANG..sup.3
Acetate.sup.- 40 Citrate.sup.- 121 Phosphate.sup.2- 49
Chloride.sup.- 21 Formiate.sup.- 29 PF.sub.6.sup.- 51
Methylsulfate.sup.- 64 Trifluoroacetate.sup.- 56 Barbituric acid 79
Pyrophosphate.sup.4- 88 Sodium.sup.+ 65 .sup.1) to 88 .sup.2)
Hydrated radii are 2, 5A and 2, 76A, respectively Potassium.sup.+
24 .sup.1) to 52 .sup.2) Hydrated radii are 1, 8A and 2, 32A,
respectively Lithium.sup.+ .sup. 164 .sup.2) Imidazolium.sup.+ 52
Morpholinium.sup.+ 69 Tris(hydroxymethyl)-aminoethan.sup.+ 91
Tris(hydroxyethyl)-aminoethan.sup.+ 130
Bis(hydroxymethyl)-aminoethan.sup.+ 74
Hydroxymethyl-aminoethan.sup.+ 50 Bis(hydroxymethyl)hydroxyethyl-
107 aminoethan.sup.+ Bis(hydroxyethyl)hydroxymethyl- 123
aminoethan.sup.+ Triethylamine.sup.+ 92
Diethyl-hydroxyethyl-amine.sup.+ 100 Arginine.sup.+ 135 Glucoronic
acid.sup.- 129 Malonic acid.sup.- 66 Tartaric acid.sup.- 97
Glucosamine.sup.+ 129 .sup.1) Gerald H. Pollack: Cells, Gels and
the Engines of Life, Ebner and Sons Publishers, 2001 .sup.2)
Hypertext transfer
protocol://www.bbc.co.uk/dna/h2g2/A1002709#footnote1
[0212] The charged polar head-groups have different representations
and the molecular volumes are given below for some individual
members of this group. These values were used for further
calculations:
TABLE-US-00007 TABLE 3 Polar head-group pK (anions) Volume in
.ANG..sup.3 (calculated/measured) Hemisuccinate (e.g., in 76 CHEMS)
Hemisuccinate (e.g., in 87 diacylglycerols) 2,3
dimethylhemisuccinate (e.g., 117 esterified to diacylglycerols)
Hemimalonate (e.g., esterified 78 to cholesterol) Hemiadipate
(e.g., esterified to 115 diacylglycerols) Sulfate (e.g., in
cholesteryl 50 sulfate) Methylsulfate (e.g., SDS) 64 Carboxyl
(e.g., in alkyl 42 carboxylic acids) Methylphosphate (e.g., in 63
Cetylphosphate, phosphatidic acid) Phosphoglycerol 115 Phosphoserin
118 Polar head-groups pK (cations) Volume in .ANG..sup.3
(calculated/measured) Trimethylammoniummethyl 67 (e.g., in
cetyltrimethyl- ammonium, DOTAP and others)
Dimethylammonium-dimethyl 66 (e.g., in DODAC)
Trimethyl-hydroxyethyl 88 ammonium (e.g., in DORIE)
Dimethyl-di-hydroxyethyl 108 ammonium (e.g., in DHDEAB)
N-(1-hydroxyprop-2-yl)-N- 102 trimethyl ammonium (e.g., in DMHMAC)
4-trimethylammonio-butenoic 128 acid methylester (e.g., in DOTB)
1-Methyl-4-choline-succinic 157 acid diester (e.g., in DOSC)
Methylimidazol (e.g., in DOIM 60 or DPIM) 3-imidazol-1-yl-propyl
121 carbamate (e.g.,, in CHIM) 2-(4-Imidazolyl)ethylamine 148
hemisuccinate (e.g., in HisChol) N-Morpholino ethylamine 166
hemisuccinate (e.g., in MoChol) N-Methylmorpholin 81 Methylamine
(e.g., in 30 Stearylamine) Ethylphosphocholine (e.g., in 161 DOEPC)
1-[2-Carboxyethyl]2-methyl-3- 157 (2-hydroxyethyl)imidazolinium
(e.g., in DOTIM) N-(a-trimethylammonioacetyl) 94 (e.g., in TMAG)
Pyridin-4-methylamine (e.g., in 83 DPAPy) N-Methyl-pyridin (e.g.,
in 87 SAINT-2) N-Methyl-4-carboxy-pyridine 110 (e.g., in SAINT
esters) N-Methyl-3,5-dicarboxy- 145 pyridine (e.g., in SAINT
diesters) Piperazine 4-N-aminoethyl 130 carbamoyl
(dimethyl)-aminoethyl 99 carbamoyl (e.g., in DC-Chol)
(trimethyl)-aminoethyl 113 carbamoyl (e.g., in TC-Chol) N-Methyl-
104 tris(hydroxy- methyl)aminomethane N-Methyl- 83.5 bis(hydroxy-
methyl)aminomethane N-Methyl- 61.8 mono(hydroxy-
methyl)aminomethane Polar head-groups (neutral or zwitterionic)
Volume in .ANG..sup.3 Phosphocholine 133 Phosphoethanolamine 97
[0213] It is possible to use other methods to determine molecular
volumes for the lipids. Also, some parameters such as the exact
split-point between membrane tail and polar head; number of water
molecules in the hydration cage or the van der Waals radii can be
varied without affecting the general applicability of the model.
With the same understanding more subtle changes in the molecular
volumes may be disregarded, in particular those arising from the
dissociation of protons or from conformational changes. In some
embodiments the molecular volumes recited in Tables 2 and 3 above
may be used in the methods of the present invention.
[0214] The counterions fall into the same category of sizes than
the actual polar head-groups. As such, it has been found that the
addition or withdrawal of counterions from lipid polar regions has
a substantial effect on the total head-group size and in
consequence on the head/tail balance .kappa.. As an example, the
CHEMS sodium salt has a head-group size of 141 A.sup.3 which is
reduced to 76 A.sup.3 in the undissociated form at pH 4. .kappa.
varies between 0.42 and 0.23, respectively. CHEMS does form a
lamellar phase at pH 7.5 and higher but adopts a hexagonal phase at
low pH.
[0215] Other lipids with known phase behaviour can be used to
select .kappa. values for discrimination between the lamellar and
hexagonal phase; an example is given in Table 4 below. PE
head-groups can form an intramolecular ring structure with hydrogen
bonding between the terminal amino group and the oxygen in the
phosphoester group (betaine structure)(e.g. Pohle et al., J. Mol.
Struct., 408/409, (1997), 273-277). PC head-groups are sterically
hindered and instead recruit counterions to their respective
charged groups.
TABLE-US-00008 TABLE 4 Lipid or mixture .kappa. Phase behaviour
POPC 0.46 Lamellar DOPE 0.19 Hexagonal
pH Induced Changes of Molecular Volumes in Amphoteric Lipid
Mixtures
[0216] In a first model, in contradistinction to the present
invention no lipid salt formation occurs between charged anionic
and cationic lipids. This reflects the assumptions of Li and Schick
(Biophys. J., 2001, 80, 1703-1711) and might be the case for lipids
that are sterically hindered to form lipid salts (independent ion
model).
[0217] The lipid species in the membrane comprise undissociated
anions and cations as well as the dissociated anions and cations,
the latter being complexed with their respective counterions. The
.kappa. value for such a mixture is assumed to be the weighted sum
of its components:
.kappa.=.kappa.(anion.sup.0)*c(anion.sup.0)+.kappa.(cation.sup.0)*c(cati-
on.sup.0)+.kappa.(anion.sup.-)*c(anion.sup.31)+.kappa.(cation.sup.+)*c(cat-
ion.sup.+); (1) [0218] wherein anion.sup.0 or cation.sup.0 denotes
the uncharged species and anion.sup.- or cation.sup.+ denotes the
respective charged species; and wherein c herein denotes
concentration.
[0219] The amounts of the individual species present under such
assumption can be calculated from known equilibrium constants K for
the acid or base dissociation:
c(anion.sup.-)=c(anion.sup.tot)/(c.sub.H+/K+1) (2)
c(anion.sup.0)=c(anion.sup.tot)-c(anion.sup.-) (3)
c(cation.sup.+)=c(cation.sup.tot)/(K/c.sub.H++1) (4)
c(cation.sup.0)=c(cation.sup.tot)-c(cation.sup.+) (5)
wherein anion.sup.0 is the undissociated anion, anion.sup.- the
negatively charged molecule and anion.sup.tot the total
concentration of the respective anion. Cations follow the same
nomenclature and c.sub.H+ and K describe the proton concentration
and the equilibrium constant for the acid or base,
respectively.
[0220] However, taking possible interaction between a cationic and
anionic amphiphile into account in accordance with the invention,
the lipid salt occurs as a fifth species in the mixture:
.kappa.=.kappa.(anion.sup.0)*c(anion.sup.0)+.kappa.(cation.sup.0)*c(cati-
on.sup.0)+.kappa.(anion.sup.-)*c(anion.sup.-)+.kappa.(cation.sup.+)*c(cati-
on.sup.+)+.kappa.(salt)*c(salt) (6)
[0221] In a lipid salt, the cationic amphiphile serves as a
counterion to the anionic amphiphile and vice versa thus displacing
the small counterions like sodium or phosphate from the head-group.
The lipid salt is net uncharged and its geometry has to be assumed
to be the sum of both parts without the small counterions.
Therefore:
.kappa.(salt)=(v.sub.head(cation)+v.sub.head(anion))/(v.sub.apolar(catio-
n)+v.sub.apolar(anion)) (7)
[0222] Salt formation is limited by the charged amphiphile that is
present in the lowest concentration:
c(salt)=MIN(c(cation.sup.+);c(anion.sup.-)) (8)
[0223] Salt formation between the two charged amphiphiles is
assumed to be complete within this model, but of course, an
incomplete salt formation may be assumed. The following
calculations further reflect the fact that the salt comprises two
lipid molecules. It is of course possible to assume further some
membrane contraction upon lipid salt formation and to put a
different weight on the contribution of k(salt).
Model Calculations
[0224] To achieve amphoteric character of a lipid mix, at least one
of the lipid ions needs to be a pH-sensitive, weak acid or base
("chargeable"). A detailed disclosure is found in WO 02/066012 the
contents of which are incorporated herein by reference. Being
different in character, three basic systems are possible and are
analysed here:
"Amphoter I" strong cation and weak anion, "Amphoter II" weak
cation and weak anion, "Amphoter III" weak cation and strong
anion.
Amphoter I Systems
[0225] Amphoter I systems need an excess of the pH-sensitive anion
to achieve amphoteric character. At pH 7 to 8 the anionic lipid is
fully charged and salt formation occurs until all cationic lipids
are consumed. In an example with 70 mol. % anionic lipid and 30
mol. % cationic lipid, all cationic lipid and a corresponding 30
mol. % of the anionic lipid would exist as lipid salt while 40 mol.
% of the anionic lipid is unbound and recruits its counterion to
the head-group.
[0226] Starting from neutral conditions, a reduction of the pH
discharges the anionic lipid, the .kappa. value becomes smaller
owing to loss of the counterion and reaches a minimum when the
portion of still-charged anionic lipid is equal to the amount of
cationic lipid. Therefore, .kappa. is minimal at the isoelectric
point of the amphoteric lipid mixture. If the pH is further
lowered, an increasingly smaller portion of the anionic lipid
remains charged. This means dissociation of the lipid salt and
recruitment of counterions, now to the cationic lipid liberated
from the lipid salt.
[0227] The left panel in FIG. 1 of the accompanying drawings
illustrates the complex behaviour of .kappa. in dependence from pH
and the amount of anionic lipid in the mixture. A "valley of
fusogenicity" appears, and any amphoteric mixture having more than
55 mol. % and less than 85 mol. % anionic lipid is expected to fuse
under slightly acidic conditions but to be stable both at
neutrality and under more acidic conditions.
[0228] Amphoter I mixtures with less than 50 mol. % anionic lipid
are no longer amphoteric since the anion can modulate, but not
overcompensate, the charge on the cationic lipid. These mixtures
might undergo a pH-dependent fusion, but do not provide a second
stable phase at low pH. A 1:1 complex adopts a lamellar phase only
at low pH and undergoes fusion at neutrality.
[0229] The parameters used for the calculations illustrated in FIG.
1 are given in Table 5 below; volumes in .ANG..sup.3.
TABLE-US-00009 TABLE 5 Anion head volume 70 Anion tail volume 400
Anion pK 5 Cation head volume 70 Cation tail volume 400 Cation pK
15 Counterion+ 70 Counterion- 70
Amphoter II Systems
[0230] Amphoter II systems have the distinct advantage to be
amphoteric over the entire range of anion:cation ratios and no
charge overcompensation for the strong ion is needed as in Amphoter
I or Amphoter III systems. A calculation for a model system is
shown in FIG. 2.
[0231] The parameters used for the calculation are given in Table 6
below; all volumes in .ANG..sup.3.
TABLE-US-00010 TABLE 6 Anion head volume 70 Anion tail volume 400
Anion pK 5 Cation head volume 70 Cation tail volume 400 Cation pK
6.5 Counterion+ volume 70 Counterion- volume 70
[0232] Again, the lipid salt model predicts stable states at
neutral to slightly alkaline pH but also at slightly acidic pH and
a pronounced valley of instability or fusogenicity in between.
[0233] In contrast to amphoter I systems, fusogenic states can be
reached across a wide range of different lipid ratios between the
anionic and cationic components. That is, the valley of
fusogenicity extends across a wider range of anion/cation ratios,
allowing a greater degree of control over the pH at which a given
system is fusogenic.
Amphoter III Mixtures
[0234] Amphoter III mixtures comprising a stable anion and a
pH-sensitive cation cannot form lipid salts at neutral pH, since
little to no charged cationic lipid exists at this pH. It needs
ongoing acidification to first create the cation which then may
undergo salt formation. Calculation for a model system is shown in
FIG. 3.
[0235] The parameters used for the calculation are given in Table 7
below; all volumes in .ANG..sup.3.
TABLE-US-00011 TABLE 7 Anion head volume 70 Anion tail volume 400
Anion pK 1 Cation head volume 70 Cation tail volume 400 Cation pK
6.5 Counterion+ volume 70 Counterion- volume 70
[0236] As can be seen from FIGS. 1 and 3, amphoter III systems
behave like the mirror image of amphoter I systems. They provide a
valley of fusogenicity as long as the weak lipid ion is present in
excess and over-compensates the constant charge on the opposite
ion. In contrast to amphoter I systems the pH for fusion locates
higher than the pK of the pH-sensitive lipid ion.
[0237] Experimental evidence for the fusion valley is given in the
Examples 1 to 4 and provides confirmation for the central
hypothesis of lipid salt formation in amphoteric liposomes. The
examples also provide two closely related amphoter III systems
(MoChol/POPG and MoChol/DOPA) where fusion is only observed in one
of the two, namely MoChol/DOPA. This may be due to steric
hindrance, as the protonated tertiary nitrogen in MoChol is
situated at the lower end of the morpholino ring and is therefore
not easily accessible. In addition, the phosphate in POPG sits
right at the lipid/water interface and is protected with a glycerol
towards the water phase. In contrast, the phosphate group in DOPA
is easily accessible. The model described above can be used to
describe both situations, if lipid salt formation is optionally
removed from the model building assumptions (equations (1) to (5)
above only). A comparison between the two scenarios is shown in
FIG. 4. The parameters used for the calculation of FIG. 4 are given
in Table 8 below, DOPA and MoChol in Na/H.sub.2PO.sub.4 were used
as model compounds; and volumes are expressed as .ANG..sup.3.
TABLE-US-00012 TABLE 8 Anion head volume 63 Anion tail volume 501
Anion pK 3 Cation head volume 166 Cation tail volume 334 Cation pK
6.5 Counterion+ volume 65 Counterion- volume 49
[0238] Whereas the lipid salt model predicts fusion with lower pH,
a deletion of salt formation leads to a system where an increase of
.kappa. is observed when the cationic lipid becomes protonated. If
at all, such situation leads to an even more lamellar and
eventually to a micellar state of the membrane. However, the limit
for micelle formation has not been determined within the context of
this model.
[0239] The monoanionic state of DOPA (pKa 1=3, pKa 2=8) was used
for the model since DOPA exists as a mono-anion when MoChol becomes
ionised (pKa=6.5). The model predicts little or no fusogenicity at
33 mol. % anion, valley type fusion behaviour at 50 mol. % anion
and monophasic fusion behaviour at 66 mol. % and more of the anion.
The entire complexity of the model is reflected in the practical
behaviour of the membrane as illustrated in Example 4 below.
[0240] The algorithm according to the present invention allows
prediction of fusion behaviour of a wide range of amphoteric lipid
mixtures. The prediction rules are derived from a simple
geometrical description of the interacting lipids and are
independent from the actual chemical representation of the
molecules. As such, existing and novel lipid combinations can be
easily tested by those skilled in the art, and the intended fusion
behaviour can be predicted in a rational way. The following key
parameters may illustrate such selection process, but other
priorities might be set dependent on the respective goals of the
application.
1. .kappa. of the Lipid Salt
[0241] .kappa. of the lipid salt is calculated in equation (7)
above and may suitably be lower than 0.34 or 0.35 to predict
reasonably a fusogenic hexagonal phase. In some embodiments .kappa.
may be lower than 0.3; preferably lower than 0.25. .kappa.(salt) is
low when the combined polar head-groups are small and the combined
hydrophobic portions are large. The preferred sum of head-group
volumes is about 300 .ANG..sup.3 or smaller; in a more preferred
embodiment this volume is smaller than 220 .ANG..sup.3, and an even
more preferred value is smaller than 170 .ANG..sup.3. According to
the selection made above, preferred sums for the tail group volumes
are larger than 650 .ANG..sup.3 and may be as large as about 1000
.ANG..sup.3, wherein combinations of proper head and tail groups
are governed by the preferred k(salt) values.
2. Amplitude of Change (d(.kappa.)/d(pH))
[0242] In a preferred embodiment of the present invention, a lipid
salt with a low value for .kappa. is stabilised below or above its
isoelectric point by recruitment of counterions. In a preferred
embodiment of the invention larger counterions are used to
stabilise either the cationic or the anionic state of the
amphoteric lipid mixture. FIG. 5 illustrates such dependence from
counterions size for an amphoter II system. The parameters used for
the calculation of FIG. 5 are given in Table 9 below.
TABLE-US-00013 TABLE 9 Anion head volume 70 Anion tail volume 400
Anion pK 5 Cation head volume 70 Cation tail volume 400 Cation pK
6.5 Counterion+ See FIG. 5 Counterion- See FIG. 5
[0243] It becomes apparent from the right panel of FIG. 5 that such
stabilisation may be asymmetric, e.g., providing rather limited
stabilisation for the cationic phase and more stabilisation of the
anionic phase of the amphoteric lipid mix. Also, counterions that
do not naturally exist in physiological body fluid may be used to
improve stability during storage; exchange of such storage ions
with the sodium ions present in the body fluids may be advantageous
for discharging the cargo from the liposomes in vivo. The selection
of proper ion volumes for the individual or common stabilisation of
a lipid phase is therefore a preferred application of the present
invention. Such stabilisation is of particular use for the
manufacturing and storage of amphoteric liposomes.
[0244] In some embodiments of the present invention larger
counter-cations are used to stabilise the amphoteric liposomes at
neutral conditions. In a preferred embodiment such counter-cations
have a molecular volume of 50 .ANG..sup.3 or more, in a more
preferred embodiment this volume exceeds 75 .ANG..sup.3 and said
neutral pH is between pH 7 and pH 8, more preferred about the
physiological pH of 7.4.
[0245] If amphoteric liposomes are produced for pharmaceutical
purposes, compatibility of the used ions with the application route
needs to be obeyed. Suitable counter-cations can be selected from
Table 2 above describing the ion sizes. Preferred counter-cations
for pharmaceutical compositions are sodium or the respective
ionized forms of tris(hydroxymethyl)aminomethane,
tris-hydroxyethylaminomethane, triethylamine, arginine, in
particular L-arginine and the like.
[0246] In an embodiment of the invention the amphoteric liposomes
may be manufactured at a low pH in their cationic state. Under
these conditions, the liposomes can bind polyanions such as
proteins, peptides or nucleic acids, whether as large plasmids or
smaller oligonucleotides. Such binding is useful for improvement of
the encapsulation efficacy of said materials into the amphoteric
liposomes.
[0247] It is advantageous to use a lipid phase with a low .kappa.
at acidic pH. Selection of large counter-anions facilitates
stabilisation of said lipid phase, e.g., for the production of such
liposomes and the encapsulation of cargo under these
conditions.
[0248] Suitable large counter-anions have a molecular volume larger
than 50 .ANG..sup.3, preferred large counterions have a molecular
volume larger than 75 .ANG..sup.3. Suitable counter-anions can be
selected from Table 2 above. Preferred counter-anions are citrate,
pyrophosphate, barbiturate, methyl sulphate and the like.
[0249] After having contacted the lipid phase with the cargo to be
encapsulated under acidic conditions, the liposomes are than
neutralized and non-encapsulated cargo can optionally be removed.
Typically, non-encapsulated cargo detaches from the lipid membrane
since both carry the same charge under neutral conditions. The
amphoteric liposomes are negatively charged above their isoelectric
point, e.g., at a pH between 7 and 8 and the cargo molecules exist
as polyanions at such a pH. This is in particular the case with
nucleic acids that carry one negative charge per nucleobase. Such
liposomes can undergo effective destabilisation when exposed to the
low pH in combination with a smaller counter-anion. This is for
example the case after systemic administration and cellular uptake
and endocytosis of such liposomes. Chloride or phosphate are the
most common counter-anions in the body fluids of animals, be it any
animal, a mammal or humans. Phosphate, but even more so chloride,
are small counterions with little or no hydration shell and
molecular volumes <60 A.sup.3.
[0250] FIG. 6 illustrates a cycle of liposome generation and use
which illustrates selective stabilisation and destabilisation of
the lipid phase under acidic conditions through asymmetric
counterion use. The parameters used for the calculation of FIG. 6
are given in Table 10 below; volumes in .ANG..sup.3.
TABLE-US-00014 TABLE 10 Anion head volume 70 Anion tail volume 400
Anion pK 5 Cation head volume 70 Cation tail volume 400 Cation pK
6.5 Counterion+ See FIG. 6 Counterion- See FIG. 6
3. Isoelectric Point
[0251] A mathematical description for the isoelectric point of
amphoteric liposomes is been given in the WO 02/066012. In
accordance with the present invention, the isoelectric point of the
amphoteric liposomes can be adjusted to a wide range of conditions,
and there is sufficient chemical representation for individual
lipids with different pK dissociation constants that allows the
skilled artisan to select useful components and combinations for
the making of amphoteric liposomes. In addition, the isoelectric
point for a given amphoteric lipid composition can be easily tuned
through the molar ratio between the anionic and the cationic lipid
as presented in Hafez et al., Biophys. J., 79, (2000),
1438-1446.
4. Relationship Between .kappa.(Lipid Salt) and Counterions
[0252] Preferred values for .kappa. and counterion size have been
given above. In addition to these criteria, the absolute molecular
volume of a lipid molecule is of importance. For large absolute
volumes the relative impact of the counterion binding becomes
smaller. This is illustrated in FIG. 7 for lipids with identical
.kappa., but different absolute volumes. Other parameters used for
the calculation of FIG. 7 are given in Table 11 below; volumes in
.ANG..sup.3.
TABLE-US-00015 TABLE 11 Anion head volume see legend in FIG. 7
Anion tail volume see legend in FIG. 7 Anion pK 5 Cation head
volume see legend in FIG. 7 Cation tail volume see legend in FIG. 7
Cation pK 6.5 Counterion+ volume 70 Counterion- volume 70
[0253] In a preferred embodiment of the present invention the
absolute molecular volumes for the lipids are small, e.g., <1000
.ANG..sup.3 for the combined volumes of an anionic and cationic
lipid. More preferred are lipid pairs with about 700 .ANG..sup.3
molecular volume.
[0254] As was the case for counterions of different volumes,
asymmetric stability of a cationic and anionic lipid phase can also
be achieved by selection of lipids with different absolute
molecular volumes, even if said lipids have equal .kappa. values.
The example in FIG. 8 illustrates such a design variant, where a
smaller anionic lipid leads to selective destabilisation under
acidic conditions. Parameters used for the calculation of FIG. 8
are given in Table 12 below; volumes in .ANG..sup.3.
TABLE-US-00016 TABLE 12 Anion head volume 50 Anion tail volume 286
Anion pK 5 Cation head volume 100 Cation tail volume 571 Cation pK
6.5 Counterion+ volume 70 Counterion- volume 70
[0255] The algorithm presented above according to the invention
therefore provides structure-activity relationships between lipid
chemistry and stability of the resulting membrane, in particular in
response to the pH of the environment. Experimental data further
illustrate the invention and justify the model predictions. There
is clear evidence for the following: [0256] 1. Amphoteric liposomes
form stable bilayers at neutral pH, but are fusogenic at
intermediate pH. Some amphoteric liposomes form bistable liposomes
that exist in a stable phase at low and neutral pHs, but undergo
fusion at intermediate pHs. Examples 2, 3, 5 and 6 and
corresponding FIGS. 10, 11, 12 and 13 show this for different
amphoteric systems and lipid geometries, e.g., lipids with dialkyl-
or cholesterol- or monoalkyl membrane anchors. [0257] 2. The
intensity of fusion may be augmented or diminished through the use
of counterions of different size, as shown in Examples 8 and 9 and
the corresponding FIGS. 16 and 17. [0258] 3. .kappa.(salt)
correlates inversely with the fusion intensity of different
amphoteric systems. This means that systems with a lower
.kappa.(salt) show enhanced fusion. Experimental evidence is given
in Examples 12 and 13. FIGS. 19 a and 19b show an inverse
correlation between fusion intensity (expressed as .SIGMA.FRET) and
.kappa.(salt) for a large number of amphoter II systems, said
systems being formed from cholesterol based pH sensitive cationic
lipids and pH sensitive anionic lipids. FIG. 19c illustrates such a
plot for the cationic lipid DmC4Mo2 in combination with different
anionic pH sensitive lipids. Likewise, FIG. 20 shows such an
inverse correlation for amphoter I systems comprising DOTAP or
DODAP and various pH sensitive anions. A clear correlation between
the value of .kappa.(salt) and the fusogenicity of the amphoteric
systems becomes apparent from these experiments. [0259] 4. The
model also applies to amphoteric lipid mixtures further comprising
neutral lipids and the quantitative impact of such admixtures is
shown in Examples 15 and corresponding FIGS. 21 to 24. A relation
between .kappa.(neutral lipid admixture) and .kappa.(min) is
implicit in the model predictions and further described below. In
brief, the inclusion of neutral lipids may decrease the fusion
intensity of a given amphoteric system whenever .kappa.(neutral) is
higher than .kappa.(min). FIGS. 21a,b and 24a,b demonstrate this
experimentally. The opposite case can also be found, as
demonstrated in the FIGS. 23a,b. Eventually, some systems are less
affected by the introduction of neutral lipids, as shown in FIGS.
22a,b. Since experimental optimisation of systems with a higher
number of components becomes increasingly difficult and laborious,
numerical predictability according to the invention becomes even
more important and allows rapid and efficacious prediction. [0260]
5. The model predicts fusion around the isoelectric point of the
lipid mixture. Such correlation can be demonstrated in the
experiment and is analyzed in FIG. 26.
[0261] The data provided above show a high degree of predictability
from model calculations. The algorithm, starting from molecular
volume considerations and rather long range interactions of
electrical charges, does not reflect steric fit or misfit of the
components; it also does not take phase transition temperatures and
the associated molecular movements into account which might occur
in isolated cases. In some cases impaired fusion behaviour (e.g.
between MoChol and POPG, but not MoChol and DOPA as described
above) or enhanced fusion behaviour can be observed (e.g. DmC4Mo2
and various anionic lipids). In cases for which an enhanced fusion
behaviour is observed k(salt) of the lipid salt may be higher than
0.35, but less than 0.45.
[0262] Following is an assessment of liposomal fusion for a wide
range of amphoteric systems and selected preferred systems for
further practical use.
[0263] The quantitative structure-activity relationships taught by
this invention facilitate in silico screening and support rational
selection and optimization. Such screening may be used on its own
or in combination with empirical verification, e.g., by the
inclusion of selected data points within a series of lipid
homologues. The following sections comprise:
Section I: In silico screening of amphoteric systems Section II: In
silico screening of amphoteric systems, further comprising neutral
lipids Section III: Experimental screening of amphoteric systems
Section IV: Experimental screening of amphoteric systems, further
comprising neutral lipids.
Section I: In Silico Screening of Amphoteric Lipids
[0264] The present invention enables the selection of amphoteric
liposomes for a number of technical purposes. A more detailed
analysis is given below of the use of such amphoteric liposomes in
pharmaceutical applications. Amongst such pharmaceutical
applications, parenteral administration and direct administration
into the blood stream of a human or non-human animal, preferably a
mammal, is of particular importance. Amphoteric liposomes have
specific applicability inter alia in the intracellular delivery of
cargo molecules. As described above, during uptake into the cells,
liposomes are exposed to an acidic environment in the endosome or
lysosome of cells. Destabilisation of the lipid phase, e.g., by
enhanced fusogenicity is known to facilitate endosome escape and
intracellular delivery. It is possible that other environments of
low pH will also trigger said fusion, e.g., the low pH conditions
found in tumors or at sites of inflammation.
[0265] Amphoteric liposomes according to the present invention with
a preferred low value of .kappa.(salt) have been found to respond
advantageously to acidification by destabilisation or formation of
a fusogenic phase as intended.
[0266] To be stable under storage conditions or while in the blood
stream, a certain difference between .kappa.(total) at neutral pH
and .kappa.(salt) is necessary. In preferred embodiments, such
difference, referred to herein as d.kappa.(pH8), may be greater
than or to equal 0.08. As noted above, .kappa.(salt) is the
dominant predictor for fusogenicity, whereas d.kappa.(pH8)>=0.08
is a necessary, but not sufficient condition. A scoring of selected
systems was done using 1/.kappa.(salt) as a metric. High values
indicate systems with good fusion and sufficient stability
amplitude.
[0267] The difference between .kappa.(salt) and .kappa.(total) for
acidic conditions is of less importance, since an unstable lipid
phase under acidic conditions does not interfere with cellular
uptake.
[0268] In addition, methods to stabilise such lipid phase for
production have been described above.
[0269] The analysis is sensitive to counter-cation size and the
proportion of anionic lipid in the mixture. As mentioned above,
larger counter-cations make the selection less stringent, since
this parameter directly improves the d.kappa.(pH8) which means that
systems with a low amplitude become more functional. It also means
that systems with a low .kappa.(salt) can be stabilized
sufficiently to yield a stable phase at neutral pH. Although
resulting in a more or less stringent selection, the counter-cation
size does not change the observed overall pattern of selected
systems. This fact effectively compensates the variability of
counter-cation sizes that can be found in the literature.
[0270] Amphoteric lipid systems with lipid head-group sizes between
40 and 190 A.sup.3 and lipid hydrophobic tail sizes of 340, 410 or
500 A.sup.3 have been analyzed in the presence of a counter-cation,
specifically sodium (65 A.sup.3). The counter-anion is of less
relevance for the presented screen, since the ion (1) does not
participate in the lipid salt and (2) does essentially not bind to
the membrane at pH8.
[0271] The following in silico screens of amphoter I and amphoter
II and III systems provide a more general and experimentally
unbiased selection of fusogenic amphoteric liposomes. The
calculations allow one skilled in the art to deduce amphiphiles
with preferred head and tail sizes and subsequently to identify
improved amphoteric lipid mixtures.
[0272] The following selections were made according to
.kappa.(salt)<0.34 and d.kappa.(pH 8)<=0.08 and identified
limits for preferred lipid systems. Other limits for selection can
be used, allowing a broader or narrower search for amphoteric lipid
systems as desired.
Amphoter I Systems
[0273] For amphoter I systems, full dissociation of the anionic
amphiphile was assumed at pH 8. A library of 324 amphoter I lipid
systems having a C/A=0.5 was constructed and preferred lipid
systems having .kappa.(salt)<0.34 and d.kappa.(pH 8)<=0.08
were selected from the entire population.
TABLE-US-00017 TABLE 13 Table 13: Highly functional amphoter I
systems (C/A = 0.5, k(salt) < 0.34, dk(pH8) >= 0.08), values
represent 1/k(salt) head 40 70 100 130 160 190 40 70 100 tail 340
340 340 340 340 340 410 410 410 Cation k 0 12 0.21 0.20 0.3 0 47
0.56 0.10 0.17 0.34 Anion head tail k 40 340 0 12 70 340 0. 1 6.82
100 340 0 29 4.86 5.36 4.41 130 340 0. 4.00 3.40 4.41 3.75 3.2 160
340 0.47 3.40 2. 6 3.75 3.2 190 340 0 50 2. 6 3.2 40 410 0.10 70
410 0 17 100 410 0. 4 130 410 0 3 4.41 4.82 160 410 0. 3.75 3.26
4.10 3.57 190 410 0.46 3.26 3.57 3.15 40 500 0.0 70 500 0.14 100
500 0 20 130 500 0.2 160 500 0 32 190 500 0. 3.96 head 130 160 190
40 70 100 130 160 190 tail 410 410 410 500 500 500 500 500 500
Cation k 0.32 0 39 0.4 0 08 0.14 0.20 0.2 0 32 0.3 Anion head tail
k 40 340 0 12 70 340 0. 1 7.64 100 340 0 29 .00 4.94 4.20 130 340
0. 4.94 4.20 3. 5 3.23 160 340 0.47 4.20 3. 5 3.23 190 340 0 50 3.
5 3.23 40 410 0.10 70 410 0 17 100 410 0. 4 .50 130 410 0 3 5.35
4.55 160 410 0. 4.55 3. 3.50 190 410 0.46 3.86 3.50 3.14 40 500 0.0
70 500 0.14 100 500 0 20 130 500 0.2 160 500 0 32 5.00 190 500 0.
4.35 3.85 indicates data missing or illegible when filed
[0274] The table shows the positively screened systems and further
reveals consistent patterns for such systems within specific
combinations of lipid tail groups.
[0275] The effect of the lipid anion content in amphoter I systems
is somewhat more complex. First of all, a lower presence of lipid
anion (e.g. C/A 0.666 or 60 mol %) results in a more stringent
selection, this is due to the smaller amplitude of such systems.
Accordingly, the presence of higher amounts of lipid anion results
in a less stringent selection. Results for the respective
calculations are presented below.
TABLE-US-00018 TABLE 14 Table 14: Highly functional amphoter I
systems (C/A = 0.666, k(salt) < 0.34, dk(pH8) >= 0.08),
values represent 1/k(salt) head 40 70 100 130 160 190 40 70 100
tail 340 340 340 340 340 340 410 410 410 Cation k 0.12 0 21 0.29
0.38 0 7 0.56 0.10 0.17 0.2 Anion head tail k 40 340 0.12 70 340
0.21 100 340 0.29 130 340 0. 160 340 0.47 190 340 0.56 2.96 3.28 40
410 0.10 70 410 0.17 100 410 0.2 130 410 0. 2 160 410 0.3 190 410 0
4 40 500 0. 70 500 0 100 500 0.20 130 500 0 2 160 500 0. 190 500 0.
head 130 160 190 40 70 100 130 160 190 tail 410 410 410 500 500 500
500 500 500 Cation k 0 32 0.39 0 46 0.0 0 14 0.20 0 26 0. 2 0.
Anion head tail k 40 340 0.12 70 340 0.21 100 340 0.29 130 340 0.
160 340 0.47 4.20 190 340 0.56 3.65 3.23 40 410 0.10 70 410 0.17
100 410 0.2 130 410 0. 2 160 410 0.3 190 410 0 4 40 500 0. 70 500 0
100 500 0.20 130 500 0 2 160 500 0. 190 500 0. indicates data
missing or illegible when filed
[0276] The presence of lower amounts of the anionic lipids results
in selection pressure towards sterol based lipid anions with large
head groups in combination with cationic lipids of any tail size,
provided that these lipids have a minimal head group with a
molecular volume between 40 and 70 A.sup.3.
[0277] Presence of higher amounts of the anionic lipid lifts said
selection pressure and makes the composition more promiscuous in
general. It also shifts the optimum size for the lipid anion head
group towards moderate values between 100 and 130 A.sup.3 as shown
in the table below.
TABLE-US-00019 TABLE 15 Table 15: Highly functional amphoter I
systems (C/A = 0.333, k(salt) < 0.34, dk(pH8) >= 0.08),
values represent 1/k(salt) head 40 70 100 130 160 190 40 70 100
tail 340 340 340 340 340 340 410 410 410 Cation k 0.12 0 21 0.29 0
38 0.47 0.56 0.10 0.17 0.24 Anion head tail k 40 340 0.12 8.50 9.38
6.82 70 340 0.21 6.18 4.86 6.82 5.36 4.41 100 340 0.2 4.86 4.00
3.40 5.36 4.41 3.75 130 340 0. 4.00 3.40 2.96 4.41 3.75 3.26 160
340 0.47 3.40 2.96 3.75 3.26 190 340 0.5 2.96 3.26 40 410 0 10 70
410 0.17 6.82 7.45 100 410 0.2 5.38 4.41 5.88 4.82 130 410 0. 4.41
3.75 3.26 4.82 4.10 3.57 160 410 0.39 3.75 3.26 4.10 3.57 3.15 190
410 0 46 3.26 3.57 3.15 40 500 0.05 70 500 0 14 100 500 0.20 6.00
6.50 130 500 0 26 4.94 5.35 4.55 160 500 0. 4.20 3.65 4.55 3.96
3.50 190 500 0 3.65 3.23 3.96 3.50 3.14 head 130 160 190 40 70 100
130 160 190 tail 410 410 410 500 500 500 500 500 500 Cation k 0 32
0 39 0 46 0.0 0.14 0.20 0. 6 0. 2 0.38 Anion head tail k 40 340
0.12 10.50 7.64 70 340 0.21 7.64 6.00 4.94 100 340 0.2 3.26 6.00
4.94 4.20 3.65 3.23 130 340 0. 4.94 4.20 3.65 3.23 160 340 0.47
4.20 3.65 3.23 190 340 0.5 3.65 3.23 40 410 0 10 11.38 70 410 0.17
8.27 6.50 100 410 0.2 6.50 5.35 4.55 130 410 0. 5.35 4.55 3.96 3.50
160 410 0.39 4.55 3.96 3.50 3.14 190 410 0 46 3.96 3.50 3.14 40 500
0.05 70 500 0 14 100 500 0.20 7.14 130 500 0 26 5.88 5.00 160 500
0. 5.00 4.35 3.85 190 500 0 4.35 3.85 3.45 3.13 indicates data
missing or illegible when filed
[0278] Preferred amphoter I systems may rapidly be identified with
the procedures described above, said systems being characterized by
[0279] .kappa.(salt) being smaller than 0.34 [0280] d.kappa.(pH8)
being greater than 0.08 More preferred systems have [0281] smaller
lipid anion tail groups with a molecular volume smaller than 420
A.sup.3, selected from the group of sterols or
dimyristoylethylenglycols, most preferred this group is a
sterol
[0282] In another embodiment, the more preferred systems have
[0283] anionic head groups with a molecular volume between 70
A.sup.3 and 190 A.sup.3, said groups are selected from but not
limited to hemimalonates, hemisuccinates, hemiglutarates,
hemiadipates, cyclohexanoic diacids, glucuronic acids and
homologues thereof. [0284] cationic head groups with small
molecular volume between 40 and 100 A.sup.3, most preferred between
40 and 70 A.sup.3, said groups being selected from but not limited
to methylamine, dimethylamine, trimethylamines, tetramethylammonium
salts, N-methylpyridinium salts, trimethyl-hydroxyethylammonium
salts, N-a trimethylammoniumacetyl salts,
dimethylaminoethylcarbamates,
N-Methyl-mono(hydroxymethyl)aminomethane,
N-Methyl-bis(hydroxymethyl)aminomethane and homologues thereof.
[0285] The most preferred amphoter I systems have low .kappa.(salt)
and high d.kappa.(pH8) values in the presence of higher amounts of
the lipid cation, thus facilitating better binding and
encapsulation of polyanionic cargo molecules such as
oligonucleotides. These systems have [0286] a sterol based lipid
anion with a polar head group that is larger than 130 A.sup.3, most
preferred about 160 to 190 A.sup.3 [0287] a dioleoylglycerol based
lipid cation with polar head group that is smaller than 100
A.sup.3, most preferred smaller than 70 A.sup.3.
Amphoter II and III Systems
[0288] For amphoter II systems, full dissociation of the anionic
amphiphile was assumed at pH 8 and essentially no dissociation of
the cationic amphiphile was assumed at this pH. Such selections
also apply to amphoter III systems, as long as they contain 50% or
less of the anionic amphiphile.
[0289] First, a library of 324 amphoter II lipid systems having a
C/A=3 was constructed and preferred lipid systems having
.kappa.(salt)<0.34 and d.kappa.(pH 8)<=0.08 were selected
from this population.
TABLE-US-00020 TABLE 16 Table 16: Highly functional amphoter II
systems (C/A = 3, k(salt) < 0.34, dk (pH8) >= 0.08), values
represent 1/k(salt) head 40 70 100 130 160 190 40 70 100 tail 340
340 340 340 340 340 410 410 410 cation k 0 12 0 21 0 0 33 0.4 0.56
0 10 0 17 0 4 anion head tail k 40 340 0 12 4. 4.00 3.40 2. 70 340
0.21 3.40 2.96 100 340 0.29 130 340 0.3 160 340 0.47 190 340 0.56
40 410 0.10 5.3 4.41 3.75 3.2 70 410 0.17 3.75 3.2 100 410 0.24 3.2
130 410 0.32 160 410 0. 190 410 0 46 40 500 0.05 6.00 4.94 4.20
3.65 6.50 70 500 0.14 4. 4 4.20 3.65 3.23 100 500 0.20 3.65 3.23
130 500 0.26 160 500 0.32 190 500 0.3 head 130 160 190 40 70 100
130 160 190 tail 410 410 410 500 500 500 500 500 500 cation k 0. 2
0.39 0.4 0 0 0.14 0. 0 0.32 0 38 anion head tail k 40 340 0 12 4.41
3.75 3.26 3.65 70 340 0.21 3.26 100 340 0.29 130 340 0.3 160 340
0.47 190 340 0.56 40 410 0.10 4.82 4.10 3.57 4.55 3.9 70 410 0.17
3.57 3.15 3.50 100 410 0.24 130 410 0.32 160 410 0. 190 410 0 46 40
500 0.05 5.35 4.55 3.9 5.00 4.35 70 500 0.14 4.55 3.96 3.50 3.85
100 500 0.20 3.50 3.14 130 500 0.26 160 500 0.32 190 500 0.3
indicates data missing or illegible when filed
[0290] Preferred cation-rich amphoter II (or amphoter III) systems
can rapidly be identified with the procedures described above, said
systems being characterized by [0291] k(salt) being smaller than
0.34 [0292] dk(pH8) being greater than 0.08 [0293] lipid cation
tail groups with a molecular volume smaller than 420 A.sup.3,
selected from the group of sterols or dimyristoylethylenglycols,
most preferred this group is a sterol [0294] lipid anion tail
groups with a molecular volume larger than 400 A.sup.3, selected
from the groups of diacylethylenglycols, most preferred
dipalmitoyl-distearoyl-palmitoyloleoyl- or
dioleoylethylenglycols.
[0295] In another embodiment, the more preferred systems have
[0296] cationic head groups with a molecular volume between 70
A.sup.3 and 160 A.sup.3, selected from but not limited to
morpholines, propylimidazols, 3-imidazol-1-yl-propyl carbamates,
piperazine 4-N-aminoethyl carbamoyls, 2-(4-Imidazolyl)ethylamine
hemisuccinates,
1-[2-carboxyethyl]2-methyl-3-(2-hydroxyethyl)imidazolinium salts,
ethylphosphocholines, N-Morpholino ethylamine hemisuccinates,
1-Methyl-4-choline-succinic acid diesters and homologues of said
compounds. [0297] anionic head groups with small molecular volume
between 40 and 100 A.sup.3, most preferred between 40 and 70
A.sup.3, said groups being selected from but not limited to
hemimalonates, hemisuccinates, hemiglutarates and homologues
thereof.
[0298] Amphoter II systems with about equimolar C/A have more
complex selection patterns, but include the aforementioned
cation-rich systems. Said systems therefore facilitate tuning of
the isoelectric point without loosing functionality. Table 17 below
presents systems that were selected in accordance with the criteria
presented above:
TABLE-US-00021 TABLE 17 Table 17: Highly functional amphoter II
systems (C/A = 1, k(salt) < 0.34, dk(pH8) >= 0.08), values
represent 1/k(salt) head 40 70 100 130 160 190 40 70 100 tail 340
340 340 340 340 340 410 410 410 cation k 0.12 0 21 0.29 0.3 0.47 0.
0.10 0.17 0.24 anion head tail k 40 340 0.12 8.50 6.18 4.86 4.00
3.40 2.96 9.38 6.82 5.36 70 340 0.21 6.18 4.86 4.00 3.40 2.96 6.82
5.36 4.41 100 340 0.29 4.86 4.00 3.40 2.96 5.36 4.41 3.75 130 340
0.39 4.00 3.40 2.96 4.41 3.75 3.26 160 340 0.47 3.40 2.96 3.75 3.26
190 340 0.5 2.96 3.26 40 410 0 10 9.38 8.82 5.36 4.41 3.75 3.26 70
410 0.17 5.36 4.41 3.75 3.26 100 410 0.24 3.75 3.26 130 410 0.32
160 410 0.39 190 410 0 40 40 500 0.0 6.00 4.94 4.20 3.65 70 500 0
14 4.20 3.65 3.23 100 500 0.20 3.65 3.23 130 500 0.26 160 500 0.32
190 500 0.38 head 130 160 190 40 70 100 130 160 190 tail 410 410
410 500 500 500 500 500 500 cation k 0. 2 0. 9 0.46 0.0 0.14 0.20
0.26 0.32 0 38 anion head tail k 40 340 0.12 4.41 3.75 10.50 7.64
6.00 4.94 70 340 0.21 3.75 3.26 7.64 6.00 4.94 4.20 3.65 100 340
0.29 3.26 6.00 4.94 4.20 3.65 3.23 130 340 0.39 4.94 4.20 3.65 3.23
160 340 0.47 4.20 3.65 3.23 190 340 0.5 3.65 3.23 40 410 0 10 11.38
70 410 0.17 8.27 6.50 100 410 0.24 6.50 5.35 4.55 130 410 0.32 5.35
4.55 3.96 3.50 160 410 0.39 4.55 3.96 3.50 3.14 190 410 0 40 3.96
3.50 3.14 40 500 0.0 4.55 3.96 70 500 0 14 3.50 100 500 0.20 130
500 0.26 160 500 0.32 190 500 0.38 indicates data missing or
illegible when filed
[0299] In addition to the selection criteria used for cation-rich
amphoter II system, the amphoter II (C/A=1) systems display a
preference for [0300] lipid anion tail groups with a molecular
volume smaller than 420 A.sup.3, selected from the group of sterols
or dimyristoylethylenglycols, most preferred this group is a
sterol
[0301] The anion-rich amphoter II systems are the least demanding
ones, as far as structural constraints are concerned. However, this
structural freedom comes at the cost of limited encapsulation
efficiency for important cargo types, such polyanions, in
particular oligonucleotides. The selection screen was applied to
amphoter II system with C/A=0.5 and results in the following
pattern:
TABLE-US-00022 TABLE 18 Table 18: Highly functional amphoter II
systems (C/A = 0.5, k(salt) < 0.34, dk(pH8) >= 0.08), values
represent 1/k(salt) head 40 70 100 130 160 190 40 70 100 tail 340
340 340 340 340 340 410 410 410 cation k 0 12 0.21 0.29 0 3 0 4 0
0.10 0 17 0.24 anion head tail k 40 340 0.12 8.50 6.18 4.86 4.00
9.38 6.82 5.36 70 340 0 21 6.18 4.86 4.00 3.40 2.96 6.82 5.36 4.41
100 340 0.29 4.86 4.00 3.40 2.96 5.36 4.41 3.75 130 340 0.3 4.00
3.40 2.96 4.41 3.75 3.26 160 340 0 4 3.40 2.96 3.75 3.26 190 340
0.5 2.96 3.26 40 410 0 10 9.38 6. 2 5.36 10.25 7.45 5.86 70 410
0.17 6. 2 5.36 4.41 3.75 7.45 5.86 4.82 100 410 0.2 5.36 4.41 3.75
3.26 5.86 4.82 4.10 130 410 0.32 4.41 3.75 3.26 4.82 4.10 3.57 160
410 0.39 3.75 3.26 4.10 3.57 3.15 190 410 0 46 3.26 3.57 3.15 40
500 0.05 10.50 11.38 70 500 0 14 7.64 6.00 8.27 6.50 100 500 0.20
6.00 4.94 6.50 5.35 4.55 130 500 0 26 4.94 4.20 3. 5 5.35 4.55 3.96
160 500 0 32 4.20 3. 5 3.23 4.55 3.96 3.50 190 500 0 8 3. 5 3.23
3.96 3.50 3.14 head 130 160 190 40 70 100 130 160 190 tail 410 410
410 500 500 500 500 503 500 cation k 0 32 0. 9 0.46 0 0 0.14 0.20
0.26 0 32 0.38 anion head tail k 40 340 0.12 4.41 10.50 7.64 6.00
4.94 70 340 0 21 3.75 3.26 7.64 6.00 4.94 4.20 3.65 3.23 100 340
0.29 3.26 6.00 4.94 4.20 3.65 3.23 130 340 0.3 4.94 4.20 3.65 3.23
160 340 0 4 4.20 3.85 3.23 190 340 0.5 3.65 3.23 40 410 0 10 11.38
8.27 6.50 70 410 0.17 4.10 8.27 6.50 5.35 4.55 100 410 0.2 3.57
3.15 6.50 5.35 4.55 3.9 3.50 130 410 0.32 3.15 5.35 4.55 3.9 3.50
3.14 160 410 0.39 4.55 3.9 3.50 3.14 190 410 0 46 3.9 3.50 3.14 40
500 0.05 12.50 70 500 0 14 9.09 7.14 100 500 0.20 7.14 5.88 5.00
130 500 0 26 5.88 5.00 4.35 3.85 160 500 0 32 3.14 5.00 4.35 3.85
3.45 3.13 190 500 0 8 4.35 3.85 3.45 3.13 indicates data missing or
illegible when filed
[0302] Amphoter II systems with anion excess have a preference for
[0303] cation head groups with a molecular volume of less than 130
A.sup.3, preferred less than 100 A.sup.3, being selected from but
not limited to imidazols, methylimidazols, ethylimidazols,
morpholins, methylmorpholins, ethylmorphlins,
N-Methyl-tris(hydroxymethyl)aminomethanes, 3-imidazol-1-yl-propyl
carbamates, piperazine 4-N-aminoethyl carbamoyls,
N-Methyl-mono(hydroxymethyl)aminomethanes,
N-Methyl-bis(hydroxymethyl)aminomethanes and homologues thereof.
[0304] anionic head groups with a molecular volume of less than 130
A.sup.3, preferred about 70 A.sup.3 or less, being selected from
but not limited to hemimalonates, hemisuccinates, hemiglutarates,
hemiadipates, cyclohexanoic diacids and homologues thereof
[0305] With more lipid anions in the mixture, the pattern of
positively selected candidates resembles this of the amphoter I
systems.
[0306] However, the latter ones are somewhat compromised in their
dk(pH8) values as the constant formation of the lipid salt reduces
this amplitude.
Section II: In Silico Screening of Amphoteric Systems, Further
Comprising Neutral Lipids
[0307] The algorithm taught by this invention also facilitates
quantitative predictions to be made on the effect of neutral lipid
admixtures to amphoteric lipid systems. Such admixtures may result
in improved stability of the liposome; they might further result in
better resistance against serum proteins or enhanced uptake into
cells. Optimization of amphoteric systems is a challenging task on
its own, owing to the large number of useful components. This task
becomes even more complicated with the addition of further
components and rational approaches are urgently needed.
[0308] The methodology developed in the previous sections above was
therefore applied to more complex systems including neutral lipid
components. With respect to the main parameters k(salt) and
dk(pH8), the addition of neutral lipids may result in [0309] an
increase of k(min) if k(neutral) is higher than k(salt) and vice
versa, said increase being proportional to the amount of neutral
lipid added. [0310] a compression of the system amplitude dk(pH8)
by addition of any neutral lipid, since these lipids do not change
their geometry upon changes in the pH.
[0311] Still, even the more complex systems need a certain k(min)
to achieve fusion and a certain dk(pH8) to maintain stability. The
corresponding values from section 1 have been used for the analysis
presented here, which are k(min) being the functional equivalent to
k(salt), said k(min)<0.34 and dk(pH8)>0.08.
Amphoter I Systems Further Comprising Neutral Lipids
[0312] Libraries of amphoter I systems (C/A=0.333) were constructed
as described previously and highly functional systems were selected
using k(min)<0.34 and dk(pH8)>0.08 as criteria. Fitness of
the selected systems is presented as 1/k(min) in the table below
for the addition of 30% cholesterol to the library.
TABLE-US-00023 TABLE 19 Table 19: Highly functional amphoter I
systems comprising 30% cholesterol. (C/A = 0.333, k(min) < 0.34
and dk(pH8) > 0.08, values represent 1/k (min) head 40 70 100
130 160 190 40 70 100 tail 340 340 340 340 340 340 415 410 410
Cation k 0.12 0.21 0.29 0.38 0 47 0. 0 10 0.17 0 24 Anion head tail
k 40 340 0.12 70 340 0.21 8.06 8.81 100 340 0.2 6.46 5.38 7.07 5.90
130 340 0. 5.38 4.62 4.04 5.90 5.06 4.44 160 340 0 47 4.62 4.04
3.59 3.23 5.06 4.44 3.95 190 340 0.56 4.04 3.59 3.23 4.44 3.95 3.55
40 410 0 10 70 410 0.17 100 410 0.24 7.67 130 410 0.32 5.90 6.41
5.51 160 410 0.39 5.06 4.44 5.51 4.83 4.30 190 410 0 4 4.44 3.95
3.55 4.83 4.30 3.87 40 500 0.0 70 500 0.14 100 500 0.20 130 500
0.26 160 500 0.32 6.07 190 500 0 38 4.94 5.33 head 130 160 190 40
70 100 130 160 190 tail 410 410 410 500 500 500 500 500 500 Cation
k 0.32 0 39 0. 0 08 0.14 0.20 0.2 0. 0.38 Anion head tail k 40 340
0.12 70 340 0.21 9.76 7.85 100 340 0.2 7.85 6.56 5.83 130 340 0.
6.56 5.63 4.94 4.40 160 340 0 47 3.55 3.23 5.63 4.94 4.40 3.96 3.60
3.31 190 340 0.56 3.23 2.96 4.94 4.40 3.96 3.60 3.31 3.05 40 410 0
10 70 410 0.17 100 410 0.24 8.44 130 410 0.32 7.06 6.07 160 410
0.39 6.07 5.33 4.74 4.28 190 410 0 4 3.52 5.33 4.74 4.28 3.89 3.57
40 500 0.0 70 500 0.14 100 500 0.20 130 500 0.26 160 500 0.32 .63
190 500 0 38 5.82 5.19 indicates data missing or illegible when
filed
[0313] In comparison with the data presented for the corresponding
amphoter I library from section I, the addition of cholesterol
results in a somewhat different selection that is more biased
towards anionic lipids with large headgroups, while maintaining
other features like the preference of amphoter I for cholesterol or
dimyristoylglycol as tail regions for the anionic lipids.
[0314] Addition of a strongly lamellar lipid such as POPC or DOPC
results in more stringent selection without qualitative impact on
the selection rules presented before.
Amphoter II Systems Further Comprising Neutral Lipids
[0315] Libraries of cation-rich amphoter II systems (C/A=3) were
constructed as described previously and highly functional systems
were selected using k(min)<0.34 and dk(pH8)>0.08 as criteria.
Fitness of the selected systems is presented as 1/k(min) in Table
20 below for the addition of 30% cholesterol to the library.
TABLE-US-00024 TABLE 20 Table 20: Highly functional amphoter II
systems comprising 30% cholesterol. (C/A = 3, k(min) < 0.34 and
dk(pH8) > 0.08, values represent 1/k(min) head 40 70 100 130 160
190 40 70 100 tail 340 340 340 340 340 340 410 410 410 cation k
0.12 0 21 0 29 0. 0 47 0 0.10 0.17 0 24 anion head tail k 40 340
0.12 4. 2 4.04 70 340 0.21 3.59 100 340 0.29 130 340 0.3 160 340 0
47 190 340 0.5 40 410 0 10 5.90 5.0 4.44 70 410 0.17 4.44 3. 5 100
410 0. 3.55 130 410 0.3 160 410 0. 190 410 0 4 40 500 0.05 .56 5.63
4.94 70 500 0 14 5.63 4. 4 4.40 100 500 0.2 4.40 3.96 130 500 0 2
3.60 160 500 0.32 3.31 190 500 0 38 head 130 160 190 40 70 100 130
160 190 tail 410 410 410 500 900 500 500 500 500 cation k 0 32 0. 9
0.46 0.0 0.14 0.20 0 2 0.32 0 8 anion head tail k 40 340 0.12 4.44
70 340 0.21 100 340 0.29 130 340 0.3 160 340 0 47 190 340 0.5 40
410 0 10 4.83 70 410 0.17 100 410 0. 130 410 0.3 160 410 0. 190 410
0 4 40 500 0.05 .07 5.33 70 500 0 14 4.74 100 500 0.2 130 500 0 2
160 500 0.32 190 500 0 38 indicates data missing or illegible when
filed
[0316] In comparison with the data presented for the corresponding
cation-rich amphoter II library from section I, the addition of
cholesterol results in a somewhat different selection that is
substantially biased towards cationic lipids with large headgroups,
while maintaining other features like the preference of amphoter I
for large tails regions such as dimyristoylglycol or dioleoylglycol
for the anionic lipids.
[0317] Addition of a lamellar lipid such as POPC or DOPC results in
more stringent selection without qualitative impact on the
selection rules presented before.
[0318] Libraries of equilibrated amphoter II systems (C/A=1) were
also constructed and introduced into the selection scheme in the
presence of 30% cholesterol in this the library.
TABLE-US-00025 TABLE 21 Table 21: Highly functional amphoter II
systems comprising 30% cholesterol. (C/A = 1, k(min) < 0.34 and
dk(pH8) > 0.08, values represent 1/k(min) head 40 70 100 130 160
190 40 70 100 tail 340 340 340 340 340 340 410 410 410 cation k
0.12 0.21 0.29 0.3 0 47 0 0.10 0 17 0.2 anion head tail k 40 340
0.12 70 340 0.21 100 340 0.29 130 340 0.3 160 340 0 47 190 340 0.
4.44 40 410 0.10 70 410 0 1 100 410 0.2 130 410 0.32 160 410 0.3
190 410 0 4 40 500 0. 70 500 0 4 100 500 0.20 130 500 0.26 160 500
0.32 190 500 0 38 head 130 160 190 40 70 100 130 160 190 tail 410
410 410 500 500 500 500 500 500 cation k 0 2 0. 9 0. 6 0. 0.14 0.20
0.26 0.32 0 8 anion head tail k 40 340 0.12 70 340 0.21 100 340
0.29 7.85 130 340 0.3 6.5 5. 3 160 340 0 47 5.63 4.94 4.40 3.9 190
340 0. 4.94 4.40 3.9 3.60 3.31 40 410 0.10 70 410 0 1 100 410 0.2
130 410 0.32 160 410 0.3 190 410 0 4 40 500 0. 70 500 0 4 100 500
0.20 130 500 0.26 160 500 0.32 190 500 0 38 indicates data missing
or illegible when filed
[0319] While the corresponding amphoter II library (C/A=1) from
section I had numerous positive systems, the addition of 30%
cholesterol resulted in a very stringent selection. This is
counterintuitive to the addition of a lipid that promotes fusion
and illustrates the impact of dk(pH8) as a selection criterium. A
close group can be identified comprising: [0320] sterol based lipid
anions with head group volumes greater than 100 A.sup.3 [0321]
diacylgylcol based cations with head group volumes smaller than 160
A.sup.3, more preferred smaller than 70 A.sup.3
[0322] In this group, the addition of a lamellar lipid such as POPC
or DOPC had similar impact than the addition of cholesterol.
[0323] Libraries of anion-rich amphoter II systems (C/A=0.33) were
also constructed and introduced into the selection scheme in the
presence of 30% cholesterol in this the library.
TABLE-US-00026 TABLE 22 Table 22: Highly functional amphoter II
systems comprising 30% cholesterol. (C/A = 0.333, k(min) < 0.34
and dk(pH8) > 0.08, values represent 1/k (min) head 40 70 100
130 160 190 40 70 100 tail 340 340 340 340 340 340 410 410 410
cation k 0.12 0.21 0 29 0. 0 47 0 0.10 0 7 0.24 anion head tail k
40 340 0.12 10.74 8.0 11.70 8.81 70 340 0.21 8.0 6.4 5.38 8.81 7.07
5.90 100 340 0. 3 6.46 5.38 4. 2 4.04 7.07 5. 0 5.08 130 340 0.3
5.38 4.82 0.04 3.59 3.23 5.90 5.0 4.44 160 340 0.47 4.62 4.04 3.59
3.23 2.94 5.0 4.44 3.95 190 340 0.56 4.04 3.59 3.23 2.94 4.44 3. 5
3.55 40 410 0.10 11.70 12. 4 70 410 0.17 .81 9.55 7. 7 100 410 0.24
7.07 5.90 7. 7 6.41 5.51 130 410 0.32 5.90 5.06 4.44 6.41 5.51 4.83
160 410 0.39 5.0 4.44 3.95 3.55 5.51 4.83 4.30 190 410 0 6 4.44
3.95 3.55 3.23 2.96 4.83 4.30 3.87 40 500 0.0 70 500 0 14 100 500
0.20 8.44 130 500 0.26 6.5 7.06 6.07 160 500 0.3 5. 3 4.94 6.07
5.33 190 500 0 8 4.94 4.40 5.33 4.74 4.28 head 130 160 190 40 70
100 130 160 190 tail 410 410 410 500 500 500 500 500 500 cation k
0.32 0. 9 0.46 0 0 1.14 0.20 0.2 0.32 0 8 anion head tail k 40 340
0.12 12.91 9.7 7.85 70 340 0.21 9.7 7.85 6.58 5.63 100 340 0. 3
4.44 3.95 7.85 6.5 5.63 4.94 4.40 130 340 0.3 3.95 3.55 3.23 6.5 5.
3 4.94 4.40 3.96 3.60 160 340 0.47 3.55 3.23 2. 5.63 4.94 4.40 3.96
3.60 3.31 190 340 0.56 3.23 2. 4.94 4.40 3.96 3. 0 3.31 3.05 40 410
0.10 13. 2 70 410 0.17 10.48 8.44 100 410 0.24 8.44 7.0 6.07 130
410 0.32 4.30 7.0 6.07 5.33 4.74 4.28 160 410 0.39 3.87 3.52 6.07
5.33 4.74 4.28 3.89 3.57 190 410 0 6 3.52 3.23 2.98 5.33 4.74 4.28
3.89 3.57 3.30 40 500 0.0 70 500 0 14 100 500 0.20 9.19 130 500
0.26 7.70 6.03 160 500 0.3 8.83 5.82 5.19 190 500 0 8 5.82 5.19
4.68 4.26 indicates data missing or illegible when filed
[0324] Here, some bias of the positive candidates towards larger
anion head groups can be observed. However, this needs to be
interpreted carefully since the fusion activity is always improving
in the presence of small anionic headgroups.
[0325] Addition of lamellar lipids such as POPC or DOPC imply more
stringent selection criteria in comparison to the systems in
section I, but do not qualitatively change the pattern of positive
candidates.
Section III: Experimental Screen of Amphoteric Lipid Systems
[0326] The fusogenicity of different amphoteric liposome mixtures
comprising charged amphiphiles can be investigated using lipid
fusion assays, particle growth or other methods known in the art,
thereby allowing the identification of preferred mixtures. Lipid
mixing can be tested with fluorescence resonance energy transfer
(FRET), and experimental details are described in Example 11
wherein the fusion of amphoteric lipid mixtures was monitored
within a pH range of between pH 2.5 and pH 7.5.
Amphoter I Systems Comprising Charged Amphiphiles Only
[0327] Amphoter I systems are characterized by a stable cation in
combination with an excess of a chargeable anion. Preferred
amphoter I systems solely comprising charged amphiphiles form
stable lamellar phases at pH 7 to pH 8 and fuse between pH 3 to pH
6, preferably between pH 4 to pH 6. Within a given amphoter I
system fusion was monitored for different ratios of cationic to
anionic lipid (C/A ratio, always <1).
[0328] Amphoter I systems that are stable at pH 7 to pH 8 and fuse
between pH 3 to pH 6, preferably between pH 4 to pH 6 may be formed
from mixtures of one or more cationic amphiphiles selected from
DOTAP, DMTAP, DPTAP, DSTAP, POTAP, DDAB, DODAC, DOEPC, DMEPC,
DPEPC, DSEPC, POEPC, DC-Chol, TC-Chol, DAC-Chol, DODAP, DMTAP,
DPDAP, DSDAP, PODAP, N-methyl-PipChol, CTAB, DOTMA with one or more
anionic amphiphile selected from diacylglycerol succinates, e.g.
DOGS, DMGS, POGS; diacylglycerolmalonates, e.g. DOGM or DMGM;
diacylglycerolglutarates, e.g. DOGG, DMGG; diacyiglyceroladipates,
e.g. DOGA, DMGA; 4-{(1,2-Diacyl-ethyl)amino}-4-oxo acids, e.g.
DOAS, DOAM, DOAG, DOAA, DMAS, DMAM, DMAG, DMAA; Diacyl-alkanoic
acids, e.g. DOS, DOM, DOG, DOA, DMS, DMM, DMG, DMA; Chems and
derivatives thereof, e.g. Chol-C3, Chol-C5 or Chol-C6; fatty
acids.
[0329] In one embodiment of the invention, the cationic amphiphiles
are selected from DODAP, DOTAP, N-methyl-PipChol, DDAB, DOEPC,
DC-Chol, DAC-Chol or TC-Chol and combined with anionic amphiphiles
selected from Chems, DMGS, DMGM, DMGG, DMGA, DMAS, DMAM, DMAG,
DMAA, DOGS, DOGM, DOGG, DOGA, DOAS, DOAM, DOAG, DOAA, DMS, DMM,
DMG, DMA, DOS, DOM, DOG, DOA, Chol-C3, Chol-C5 or Chol-C6.
[0330] In some embodiments of the invention the following amphoter
I mixtures are preferred which comprise charged amphiphiles only,
are stable at pH 7 to pH 8 and fuse between pH 3 to pH 6,
preferably between pH 4 to pH 6 (Table 23):
TABLE-US-00027 TABLE 23 C/A Cation Anion (molar ratio) DODAP DMGS
>0-<1 DODAP DOGS >0-<1 DODAP Chems >0-<1 DOTAP OA
>0-0.33 N-methyl- DMGS >0-<1 PipChol N-methyl- DOGS
>0-<1 PipChol N-methyl- Chems >0-<1 PipChol DDAB DMGS
>0-0.5 DDAB DOGS >0-0.5 DOEPC DMGS >0-0.5 DOEPC DOGS
>0-0.5 DOEPC Chems >0-0.5 DC-Chol DOGS >0-<1 DOTAP DMGS
>0-0.5 DC-Chol DMGS >0-<1 DC-Chol Chems >0-<1 CTAB
DMGS >0-0.5 TC-Chol DMGS >0-0.67 TC-Chol DOGS >0-0.67
TC-Chol Chems >0-0.67 DOTAP Chol-C3 .gtoreq.0.5-0.67 DOTAP
Chol-C5 >0-0.4 DOTAP Chems >0-0.4 DOTAP DOGS >0-0.4 DOTAP
Chol-C6 >0-0.17 DDAB Chems >0-0.17
[0331] Less preferred are amphoter I systems that are not stable at
pH 7 to pH 8, for example the amphoter I systems DOTAP/stearic acid
or DOTAP/oleic acid at C/A>0.33.
[0332] More versatile amphoter I systems of Table 23 are those that
are fusogenic over a wide range of C/A ratios, thus allowing
adjustment of the fusion pH without changing the chemistry of the
system. Some versatile systems are fusogenic over C/A ratios that
differ by .gtoreq.0.4, more versatile systems retain fusogenicity
for C/A ratios that differ by >0.6 and some system are fusogenic
over the entire range of C/A ratios. For example, the amphoter I
system DDAB/DMGS shows fusion from C/A>0 to C/A=0.5. The range
of C/A ratios for which this system is fusogenic is about 0.5.
[0333] Preferred amphoteric mixtures of Table 23 are fusogenic at
higher C/A ratios, preferably at C/A.gtoreq.0.4, and more
preferably at C/A ratios.gtoreq.0.5, thus facilitating the
encapsulation of higher amounts of polyanionic cargoes, such as
nucleic acids.
[0334] In another embodiment of the invention, the amphoter I
systems may form a second stable phase at an acidic pH of between
pH 2 to pH 4 in addition to the aforementioned stable lamellar
phase at pH 7 to pH 8. However, as described before a stable
lamellar phase at low pH is not mandatory and, e.g., for the
production of amphoteric liposomes and the encapsulation of cargo
under acidic conditions large counter-anions may stabilize the
lipid phase at this pH range.
[0335] Preferred fusogenic amphoter I systems comprising charged
amphiphiles only and having stable lamellar phases at pH 7 to pH 8
and at pH 2 to pH 4 and fusing between pH 3 and pH 6, preferably
between pH 4 and pH 6 may comprise the following specific mixtures
(Table 24):
TABLE-US-00028 TABLE 24 C/A Cation Anion (molar ratio) DODAP DMGS
.gtoreq.0.5-<1 DOTAP OA 0.17 N-methyl- DMGS .gtoreq.0.5-<1
PipChol DDAB DMGS 0.5 DOEPC DMGS .gtoreq.0.33-0.5 DC-Chol DOGS
.gtoreq.0.33-<1 DOTAP DMGS .gtoreq.0.4-0.5 DC-Chol DMGS
.gtoreq.0.5-<1 DC-Chol Chems .gtoreq.0.4-<1 CTAB DMGS 0.5
TC-Chol DMGS .sup. .gtoreq.0.4-0.67 DOTAP Chol-C3 .sup.
.gtoreq.0.5-0.67 DOTAP Chol-C5 .gtoreq.0.33-0.4 DOTAP Chems
.gtoreq.0.33-0.4 DOTAP DOGS >0-0.4 DDAB Chems 0.17
[0336] Versatile amphoter I systems of Table 14 are fusogenic over
a wide range of C/A ratios, preferably over a range of C/A ratios
of .gtoreq.0.4, and more preferably over a range of C/A ratios of
.gtoreq.0.6.
[0337] Furthermore, preferred are the amphoteric mixtures of Table
24 above that are fusogenic at higher C/A ratios, preferably at
C/A.gtoreq.0.4, and more preferably at C/A ratios.gtoreq.0.5.
Amphoter II Systems Comprising Charged Amphiphiles Only
[0338] Amphoter II systems comprise chargeable anions and
chargeable cations and have therefore the advantage of being
amphoteric over the entire range of anion:cation ratios. No charge
overcompensation for the strong ion is needed as in Amphoter I or
Amphoter III systems.
[0339] Amphoter II systems that are stable at pH 7 to pH 8 and fuse
between pH 3 to pH 6, preferably between pH 4 to pH 6 may include
mixtures of one or more cationic amphiphiles selected from MoChol,
HisChol, Chim, MoC3Chol, DmC4Mo2, DmC3Mo2, C3Mo2, C5Mo2, C6Mo2,
C8Mo2, C4Mo4, DOIM or DPIM with one or more anionic amphiphile
selected from diacylglycerol succinates, like DOGS, DMGS, POGS;
diacylglycerolmalonates, like DOGM or DMGM;
diacylglycerolglutarates, e.g. DOGG, DMGG; diacylglyceroladipates,
e.g. DOGA, DMGA; 4-{(1,2-Diacyl-ethyl)amino}-4-oxo acids, e.g.
DOAS, DOAM, DOAG, DOAA, DMAS, DMAM, DMAG, DMAA; Diacyl-alkanoic
acids, like DOS, DOM, DOG, Chems and derivatives thereof, e.g.
Chol-C3, Chol-C5 or Chol-C6; fatty acids.
[0340] In one embodiment of the invention, the cationic amphiphiles
are selected from MoChol, HisChol, Chim, MoC3Chol, DmC4Mo2,
DmC3Mo2, C3Mo2, C5Mo2, C6Mo2, C8Mo2, C4Mo4, DOIM or DPIM and
combined with anionic amphiphiles selected from Chems, DMGS, DMGM,
DMGG, DMGA, DMAS, DMAM, DMAG, DMAA, DOGS, DOGM, DOGG, DOGA, DOAS,
DOAM, DOAG, DOAA, DMS, DMM, DMG, DMA, DOS, DOM, DOG, DOA, Chol-C3,
Chol-C5 or Chol-C6.
[0341] In some embodiments of the invention, amphoter II systems
comprising charged amphiphiles only that are stable at pH 7 to pH 8
and fuse between pH 3 to pH 6, preferably between pH 4 to pH 6, are
preferred (Table 25):
TABLE-US-00029 TABLE 25 C/A Cation Anion (molar DmC4Mo2 DOGS >0
DmC4Mo2 Chems >0 DmC4Mo2 DMGS >0 DmC4Mo2 Chol-C5 >0-2.sup.
DmC4Mo2 Chol-C3 .gtoreq.0.7 DmC4Mo2 Chol-C6 >0-2.sup. Chim DMGS
>0-2.sup. Chim Chems >0-1.5 Chim DOGS >0-1.sup. HisChol
DOGS >0-0.7 HisChol Chems >0-0.7 HisChol DMGS >0-0.7
MoC3Chol DOGS >0-0.7 MoC3Chol Chems >0-0.7 MoC3Chol DMGS
>0-0.7 DmC3Mo2 DOGS >0-1.5 DmC3Mo2 Chems >0-1.sup. DmC3Mo2
DMGS >0-1.5 C3Mo2 DOGS >0-2.sup. C3Mo2 Chems >0-1.sup.
C3Mo2 DMGS >0-2.sup. C3Mo3 DOGS >0-0.7 C3Mo3 Chems >0-0.7
C3Mo3 DMGS >0-0.7 C4Mo4 DOGS >0-0.7 C4Mo4 Chems >0-0.5
C4Mo4 DMGS >0-0.7 C5Mo2 DOGS >0-1.sup. C5Mo2 Chems >0-0.7
C5Mo2 DMGS >0-1.sup. C6Mo2 DOGS >0-1.sup. C6Mo2 Chems
>0-1.sup. C6Mo2 DMGS >0-1.sup. C8Mo2 DOGS >0-1.sup. C8Mo2
Chems >0-0.7 C8Mo2 DMGS >0-1.sup. MoChol DOGS >0-1.sup.
MoChol Chems >0-0.7 MoChol Chol-C3 >0-0.7 MoChol Chol-C5
>0-0.7 MoChol DMGS >0-0.7 MoChol Chol-C6 >0-0.5 DOIM DOGS
>0-0.7 DOIM Chems >0-0.7 DOIM DMGS >0-0.7
[0342] More versatile amphoter II systems of Table 25 are those
that are fusogenic over a wide range of C/A ratios, thus allowing
adjustment of the fusion pH without changing the chemistry of the
system. Some versatile systems are fusogenic over C/A ratios that
differ by .gtoreq.0.7, more versatile systems retain fusogenicity
for C/A ratios that differ by .gtoreq.1 and some systems are
fusogenic over the entire range of C/A ratios. For example, the
amphoter I system DDAB/DMGS shows fusion from C/A>0 to C/A=0.5.
The range of C/A ratios for which this system is fusogenic is about
0.5. For example, the mixture Chim/Chems may fuse between C/A>0
and C/A=1.5, resulting in a range of C/A ratios of >1.
[0343] Preferred amphoter II mixtures of Table 25 are fusogenic at
higher C/A ratios, preferably at C/A.gtoreq.0.7, and more
preferably at C/A ratios.gtoreq.1, thus facilitating the
encapsulation of higher amounts of polyanionic cargoes such as
nucleic acids.
[0344] In another embodiment of the invention, amphoter II systems
may form a second stable phase at an acidic pH of between pH 2 and
pH 4 in addition to the aforementioned stable lamellar phase at pH
7 to pH 8. However, as described before, a stable lamellar phase at
low pH is not mandatory and, e.g., for the production of amphoteric
liposomes and the encapsulation of cargo under acidic conditions,
large counter-anions may stabilize the lipid phase at this pH
range.
[0345] Further preferred are the following fusogenic amphoter II
systems comprising charged amphiphiles only and having both a
stable lamellar phase at pH 7 to pH 8 and at pH 2 to pH 4 and
fusing between pH 3 and pH 6, preferably between pH 4 and pH 6
(Table 26):
TABLE-US-00030 TABLE 26 C/A Cation Anion (molar ratio) DmC4Mo2 DOGS
.gtoreq.0.7 .sup. HisChol DOGS 0.7 Chim DMGS .gtoreq.0.5-2.sup.
DmC4Mo2 Chol-C3 .gtoreq.1.5 .sup. DmC4Mo2 Chems .gtoreq.1.sup. Chim
Chems .gtoreq.0.5-1.5 Chim DOGS >0-1 C3Mo2 DOGS
.gtoreq.0.5-2.sup. DmC4Mo2 DMGS .gtoreq.1.sup. DmC4Mo2 Chol-C5
.gtoreq.1-2 DmC3Mo2 DOGS >0-1.5 MoC3Chol DOGS 0.7 MoChol DOGS
.gtoreq.0.5-1.sup. DmC4Mo2 Chol-C6 .gtoreq.0.5-2.sup. C3Mo2 Chems
.gtoreq.0.7-1.sup. C8Mo2 DOGS >0-1 HisChol Chems >0-0.7 C5Mo2
DOGS >0-1 C6Mo2 Chems .gtoreq.0.7-1.sup. C5Mo2 Chems 0.7 C6Mo2
DOGS >0-1 MoChol Chems .gtoreq.0.5-0.7 C3Mo3 Chems
.gtoreq.0.5-0.7 DmC3Mo2 Chems .gtoreq.0.5-1.sup. MoChol Chol-C3 0.7
C8Mo2 Chems .gtoreq.0.5-0.7 C3Mo3 DOGS >0-0.7 C4Mo4 DOGS
>0-0.7 MoChol Chol-C5 .gtoreq.0.5-0.7 MoChol DMGS
.gtoreq.0.5-0.7 C4Mo4 Chems 0.5 DOIM DOGS >0-0.7 MoChol Chol-C6
>0-0.5
[0346] Versatile amphoter II systems of Table 26 are fusogenic over
a wide range of C/A ratios, preferably over a range of C/A ratios
of .gtoreq.0.7, more preferably over a range C/A ratios of
.gtoreq.1.
[0347] Furthermore, preferred are the amphoteric mixtures of Table
26 that are fusogenic at higher C/A ratios, preferably at
C/A.gtoreq.0.4, and more preferably at C/A ratios.gtoreq.0.5.
Amphoter III Systems of Solely Charged Amphiphiles
[0348] Amphoter III systems are characterized by a stable anion and
a pH-sensitive cation. Thus amphoter III systems cannot form lipid
salts at neutral pH, since little to no charged cationic lipid
exists at this pH. Ongoing acidification is needed first to create
the cation which then may undergo salt formation.
[0349] Amphoter III systems that are stable at pH 7 to pH 8 and
fuse between pH 3 to pH 6, preferably between pH 4 to pH 6 may
include mixtures of one or more cationic amphiphiles selected from
MoChol, HisChol, Chim, MoC3Chol, DmC4Mo2, DmC3Mo2, C3Mo2, C5Mo2,
C6Mo2, C8Mo2, C4Mo4, DOIM or DPIM with one or more anionic
amphiphile selected from DOPA, DMPA, DPPA, POPA, DSPA, Chol-SO4,
DOPG, DMPG, DPPG, POPG, DSPG or DOPS, DMPS, DPPS, POPS and
DSPS.
[0350] Less preferred are amphoter III systems which do not show
fusion owing to steric hindrance. One example that has been
described above is the amphoter III system MoChol/POPG.
[0351] Preferred amphoter III systems comprising charged
amphiphiles only that are stable at pH 7 to pH 8 and fuse between
pH 3 and pH 6, preferably between pH 4 and pH 6 include the
following specific mixtures (Table 27):
TABLE-US-00031 TABLE 27 C/A Cation Anion (molar ratio) MoChol DOPA
>1-1.5 HisChol DOPA >1-2 Chim DOPS >1-2
[0352] Among these amphoter III mixtures further preferred are the
following mixtures having stable lamellar phase at pH 7 to pH 8 as
well as stable phase at an acidic pH of between pH 2 and pH 4
(Table 28):
TABLE-US-00032 TABLE 28 C/A Cation Anion (molar ratio) MoChol DOPA
>1-1.5 HisChol DOPA 2 Chim DOPS >1-2
Section IV: Amphoteric Systems Further Comprising Neutral or
Zwitterionic Lipids
[0353] Neutral lipids comprise structures such as
phosphatidylcholine, phosphatidyl ethanolamine, sphingolipids or
cholesterol and the like. As these lipids do not have pH responsive
elements that would react between pH 3 and 8, no changes in the
molecular geometry occur in this range. Depending on the individual
.kappa. values of the neutral lipids, dilution of the bistable
behaviour of the amphoteric lipid pair occurs and the steepness of
d(.kappa.)/d(pH) becomes smaller, as shown in FIG. 9. In addition,
the curve in the phase diagram is shifted towards lower or higher
values of .kappa., depending on the neutral lipid used for dilution
of the charged lipids. The parameters used for the calculation of
FIG. 9 are given in Table 29 below; volumes in .ANG..sup.3.
TABLE-US-00033 TABLE 29 Anion head volume 70 Anion tail volume 400
Anion pK 5 Cation head volume 70 Cation tail volume 400 Cation pK
6.5 Counterion+ volume 70 Counterion- volume 70
[0354] FIG. 9 illustrates this behaviour for the addition of
different neutral lipids with .kappa. values of 0.5, 0.3 or 0.19,
respectively, in combination with the amphoter II model system
described above. The amplitude of the system is reduced from
.kappa.=0.089 to 0.044, while the minimum value follows the .kappa.
for the individual neutral components.
[0355] Thus, in some embodiments of the invention, 65 mol. % or
less neutral lipids may be added to the salt forming charged
lipids. More preferred are additions of 50 mol. % or less and even
more preferred are additions of 35 mol. % or less neutral lipid.
The addition of neutral lipids may stabilise further the lipid
bilayer, and preferred lipids for such purpose have higher .kappa.
values, e.g., .kappa.>0.4 or even about 0.5. Typical examples of
such lipids are the phosphatidylcholines with C14 to C18 alkyl
chains in the hydrophobic region. As with most polar regions of
lipids, the head-groups of phosphatidylcholines recruit
counterions.
[0356] The addition of neutral lipids may also extend the zone of
fusogenic behaviour and to this end neutral lipids with low values
of .kappa. may be employed. Such preferred lipids have .kappa.
values of 0.3 or less; more preferred lipids have .kappa. values of
about 0.2. Typical examples of such lipids are
phosphatidylethanolamines. Phosphatidylethanolamines are assumed to
form internal salt bridges (betaine structures) between the
terminal amino group and the phosphate; therefore no counterions
are recruited to the head-groups.
[0357] Phosphatidylethanolamines with C14 to C18 alkyl chains are
preferred lipids to modulated the fusogenicity of the amphoteric
liposomes.
[0358] Cholesterol is another example of a lipid having low .kappa.
and might therefore extend the fusogenic behaviour of an amphoteric
lipid system.
[0359] It is of course possible to use mixtures of different
neutral lipids to optimize the balance between fusogenicity and
stability of such systems.
[0360] In practical terms, the presence of neutral lipids in the
membrane of amphoteric liposomes has an effect on the fusogenicity
of the liposomes and may, as predicted by the presented algorithm
of the present invention, improve or impair the fusion of the
liposomes. It is apparent from the algorithm, that the nature of
such effect is largely dictated by the relation between k(salt) of
the amphoteric system and k(neutral), the membrane constant of the
neutral lipid or a mixture of neutral lipids. If, for example
k(salt), is higher than k(neutral), then the addition of such
neutral lipids may stimulate fusion or expand the width of the
fusion zone. Of course, k(total) has to reach a certain minimum for
this. In some embodiments, such minimum is smaller than 0.34 or
0.35, more preferred smaller than 0.3 and even more preferred such
minimum is smaller than 0.25.
[0361] Experimental evidence is given in Example 15 and FIG. 25,
where different neutral lipids in different amounts were mixed into
the membrane of an amphoter II system (MoChol/DOGS). Furthermore,
the influence of neutral lipids on the fusogenicity of other
amphoteric systems was tested in Example 15.
[0362] Neutral lipids may also have impact on other characteristics
of amphoteric liposomes, such as colloidal stability or stability
in body fluids. For example, the use of amphoteric liposomes in
pharmaceutical applications requires stability of the liposomes
during storage and travelling through the bloodstream.
[0363] It becomes apparent that the presence of
phosphatidylcholines as single neutral lipid in the membrane of
amphoteric lipid mixtures lowers the fusability of amphoteric lipid
mixtures. Phosphatidylcholines are zwitterionic lipids having high
k values of >0.4.
[0364] In one embodiment of the invention less than 40 mol %,
preferably less than 30 mol % and more preferably less than 20 mol
% of neutral or zwitterionic lipids with k>0.4 as single neutral
component are present in the amphoteric lipid mix. Such neutral or
zwitterionic lipids include, but are not limited to,
phosphatidylcholines, sphingomyelins or ceramides.
[0365] Cholesterol as neutral lipid has either no effect on the
fusogenicity of amphoteric lipid systems or may even lead to an
improvement in fusability. Similar behaviour was observed for the
lipid DOPE. Cholesterol and phosphatidylethanolamines are neutral
or zwitterionic lipids that have k values below 0.3 and adopt
hexagonal phases.
[0366] In a further embodiment of the invention, cholesterol or
phosphatidylethanolamines or a mix of both lipids may be present in
the amphoteric lipid mix as single neutral or zwitterionic lipids.
Preferably not more than 65 mol %, more preferably not more than 50
mol %, of these lipids is used as single neutral or zwitterionic
lipids in the amphoteric liposomes. In other embodiments of the
present invention, where cholesterol is essentially the only
neutral lipid, cholesterol may comprise more than 80 mol % of the
total neutral lipids of the amphoteric liposomes described herein.
In another embodiment, cholesterol is the only neutral lipid in the
amphoteric liposomes.
[0367] For optimising the balance between fusogenicity and
stability it may be advantageous to use a mix of neutral or
zwitterionic lipids as neutral component in the amphoteric
liposomes.
[0368] In a still further embodiment of the invention a mixture of
neutral lipids, such as phosphatidylcholines (PC), sphingomyelins
or ceramides and phosphatidylethanolamines (PE) or a mixture of
phosphatidylcholines (PC), sphingomyelins or ceramides and
cholesterol (Chol) may be used as neutral components in the
amphoteric liposomes. Preferably the ratio of PC/PE or PC/Chol is
between 4 and 0.25, more preferably between 3 and 0.33. These
neutral lipid mixes may be added to the salt-forming charged lipids
in the amount 80 mol % or less, preferably 65 mol % or less. Most
preferred are additions of 50 mol % or less.
[0369] It has also been found that neutral lipids may extend
fusability to further C/A ratios as compared to mixture solely of
charged amphiphiles. For example, the addition of 40 mol %
POPC/DOPE=0.33 to a HisChol/DOGS mixture leads to an extension of
the range of fusogenicity from C/A=>0-0.7 to C/A=>0-1.
Similarly, the addition of 40 mol % cholesterol expands the C/A
ratio of DOTAP/Chems for fusion to occur from C/A=>0-0.4 to
C/A=>0-0.67.
[0370] In summary, the algorithm disclosed herein is suitable to
describe phase transitions in amphoteric liposomes. Essential
elements of the algorithm are (i) the lipid shape theory, (ii) the
notion that counterions form part of the head-group volume and
(iii) that lipid salt formation may occur in bilayers, leading to
dissociation of the counterions. The simplicity of the algorithm
makes it easy for the person skilled in the art to reproduce the
calculations and adopt the system to the individual goals. It is
possible to use other tools well-known to those skilled in the art
than indicated to calculate molecular volumes. The qualitative
prediction would not even change if molecular cross-sections were
used instead of the volumes. Of course, one would have to
re-calibrate the results in such a case.
[0371] As mentioned above, steric hindrance may interfere with salt
formation in isolated cases. In such cases, the phase behaviour may
differ substantially from what has been hereinbefore described.
Most importantly, no dual stability at low and neutral pH is
observed. Instead, a saddle of stability is observed for an
intermediate pH with zones of instability to one or either side,
depending on the type of amphoter system. Typical examples are
included into the examples below.
[0372] The molecular volume calculations disclosed herein are
silent on chain saturation in the hydrophobic parts. Use of
unsaturated lipids may have specific advantages, since lipid
membranes comprising such lipids have higher fluidity at ambient
temperature which may improve fusion behaviour. It is also known
that unsaturated lipids exert lateral pressure in the membrane,
thus a correction factor can be inserted to reflect the apparent
volume of these components. Such correction factor is higher than
1.
[0373] The algorithm of the present invention assumes the formation
of lipid salts with a 1:1 stoichiometry between the two charged
partners. It is possible to extend this concept to more complex
situations, e.g., binding of multiple, monocharged lipids to a
single other lipid with a plurality of charged groups. Binding of
CHEMS, DOGS or oleic acid to amphiphilic derivatives of spermin may
provide an example of this, but many other combinations exist. In
another embodiment, the charged groups on the lipids might be more
complex and comprise different charged groups, e.g., as in
HistChol.
[0374] In such a case, 1:1 complexes might be formed with either
other lipid anions or cations. Applying the concept of this
invention to this example, counterion displacement can occur
between the imidazolium cation and a separate lipid anion, e.g.,
CHEMS, DOGS or oleic acid anions; in addition, parallel counterion
dissociation from the carboxyl of the histidine moiety may further
support the formation of a hexagonal phase.
[0375] It will be apparent to those skilled in the art that such
more complex arrangements of lipids put an extra burden on the
steric compatibility of the interaction partners which may lead to
lack of experimental success or mixed forms of possible
interactions where not every possible binding site is engaged in
salt formation.
[0376] The methods disclosed herein substantially reduce the number
of variables involved with the optimisation of the system.
[0377] The present invention has been exemplified with various
calculations in the detailed description. Further experimental work
is described in the following examples. Examples are given with the
understanding of further detailing certain aspects of practising
the current invention. The examples by no means limit the scope of
this disclosure.
EXAMPLES
Example 1
Preparation of Liposomes and pH-Dependent Fusion Experiment
[0378] Buffer System 100 mM sodium citrate and 200 mM sodium
hydrogen phosphate were prepared as stock solutions and variable
amounts of both solutions were mixed to adjust for the pH needed.
CiP 7.0 as an example specifies a buffer from that series having a
pH of 7.0 and is made from citrate and phosphate.
Liposome Production
[0379] Liposomes were formed from a dried lipid film. In brief, 20
.mu.mol of the respective lipid composition was dissolved in 1 mL
chloroform/methanol 3:1 and dried in vacuum using a rotary
evaporator. The resulting film was hydrated for 45 min in 1 mL of
CiP 8.0 with gentle agitation. The resulting liposome suspension
was frozen, sonicated after thawing and eventually extruded through
200 nm polycarbonate filters.
pH-Jump Experiment
[0380] 10 .mu.l liposomes in CiP 8.0 were placed into a glass tube
and mixed rapidly with 1 mL of CiP buffer of the pH needed. Samples
were allowed to stand for 1 h at room temperature and 3 mL of 200
mM sodium hydrogen phosphate were rapidly mixed with the sample.
Liposomes were analyzed for size using a MALVERN Zetasizer 3000HS
and sizes were recorded as Z-average.
Example 2
Fusion of Amphoter I Lipid Mixtures
[0381] Liposomes were prepared from DOTAP and CHEMS in sodium
citrate/sodium phosphate pH 8.0 (CiP 8.0) and small amounts were
injected into a CiP buffer with a lower pH (see Example 1 for
details). Any larger structures observed at the lower pH might be
either due to aggregate formations and generation of multicentric
honeycomb structures or such structures might result from genuine
fusion. To separate between these two outcomes we readjusted the pH
to neutrality using 200 mM sodium hydrogen phosphate. Electrostatic
repulsion dissociates multicentric vesicles but not fusion
products. The results are illustrated in FIG. 10.
[0382] As predicted in the mathematical salt bridge model, a valley
of instability exists at slightly acidic conditions and fusion to
larger particles was observed starting from pH 6.5. However, fast
addition of the liposomes into low pH resulted in stabilisation of
the particles as long as some DOTAP was present in the mixture.
Liposomes from 100 mol. % CHEMS enter a fusogenic state below pH
4.5 and do not get stabilised at the lower pH.
[0383] Noteworthy, a 1:1 mixture of DOTAP/CHEMS cannot form
liposomes in CiP 8.0 which is in good agreement with the
mathematical model that predicts a non-lamellar phase for these
parameters.
Example 3
Fusion of Amphoter II Systems
[0384] Liposomes were prepared from MoChol and CHEMS in sodium
citrate/sodium phosphate pH 8.0 (CiP 8.0) and small amounts were
injected into a CiP buffer with a lower pH (see Example 1 for
details). Any larger structures observed at the lower pH might be
either due to aggregate formations and generation of multicentric
honeycomb structures or such structures might result from genuine
fusion. To separate between these two outcomes we readjusted the pH
to neutrality using 200 mM sodium hydrogen phosphate. Electrostatic
repulsion dissociates multicentric vesicles but not fusion
products.
[0385] Experimental evidence supports the salt bridge model. (See
FIG. 11). The fusion zone is inclined towards high anion content
due to the large head-group size of MoCHol Consequently, no fusion
occurs with 33 mol. % or 50 mol. % CHEMS in the mixture, whereas
mixtures containing 66 mol. % or 75 mol. % CHEMS undergo fusion
when exposed to a pH between 4 and 6. As predicted, the onset of
fusion is shifted to lower pH values with higher amounts of CHEMS.
Again, 100 mol. % CHEMS is fusogenic with low pH but has no stable
state at low pH.
[0386] The parameters used for the calculation are given in Table
30 below; CHEMS and MoChol in Na/H.sub.2PO.sub.4 were used as model
compounds; all volumes in .ANG..sup.3.
TABLE-US-00034 TABLE 30 Anion head volume 76 Anion tail volume 334
Anion pK 5.8 Cation head volume 166 Cation tail volume 371 Cation
pK 6.5 Counterion+ volume 65 Counterion- volume 49
Example 4
Fusion in Amphoter III Systems with Steric Hindrance
[0387] Liposomes were prepared from POPG and MoChol in sodium
citrate/sodium phosphate pH 8.0 (CiP 8.0) and small amounts were
injected into a CiP buffer with a lower pH (see Example 1 for
details). Any larger structures observed at the lower pH might be
either due to aggregate formations and generation of multicentric
honeycomb structures or such structures might result from genuine
fusion. To separate between these two outcomes we readjusted the pH
to neutrality using 200 mM sodium hydrogen phosphate. Electrostatic
repulsion dissociates multicentric vesicles but not fusion
products.
[0388] Experimental evidence supports only a situation where no
salt bridge formation occurs. A mixture between MoChol and POPG
does not undergo fusion in the pH-jump experiment (data not shown).
This is quite possibly due to steric hindrance, as the protonated
nitrogen in Mo-Chol is situated at the lower end of the morpholino
ring and is therefore not easily accessible. In addition, the
phosphate in POPG sits right at the lipid/water interface and is
protected with a glycerol towards the water phase.
Example 5
Fusion in Amphoter III Systems, No Steric Hindrance
[0389] Amphoter III systems from POPG/MoChol do not undergo fusion
(see Example 4 above). It was therefore questioned whether the
removal of the protecting glycerol and exchange of POPG with DOPA
would avoid such steric hindrance. In fact, such system undergoes
fusion, as illustrated in the FIG. 12.
[0390] Details as per Example 1 above. The parameters used for the
calculation are given in Table 31 below; DOPA and MoChol in
Na/H.sub.2PO.sub.4 were used as model compounds, and volumes are
expressed as .ANG..sup.3.
TABLE-US-00035 TABLE 31 Anion head volume 63 Anion tail volume 501
Anion pK 3 Cation head volume 166 Cation tail volume 371 Cation pK
6.5 Counterion+ volume 65 Counterion- volume 49
[0391] The model calculation reflects the full complexity of the
experimental fusion behaviour: no fusion for mixtures with less
than 50% MoCHol, strong fusion for MoChol=DOPA and ongoing fusion
with no stable phase under acidic conditions for mixtures with
excess DOPA.
Example 6
Lipid Salt Formation with Monoalkyl Lipids
[0392] Oleic acid was chosen as a known and popular pH-sensitive
membrane component. As the lipid tail is relatively small in
volume, any change in the head-group has more pronounced
consequences for the membrane stability. As shown in FIG. 13,
modelling predicts oleic acid to be a strong driver for fusion in
an amphoter II system with MoChol. This is confirmed
experimentally. Mixtures of oleic acid do form liposomes with
Mo-Chol and particles rapidly undergo fusion when exposed to
different conditions. As expected from the algorithm, the extent of
fusion is limited for smaller amounts of OA in the mixture, but 50
mol. % of the anion results in the classic valley type fusion
pattern. Since the fusion tendency is much stronger with OA, a
bigger portion of that anion in the mix results in extensive fusion
over a wide range of pH values. Still, mixtures can always be
stabilised at low pH. Details as per Example 1.
TABLE-US-00036 TABLE 32 MoChol head-group volume 166 MoChol tail
volume 371 MoChol pK 6.5 Oleic acid head volume 42 Oleic acid tail
volume 208 Oleic acid pK 4.5 Counterion citrate volume 121
Counterion sodium volume 65
Example 7
Influence of Neutral Lipids
[0393] DOTAP and CHEMS were chosen as an amphoter I charged lipid
pair and POPC (.kappa. .about.0.5) was added as a neutral lipid. As
expected, the pure mixture of the charged components undergoes
aggregation or fusion at and below the isoelectric point. However,
as shown in FIG. 14, the addition of only 20 mol. % POPC
ameliorated such aggregation tendency to a great extent for all
cation:anion ratios ranging from pure anion through to the 1:1
mixture that is no longer amphoteric.
[0394] As shown in FIG. 15, the addition of the same amount of DOPE
(.kappa. .about.0.19) to the amphoter I mixture from DOTAP and
CHEMS maintains the fusion behaviour independent of the ratio
between DOTAP and CHEMS.
Example 8
Large Countercations can Reduce Fusion in Amphoter I Systems
[0395] In order to investigate the impact of different cations on
the fusion behaviour of an amphoter I system, lipid films were
prepared from 20 mol % DOTAP and 80 mol % CHEMS. A set of buffers
was created starting from 20 mM citric acid and 40 mM phosphoric
acid which were neutralized using KOH, NaOH, LiOH,
Tris-(hydroxymethyl)aminomethane (TRIS, free base) or L-Arginine
(free base).
[0396] The lipid films were hydrated at pH 8.0 and small amounts
were injected into corresponding buffers with a lower pH (see
Example 1 for details). After one hour incubation, the pH was
readjusted to neutrality using the corresponding bases. The results
are illustrated in FIG. 16.
[0397] As predicted in the model, fusion of liposomes from 20 mol %
DOTAP and 80 mol % CHEMS can be observed around pH 4 to 5 as long
as rather small countercations like potassium or sodium are used.
Larger countercations like L-arginine or TRIS effectively stabilize
the formulation and reduce or completely suppress fusion of the
liposomes.
Example 9
Large Countercations can Reduce Fusion in Amphoter II Systems
[0398] In order to investigate the impact of different cations on
the fusion behaviour of amphoter II systems, lipid films were
prepared from 20 mol % MoCHOL and 80 mol % CHEMS. A set of buffers
was created starting from 20 mM citric acid and 40 mM phosphoric
acid which were neutralized using KOH, NaOH, LiOH,
Tris-(hydroxymethyl)aminomethan (TRIS, free base) or L-Arginine
(free base).
[0399] The lipid films were hydrated at pH 8.0 and small amounts
were injected into corresponding buffers with a lower pH (see
Example 1 for details). After one hour incubation, the pH was
readjusted to neutrality using the corresponding bases. The results
are illustrated in FIG. 17.
[0400] As predicted in the model, fusion of liposomes from 20 mol %
MoChol and 80 mol % CHEMS can be observed between pH 3.5 to 4.5 as
long as rather small countercations like potassium or sodium are
used. Larger countercations like L-arginine or TRIS effectively
stabilize the formulation and reduce or completely suppress fusion
of the liposomes.
Example 10
Countercations have Little or No Effect on Fusion in Amphoter III
Systems
[0401] In order to investigate the impact of different cations on
the fusion behaviour of amphoter III systems, lipid films were
prepared from 50 mol % MoCHOL and 50 mol % DOPA. A set of buffers
was created starting from 20 mM citric acid and 40 mM phosphoric
acid which were neutralized using KOH, NaOH, LiOH,
Tris-(hydroxymethyl)aminomethan (TRIS, free base) or L-Arginine
(free base).
[0402] The lipid films were hydrated at pH 8.0 and small amounts
were injected into corresponding buffers with a lower pH (see
Example 1 for details). After one hour incubation, the pH was
readjusted to neutrality using the corresponding bases. The results
are illustrated in FIG. 18.
[0403] As predicted in the model, fusion of liposomes from 50 mol %
MoChol and 50 mol % DOPA can be observed between pH 3.5 to 5 and
there is little impact of the various cations on an amphoter III
system. This is expected from the model, since MoCHOL and DOPA form
a lipid salt at low pH conditions, thereby excluding countercations
from the membrane. Consequently, once excluded from the lipid
membrane, the countercations cannot contribute to membrane
stability or instability.
Example 11
Fusion Assay Based on Fluorescence Resonance Energy Transfer
(FRET)
[0404] To investigate the fusability of different amphoteric lipid
mixtures a lipid mixing assay, based on FRET was used. Liposomes,
single labelled with 0.6 mol % NBD-PE
(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-ph-
osphoethanol-amine, triethylammonium salt) or Rhodamine-PE
(Lissamine.TM. rhodamine B
1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,
triethylammonium salt), respectively, were prepared to monitor
lipid fusion through the appearance of a FRET signal.
[0405] Lipids were dissolved in isopropanol (final lipid
concentration 16 mM) and mixed. Liposomes were produced by adding
buffer (acetic acid 10 mM, phosphoric acid 10 mM, NaOH, pH 7.5) to
the alcoholic lipid mix, resulting in a final lipid concentration
of 1.95 mM and a final isopropanol concentration of 12.2%. For the
preparation of the liposomes a liquid handling robot (Perkin Elmer,
Multiprobe II Ex) was used. The NBD-labelled and Rh-labelled
amphoteric liposomes were combined in a ratio 1:1 and subsequent
diluted 1:1 with the buffer mentioned above. Finally small aliquots
of this mixed sample were brought to decreasing specific pH (HAc 50
mM, Phosphoric acid 50 mM, NaOH, pH 7.5-2.5) and incubated at
37.degree. C. for 2 h. Liposomes were diluted again 1:1 in this
step.
[0406] Samples were measured for fluorescence using two sets of
filters: NBD/Rhodamine: 460/590 nm and NBD/NBD: 460/530 nm. FRET as
a signal for membrane fusion was expressed as the ratio of emission
(590 nm)/emission (530 nm). A background of 0.4 indicates
background fluorescence and was therefore subtracted from the FRET
signals.
[0407] To discriminate between fusion and mere aggregation the
suspension was neutralized to pH 7.5 and FRET signals were measured
again. A possible interference of the remaining alcohol content of
3% on the fusion of the liposomes was excluded by
pre-experiments.
Example 12
Fusion Assay of Amphoter I Lipid Mixtures Comprising Solely Charged
Amphiphiles
[0408] Fusion assays were performed as described in Example 8.
Lipid pairs as indicated in Table 33 were tested for fusion in
cation/anion molar ratios (C/A ratio) of 0.17, 0.33, 0.40, 0.50,
0.67, 0.75 and pure anionic liposomes were prepared as
controls.
[0409] Table 33 shows the lipid pairs tested in the experiment. For
each lipid pair, the ranges of C/A ratios for which stability at pH
7 to pH 8 and fusion between pH 4 to pH 6 was observed are included
in the table. In addition, for each lipid pair, the ranges of C/A
ratios having a stable lamellar phase both at pH 7 to pH 8 and at
pH 2 to pH 4 and fusion between pH 4 to pH 6 are shown.
[0410] The fusion can be expressed as .SIGMA. FRET, the sum of all
measured FRET signals of the complete matrix C/A=0.17-0.75 vs.
pH.
TABLE-US-00037 TABLE 33 Fusion Zone Fusion Zone and stablility at
and stability at pH 8 to pH 7 and pH 8 to pH 7 at pH 2 to pH 4 C/A
C/A Cation Anion .SIGMA. FRET (molar ratio) (molar ratio) DOTAP
CHEMS 17 >0-0.4 .gtoreq.0.33-0.4 DOTAP Chol C3 20
.gtoreq.0.5-0.67 .sup. .gtoreq.0.5-0.67 DOTAP Chol C5 17 >0-0.4
.gtoreq.0.33-0.4 DOTAP Chol C6 12 >0-0.17 not stable at pH 2 to
pH 4 DOTAP DMGS 33 >0-0.5 .gtoreq.0.4-0.5 DOTAP DOGS 17
>0-0.4 >0-0.4 DOTAP OA ND >0-0.33 0.17 DOTAP SA ND not
stable at -- pH 7.5-6.5 DC-Chol Chems 31 >0-<1
.gtoreq.0.4-<1 DC-Chol DMGS 32 >0-<1 .gtoreq.0.5-<1
DC-Chol DOGS 35 >0-<1 .gtoreq.0.33-<1 TC-Chol DMGS 28
>0-0.67 .sup. .gtoreq.0.4-0.67 DDAB Chems 6 >0-0.17 0.17 DDAB
DMGS 35 >0-0.5 0.5 DODAP DMGS 42 >0-<1 .gtoreq.0.5-<1
CTAB DMGS 29 >0-0.5 0.5 DOEPC DMGS 37 >0-0.5 .gtoreq.0.33-0.5
Stearylamine DOGS ND Not stable at -- pH 7.5-6.5 N-methyl- DMGS 38
>0-<1 .gtoreq.0.5-<1 PipChol
[0411] The following Tables 34 a-d show the fusion profiles for
tour selected amphoter I systems as matrix C/A vs. pH. In addition
the fusion of liposomes of pure anionic lipid is shown (C/A=0). For
example, it is known that liposomes of 100% Chems fuse at a pH of
about 4.2 (Hafez et al, Biophys. J., 79, (2000), 1438-1446). This
is confirmed by the present experiment. In addition the fusion
profiles show the amphoter I mixtures having a stable lamellar
phase both at pH 7 to pH 8 and at pH 2 to pH 4 and fuse between pH
4 to pH 6.
TABLE-US-00038 TABLES 34 a-d DC-Chol/Chems .fwdarw. fusion over a
broad range of C/A ratios pH .fwdarw. C/A 7.50 6.50 5.50 4.50 3.50
2.50 0.00 0.01 0.01 0.05 0.47 1.54 1.63 0.17 0.00 0.00 0.14 2.61
3.65 1.23 0.33 0.00 0.00 0.91 2.87 1.06 0.75 0.40 0.00 0.00 1.80
2.89 0.88 0.62 0.50 0.00 0.00 2.27 2.26 0.58 0.36 0.67 0.00 0.00
2.07 0.63 0.24 0.14 0.75 0.00 0.00 1.84 0.18 0.07 0.03
DOTAP/Chol-C6 .fwdarw. fusion over a narrow range of C/A ratios pH
.fwdarw. C/A 7.5 6.5 5.5 4.5 3.5 2.5 0 0.00 0.00 0.08 0.85 2.27
2.98 0.17 0.00 0.00 1.64 2.61 1.02 0.63 0.33 0.00 0.00 0.94 0.81
0.25 0.21 0.4 0.00 0.00 0.55 0.38 0.16 0.12 0.5 0.00 0.15 0.37 0.04
0.00 0.01 0.67 0.00 0.34 0.20 0.00 0.00 0.00 0.75 0.00 0.47 0.01
0.00 0.00 0.00 DOTAP/DOGS .fwdarw. fusion range at C/A ratios >
0-0.4 pH .fwdarw. C/A 7.5 6.5 5.5 4.5 3.5 2.5 0 0.05 0.08 0.32 0.81
1.63 1.44 0.17 0.00 0.00 0.70 4.08 0.80 0.17 0.33 0.00 0.03 1.38
1.98 0.20 0.06 0.4 0.00 0.05 1.35 1.28 0.14 0.04 0.5 0.08 0.21 0.73
0.27 0.00 0.00 0.67 0.02 0.05 0.04 0.00 0.00 0.00 0.75 0.09 0.25
0.28 0.01 0.00 0.00 DODAP/DMGS .fwdarw. fusion over a broad range
of C/A ratios pH .fwdarw. C/A 7.50 6.50 5.50 4.50 3.50 2.50 0 0.27
0.62 1.75 1.71 2.45 3.60 0.17 0.04 0.15 2.35 4.28 4.16 2.36 0.33
0.01 0.03 1.39 4.17 2.12 0.72 0.4 0.00 0.00 1.37 3.77 1.10 0.45 0.5
0.00 0.00 1.42 2.67 0.52 0.26 0.67 0.00 0.00 1.26 1.19 0.29 0.17
0.75 0.00 0.00 1.27 0.89 0.23 0.14
Example 13
Fusion Assay of Amphoter II Lipid Mixtures Comprising Solely
Charged Amphiphiles
[0412] A fusion assay was performed as described in Example 8.
Lipid pairs as indicated in Table 35 were tested for fusion in
cation/anion molar ratios (C/A ratio) of 0.33, 0.5, 0.67, 1, 1.5,
2, 3 and pure anionic liposomes were prepared as controls.
[0413] Table 35 shows the lipid pairs tested in the experiment. For
each lipid pair the ranges of C/A ratios for which stability at pH
7 to pH 8 and fusion between pH 4 to pH 6 was observed are included
in the table. In addition, for each lipid pair the ranges of C/A
ratios having a stable lamellar phase both at pH 7 to pH 8 and at
pH 2 to pH 4 and fuse between pH 4 to pH 6 are shown.
[0414] The fusion can be expressed as .SIGMA. FRET, the sum of all
measured FRET signals in the matrix C/A=0.33-3 vs. pH.
TABLE-US-00039 TABLE 35 Fusion Zone Fusion Zone and stablility at
and stability at pH 8 to pH 7 and pH 8 to pH 7 at pH 2 to pH 4 C/A
C/A Cation Anion .SIGMA. FRET (molar ratio) (molar ratio) MoChol
Chems 19 >0-0.7 .gtoreq.0.5-0.7 MoChol DOGS 25 >0-1.sup.
.gtoreq.0.5-1.sup. MoChol DMGS 15 >0-0.7 .gtoreq.0.5-0.7 MoChol
Chol-C3 17 >0-0.7 0.7 MoChol Chol-C5 15 >0-0.7
.gtoreq.0.5-0.7 MoChol Chol-C6 11 >0-0.5 >0-0.5 MoChol Oleic
ND not stable at -- acid pH 7.5-6.5 DmC3Mo2 Chems 17 >0-1.sup.
.gtoreq.0.5-1.sup. DmC3Mo2 DOGS 26 >0-1.5 >0-1.5 C4Mo4 Chems
14 >0-0.5 0.5 C4Mo4 DOGS 15 >0-0.7 >0-0.7 DmC4Mo2 Chems 33
>0 .gtoreq.1 DmC4Mo2 DMGS 28 >0 .gtoreq.1 DmC4Mo2 DOGS 45
>0 .gtoreq.0.7.sup. DmC4Mo2 Chol-C3 36 .gtoreq.0.7
.gtoreq.1.5.sup. DmC4Mo2 Chol-C5 27 >0-2.sup. .gtoreq.1-2
DmC4Mo2 Chol-C6 23 >0-2.sup. .gtoreq.0.5-2.sup. C3Mo3 Chems 18
>0-0.7 .gtoreq.0.5-0.7 C3Mo3 DOGS 16 >0-0.7 >0-0.7 C3Mo2
Chems 23 >0-1.sup. .gtoreq.0.7-1.sup. C3Mo2 DOGS 30 >0-2.sup.
.gtoreq.0.5-2.sup. C5Mo2 Chems 20 >0-0.7 0.7 C5Mo2 DOGS 22
>0-1.sup. >0-1 C6Mo2 Chems 21 >0-1.sup. .gtoreq.0.7-1.sup.
C6Mo2 DOGS 20 >0-1.sup. >0-1 C8Mo2 Chems 16 >0-0.7
.gtoreq.0.5-0.7 C8Mo2 DOGS 23 >0-1.sup. >0-1 Chim Chems 30
>0-1.5 .gtoreq.0.5-1.5 Chim DMGS 36 >0-2.sup.
.gtoreq.0.5-2.sup. Chim DOGS 30 >0-1.sup. >0-1 Chim Oleic ND
not stable at -- acid pH 7.5-6.5 MoC3Chol DOGS 26 >0-0.7 0.7
DOIM DOGS 14 >0-0.7 >0-0.7 HisChol Chems 22 >0-0.7
>0-0.7 HisChol DOGS 37 >0-0.7 0.7 HisChol OA ND not stable at
pH 7.5-6.5
[0415] The following Tables 36 a-d show exemplary the matrix of the
fusion profile for four selected amphoter II systems. In addition
the fusion of liposomes of pure anionic lipid is shown (C/A=0). The
fusion profiles indicate the amphoter II mixtures having a stable
lamellar phase both at pH 7 to pH 8 and at pH 2 to pH 4 and fuse
between pH 4 to pH 6.
TABLE-US-00040 TABLES 36 a-d DmC4Mo2/Chems .fwdarw. fusion over a
broad range of C/A ratios pH .fwdarw. C/A 7.5 6.5 5.5 4.5 3.5 2.5
0.00 0.00 0.00 0.00 0.32 1.46 1.28 0.33 0.00 0.00 0.11 3.31 3.49
1.08 0.50 0.00 0.00 0.39 3.02 1.74 0.85 0.66 0.00 0.00 0.78 2.59
1.28 0.66 1.00 0.00 0.00 2.37 2.32 0.78 0.43 1.50 0.00 0.00 2.03
1.19 0.27 0.16 2.00 0.00 0.00 1.62 0.35 0.15 0.08 3.00 0.00 0.00
1.42 0.13 0.03 0.00 MoChol/Chol-C6 .fwdarw. fusion over a narrow
range of C/A ratios pH .fwdarw. C/A 7.5 6.5 5.5 4.5 3.5 2.5 0.00
0.00 0.00 0.02 0.81 2.95 3.54 0.33 0.00 0.00 2.62 2.62 0.98 0.61
0.50 0.00 0.00 2.45 0.42 0.17 0.10 0.66 0.00 0.00 0.99 0.06 0.03
0.04 1.00 0.00 0.00 0.09 0.00 0.00 0.00 1.50 0.00 0.00 0.00 0.00
0.00 0.00 2.00 0.00 0.00 0.00 0.00 0.00 0.00 3.00 0.00 0.00 0.00
0.00 0.00 0.00 Chim/DMGS .fwdarw. fusion range at C/A ratios >
0-2 pH .fwdarw. C/A 7.5 6.5 5.5 4.5 3.5 2.5 0.00 0.33 0.55 2.00
2.16 2.03 1.99 0.33 0.05 0.10 1.50 4.53 2.20 0.83 0.50 0.00 0.04
4.19 4.13 0.67 0.38 0.66 0.00 0.03 3.52 2.25 0.42 0.30 1.00 0.00
0.02 3.15 0.45 0.25 0.21 1.50 0.00 0.29 2.12 0.18 0.13 0.09 2.00
0.00 0.80 1.28 0.07 0.05 0.04 3.00 0.01 1.68 0.32 0.00 0.00 0.00
C3Mo2/DOGS .fwdarw. fusion range at C/A ratios > 0-2 pH .fwdarw.
R C:A 7.5 6.5 5.5 4.5 3.5 2.5 0.00 0.00 0.01 0.14 0.46 1.36 1.66
0.33 0.00 0.02 1.89 3.58 1.37 0.85 0.50 0.00 0.01 2.71 2.68 0.93
0.66 0.66 0.00 0.04 2.87 1.99 0.95 0.80 1.00 0.00 0.06 2.25 0.65
0.52 0.49 1.50 0.00 0.12 1.48 0.38 0.35 0.30 2.00 0.00 0.16 1.03
0.21 0.16 0.14 3.00 0.00 0.20 0.21 0.00 0.00 0.01
Example 14
Fusion Assay of Amphoter III Lipid Mixtures Comprising Solely
Charged Amphiphiles
[0416] Fusion assays were performed as described in Example 8.
Lipid pairs as indicated in Table 37 were tested for fusion in
cation/anion molar ratios (C/A ratio) of 1.5, 2, 3.
[0417] Table 37 shows the lipid pairs tested in the experiment. For
each lipid pair the ranges of C/A ratios for which stability at pH
7 to pH 8 and fusion between pH 4 to pH 6 was observed are included
in the table. In addition for each lipid pair the ranges of C/A
ratios having a stable lamellar phase both at pH 7 to pH 8 and at
pH 2 to pH 4 and fuse between pH 4 to pH 6 are shown.
TABLE-US-00041 TABLE 37 Fusion Zone Fusion Zone and stablility at
and stability at pH 8 to pH 7 and at pH 8 to pH 7 pH 2 to pH 4 C/A
C/A Cation Anion (molar ratio) (molar ratio) MoChol DOPA >1-1.5
>1-1.5 HisChol DOPA >1-2 2 MoChol POPG no fusion no fusion
MoChol Chol-SO4 no fusion no fusion
[0418] The following Tables 37 a-d show by way of example the
matrix of the fusion profile for the four tested amphoter III
systems. In addition the fusion of liposomes of pure anionic lipid
is shown (C/A=0). The fusion profiles indicate the amphoter III
mixtures having a stable lamellar phase both at pH 7 to pH 8 and at
pH 2 to pH 4 and fuse between pH 4 to pH 6. As described in Example
4 a mixture between MoChol and POPG does not undergo fusion owing
to steric hindrance. This can be observed in the present FRET
experiment as well. The same might be the case for the lipid pair
MoChol/CholSO4.
TABLE-US-00042 TABLES 37 a-d pH .fwdarw. C/A 7.5 6.5 5.5 4.5 3.5
2.5 MoChol/DOPA 1.50 0.00 0.10 1.41 0.40 0.24 0.22 2.00 0.00 0.04
0.34 0.02 0.02 0.03 3.00 0.00 0.00 0.00 0.00 0.00 0.00 HisChol/DOPA
1.50 0.00 0.23 2.85 2.43 1.05 0.68 2.00 0.01 0.56 1.31 0.61 0.23
0.12 3.00 0.12 0.74 0.77 0.02 0.01 0.00 MoChol/POPG 1.50 0.13 0.16
0.31 0.34 0.50 0.46 2.00 0.00 0.03 0.12 0.10 0.13 0.11 3.00 0.00
0.00 0.00 0.00 0.00 0.01 MoChol/Chol-SO4 1.50 0.00 0.00 0.04 0.02
0.02 0.01 2.00 0.00 0.00 0.00 0.00 0.00 0.00 3.00 0.00 0.00 0.00
0.00 0.00 0.00
Example 15
Impact of Neutral or Zwitterionic Lipids on the Fusion of
Amphoteric Lipid Mixtures
[0419] Amphoteric liposomes with increasing amounts of neutral or
zwitterionic lipids were prepared as described in Example 8.
[0420] Initially, an amphoter I system (DOTAP/DMGS) and an amphoter
II (MoChol/DOGS) were prepared with the addition of 10-50%
different neutral or zwitterionic lipids or mixtures thereof.
Fusion was measured for a series of liposomes having different C/A
ratios. Systems can be characterized using the sum of all such
measurements in the entire matrix. The effect of the neutral or
zwitterionic lipids was then analyzed using this global parameter
(.SIGMA. FRET).
[0421] FIG. 25 shows the influence of different neutral or
zwitterionic lipids on the fusogenicity of the amphoteric lipid
mixture MoChol/DOGS. It is apparent that neutral lipids having a
high .kappa., such as POPC or DOPC, decrease the fusogenicity of
the amphoteric liposomes, whereas the lipids having a lower
.kappa., such as DOPE or cholesterol, have little impact on the
fusogenicity or may even improve the fusion. Mixtures of POPC and
DOPE and mixtures of POPC or DOPC and cholesterol may have little
impact or decrease the fusion ability, depending of the ratio of
the two lipids. The presence of DOPE and cholesterol in the
membrane of the amphoteric liposomes does not change the
fusogenicity or even leads to an increase of it. These findings
correlate very well with the model as shown in FIGS. 21-24 for the
neutral lipids POPC, DOPE, Cholesterol and mixtures of POPC/Chol=1.
In the figures FRET of liposomes from DOTAP/DMGS (C/A=0.17-0.75) or
MoCHol/DOGS (C/A 0.33-3) was plotted against k(min) for mixtures
with 0%-50% neutral lipid. The reference K(min) was modelled for
C/A=0.66 (DOTAP/DMGS) or C/A=1 (MoChol/DOGS).
[0422] In a further experiment the effect of different neutral or
zwitterionic lipid systems (POPC, cholesterol or POPC/DOPE=0.33) on
the fusogenicity of other amphoteric lipid systems were determined.
Tables 38 and 39 summarize these data and confirm the results of
the first part of the experiment. Tables 38 and 39 show the .SIGMA.
Fret and range of C/A ratios for which the amphoteric liposomes are
stable at pH 7 to pH 8 and fuse between pH 3 to pH 6, preferably
between pH 4 to pH 6.
[0423] It becomes apparent that amphoteric lipid systems having low
fusogenicity can be clearly improved by the addition of neutral or
zwitterionic lipids. Furthermore the results indicate that neutral
or zwitterionic lipids may have also an impact on the range of
fusogenicity. This means that the range of C/A ratios can be
broadened or narrowed depending on the neutral or zwitterionic
lipid used in the mixtures.
TABLE-US-00043 TABLE 38 .SIGMA. Fret .SIGMA. Fret .SIGMA. Fret
.SIGMA. Fret .SIGMA. Fret .SIGMA. Fret .SIGMA. Fret 0% neutral 20%
40% 20% 40% 40% POPC/DOPE 40% POPC/DOPE Cation Anion K(salt) lipid
POPC POPC Chol Chol 0.33 0.33 C/A C/A C/A C/A C/A ratio ratio ratio
ratio ratio C/A ratio C/A ratio DODAP DMGS 0.157 42 31 21 40 42 30
29 DODAP DMGS >0-<1 >0-<1 >0- >0-<1
>0-<1 >0-0.67 >0-<1 N-methyl- DMGS 0.271 38 16 3 40
44 40 31 PipChol N-methyl- DMGS >0-<1 >0- -- >0-<1
>0-<1 >0-<1 >0-<1 PipChol 0.5 DDAB DMGS 0.182 35
7 4 16 21 27 30 DDAB DMGS >0-0.5 >0- -- >0- >0-
>0-0.67 >0-0.67 DOEPC DMGS 0.269 37 ND ND ND ND 26 23 DOEPC
DMGS >0-0.5 ND ND ND ND >0-0.67 >0-0.67 DC-Chol DOGS 0.225
35 20 10 25 24 28 39 DC-Chol DOGS >0-<1 >0- >0- >0-
>0-<1 >0-<1 >0-<1 DOTAP DMGS 0.169 33 28 24 31 32
30 33 DOTAP DMGS >0-0.5 >0- >0- >0- >0- >0-0.67
>0-0.67 DC-Chol DMGS 0.254 32 22 13 35 42 36 29 DC-Chol DMGS
>0-<1 >0- >0- >0-<1 >0-<1 >0-<1
>0-<1 DC-Chol Chems 0.265 31 8 1 33 33 25 19 DC-Chol Chems
>0-<1 >0- -- >0-<1 >0-<1 >0-<1
>0-0.67 CTAB DMGS 0.271 29 ND ND ND ND 24 16 CTAB DMGS >0-0.5
ND ND ND ND >0-0.5 >0-0.5 TC-Chol DMGS 0.271 28 ND ND ND ND
33 28 TC-Chol DMGS >0-0.67 ND ND ND ND >0-<1 >0-<1
DOTAP Chol- 0.156 20 ND ND ND ND 25 29 DOTAP Chol- .gtoreq.0.5-0.67
ND ND ND ND .gtoreq.0.33-<1 >0-<1 DOTAP Chol- 0.186 17 ND
ND ND ND 17 17 DOTAP Chol- >0-0.4 ND ND ND ND >0-0.4
>0-0.4 DOTAP Chems 0.171 17 11 3 21 25 19 25 DOTAP Chems
>0-0.4 >0- >0- >0- >0- >0-0.4 >0-0.67 DOTAP
DOGS 0.153 17 17 17 36 37 ND ND DOTAP DOGS >0-0.4 >0- >0-
>0- >0- ND ND DOTAP Chol- 0.202 12 ND ND ND ND 13 16 DOTAP
Chol- >0-0.17 ND ND ND ND >0-0.17 >0-0.4 DDAB Chems 0.186
6 7 0 33 52 10 9 DDAB Chems >0-0.17 >0- -- >0- >0-<1
>0-0.33 >0-0.4
TABLE-US-00044 TABLE 39 .SIGMA. Fret .SIGMA. Fret .SIGMA. Fret
.SIGMA. Fret .SIGMA. Fret .SIGMA. Fret .SIGMA. Fret 0% neutral 20%
40% 20% 40% 40% POPC/DOPE 40% POPC/DOPE Cation Anion K(salt) lipid
POPC POPC Chol Chol 0.33 0.33 C/A C/A C/A C/A C/A ratio ratio ratio
ratio ratio C/A ratio C/A ratio DmC4Mo2 DOGS 0.336 45 ND ND ND ND
35 36 DmC4Mo2 DOGS >0 ND ND ND ND >0 >0 Chim DOPS 0.284 39
ND ND ND ND 31 33 Chim DOPS .gtoreq.1-2 ND ND ND ND .gtoreq.1-1.5
.gtoreq.1-1.5 HisChol DOGS 0.282 37 19 9 33 50 34 29 HisChol DOGS
>0-0.7 >0- >0- >0- <0- >0-0.7 <0-1 Chim DMGS
0.278 36 12 5 37 42 26 23 Chim DMGS >0-2 >0- >0- >0-
>0-1 >0-1 >0-1 DmC4Mo2 Chol- 0.385 36 ND ND ND ND 21 18
DmC4Mo2 Chol- .gtoreq.0.7 ND ND ND ND .gtoreq.0.5 >0 DmC4Mo2
Chems 0.403 33 10 0 32 23 23 20 DmC4Mo2 Chems >0 >0- -- >0
.gtoreq.1 >0-2 >0-2 Chim Chems 0.292 30 ND 0 33 27 15 12 Chim
Chems >0-1.5 ND -- >0- >0- >0-1.0 >0-0.7 Chim DOGS
0.247 30 16 14 29 29 24 22 Chim DOGS >0-1 >0- >0- >0-1
>0- >0-1 >0-1 C3Mo2 DOGS 0.288 30 ND ND ND ND 28 24 C3Mo2
DOGS >0-2 ND ND ND ND >0-1.5 >0-1 DmC4Mo2 DMGS 0.378 28 22
9 33 41 36 38 DmC4Mo2 DMGS >0 >0-0.7 >0- >0 >0
>0-2 >0 DmC4Mo2 Chol- 0.423 27 ND ND ND ND 19 17 DmC4Mo2
Chol- >0-2 ND ND ND ND >0-2 >0-1.5 DmC3Mo2 DOGS 0.319 26
ND ND ND ND 20 19 DmC3Mo2 DOGS >0-1.5 ND ND ND ND >0-1
>0-1 MoC3Chol DOGS 0.269 26 15 9 30 34 21 19 MoC3Chol DOGS
>0-0.7 >0- >0- >0- >0- >0-0.7 >0-0.7 MoChol
DOGS 0.303 25 17 11 24 ND 21 20 MoChol DOGS >0-1 >0- >0-
>0-1 ND >0-1 >0-1 DmC4Mo2 Chol- 0.442 23 ND ND ND ND 17 14
DmC4Mo2 Chol- >0-2 ND ND ND ND >0-1.5 >0-1 C3Mo2 Chems
0.344 23 ND ND ND ND 13 9 C3MO2 Chems >0-1 ND ND ND ND >0-0.7
>0-0.7 C8Mo2 DOGS 0.365 23 ND ND ND ND 19 24 C8Mo2 DOGS >0-1
ND ND ND ND >0-1 >0-1 HisChol Chems 0.336 22 9 1 22 25 18 16
HisChol Chems >0-0.7 >0- -- >0-1 >0- >0-1 >0-0.7
C5Mo2 DOGS 0.318 22 ND ND ND ND 23 18 C5Mo2 DOGS >0-1 ND ND ND
ND >0-1 >0-1 MoChol DOPS 0.340 21 ND ND ND ND 22 21 MoChol
DOPS 1 ND ND ND ND 1 1 C6Mo2 Chems 0.401 21 ND ND ND ND 12 8 C6Mo2
Chems >0-1 ND ND ND ND >0-0.7 >0-0.7 C5Mo2 Chems 0.382 20
ND ND ND ND 11 8 C5Mo2 Chems >0-0.7 ND ND ND ND >0-0.7
>0-0.7 C6Mo2 DOGS 0.334 20 ND ND ND ND 19 19 C6Mo2 DOGS >0-1
ND ND ND ND >0-1 >0-1 MoChol Chems 0.363 19 3 0 20 24 13 10
MoChol Chems >0-0.7 >0- -- >0-1 >0-1 >0-0.7
>0-0.7 MoChol DMPS 0.383 19 ND ND ND ND 11 13 MoChol DMPS 1 ND
ND ND ND -- -- C3Mo3 Chems 0.363 18 ND ND ND ND 10 8 C3Mo3 Chems
>0-0.7 ND ND ND ND >0-0.7 >0-0.5 DmC3Mo2 Chems 0.382 17 ND
ND ND ND 10 9 DmC3Mo2 Chems >0-1 ND ND ND ND >0-0.7 >0-0.7
MoChol Chol- 0.345 17 ND ND ND ND 13 9 MoChol Chol- >0-0.7 ND ND
ND ND >0-0.7 >0-0.7 C8Mo2 Chems 0.440 16 ND ND ND ND 10 8
C8Mo2 Chems >0-0.7 ND ND ND ND >0-0.33 >0-0.7 C3Mo3 DOGS
0.304 16 ND ND ND ND 21 17 C3Mo3 DOGS >0-0.7 ND ND ND ND >0-1
>0-1 C4Mo4 DOGS 0.334 15 ND ND ND ND 14 13 C4Mo4 DOGS >0-0.7
ND ND ND ND >0-0.7 >0-0.7 MoChol Chol- 0.382 15 ND ND ND ND 7
6 MoChol Chol- >0-0.7 ND ND ND ND >0-0.7 >0-0.5 MoChol
DMGS 0.342 15 8 4 18 23 15 14 MoChol DMGS >0-0.7 >0- --
>0-1 >0-1 >0-0.7 >0-0.7 C4Mo4 Chems 0.401 14 ND ND ND
ND 6 4 C4Mo4 Chems >0-0.5 ND ND ND ND >0-0.5 >0-0.3 DOIM
DOGS 0.145 14 9 10 13 26 15 13 DOIM DOGS >0-0.7 >0- >0-
>0- >0- >0-0.7 >0-0.7 MoChol Chol- 0.401 11 ND ND ND ND
6 5 MoChol Chol- >0-0.5 ND ND ND ND >0-0.5 >0-0.5
Example 16
Synthesis of Cationic Amphiphiles
Synthesis of DmC4Mo2
[0424] A. Synthesis of 2,3-Dimethylsuccinic-anhydride
[0425] 2,3-Dimethyl succinic anhydride was prepared as described in
Sutton, et al. OPPI 24 (1992) 39. Briefly, 7.1 g 2,3-dimethyl
succinic acid and 6.9 ml acetanhydride were slowly heated up to
50.degree. C. for 3 hours. Then acetanhydride was removed by
distillation. The product was recrystallized from ethanol abs. and
characterized by the melting point.
[0426] B. Synthesis of
2,3-Dimethylsuccinicacid-monocholesterylester
[0427] 2,3-Dimethylsuccinic acid-monocholesterylester was prepared
according to the Chems synthesis in J. T. Kley et al., Monatshefte
Chem 129 (1998) 319. 2.9 g 2,3-dimethylsuccinic-anhydride, 6.3 ml
triethylamine, 0.06 g dimethylaminopyridine and 50 ml chloroform
were combined in a round-bottomed flask and refluxed. 7.8 g
cholesterol was added to the mixture in two steps during 45 min.
The mixture was refluxed for 6 days. Finally the solvent was
evaporated and 100 ml toluol and 1.8 g pyridine were added. The
mixture was refluxed again for 1.5 days. The solvent was removed at
a rotovap and the crude product was first purified by column
chromatography on silica gel (eluent: dichloromethane/methanol
96:4) followed by a recrystallization from ether and a second
column chromatography on silica gel (eluent: acetic acid ethyl
ester:petrol ether 1:1). Purity of the product was judged by thin
layer chromatography.
Synthesis of DmC4Mo2
[0428] 5.9 g 2,3-Dimethylsuccinic acid-monocholesterylester and 300
ml tetrahydrofurane were stirred under N.sub.2 atmosphere and
cooled down to -10.degree. C. 1.9 ml N-methylmorpholine was added
to the mixture. Then 1.6 ml isobutylchloroformiate was added
dropwise to the mixture. After on hour the mixture was brought to
0.degree. C. and about 2 hours later to room temperature. Then the
mixture was again cooled down to -10.degree. C., 1.5 ml
4-(2-Aminoethyl)morpholine was added dropwise and the reaction was
allowed to stir overnight at room temperature. The mixture was
filtered, then the solvent was evaporated and the product was
purified by column chromatography on silica gel (eluent:
dichloromethane/methanol 96:4) and recrystallization from hexane.
The product was characterized by thin layer chromatography,
.sup.1H-NMR and HPLC.
Synthesis of DmC3Mo2
[0429] A. Synthesis of 2,2-Dimethyl-malonic acid monoethyl
ester
[0430] 25 g 2,2-Dimethyl-malonic acid diethyl ester, 7.8 g
potassium hydroxide and 500 ml ethanol were mixed in a
round-bottomed flask and refluxed for 3 hours. Then again 2.2 g
potassium hydroxide were added and the mixture was refluxed
overnight. The solvent was removed on a rotovap, 250 ml H.sub.2O
were added and the mixture was washed with ether. The aqueous phase
was acidified with HCl to pH 3-4 followed by two extractions with
dichloromethane. The organic solvent was dried and evaporated and
the resulting product was characterized by .sup.1H-NMR.
[0431] B. Synthesis of
2,2-Dimethyl-N-(2-morpholin-4-yl-ethyl)-malonamic acid ethyl
ester
[0432] 19 g 2,2-Dimethyl-malonic acid monoethyl ester were weighed
into a round-bottomed flask and under N.sub.2 atmosphere and room
temperature, 200 ml tetrahydrofurane, 15.6 ml
4-(2-Aminoethyl)-morpholine and 32.6 ml N-Methylmorpholine were
added. The reaction was stirred and cooled down to 5.degree. C.
Then, 43.8 g TBTU were added and the mixture was allowed to stir
for another 1.5 hours. Finally the solvent was removed and the
residue dissolved in 400 ml dichloromethane. This organic phase was
washed two times with 500 ml NaHCO.sub.3 solution. The
dichloromethane phase was dried then the solvent was evaporated.
Purity of the product was judged by gas chromatography.
[0433] C. Synthesis of
2,2-Dimethyl-N-(2-morpholin-4-yl-ethyl)-malonamic acid (HCl
salt)
[0434] 34.1 g 2,2-Dimethyl-N-(2-morpholin-4-yl-ethyl)-malonamic
acid ethyl ester, 8.4 g potassium hydroxide, 200 ml ethanol and 4.5
ml H.sub.2O were stirred at 80.degree. C. for 6 hours. Then most of
the solvent was removed and the pH was adjusted to pH 3-4 with
about 100 ml 2N HCl. The solvent was evaporated and toluol was
added and then removed. To the final residue methanol was added and
the suspension was filtered to remove salts. The solvent was
removed and the residue was dissolved in H.sub.2O and lyophilized.
The product was characterized by .sup.1H-NMR.
[0435] C. Synthesis of DmC3Mo2
[0436] 10.7 g 2,2-Dimethyl-N-(2-morpholin-4-yl-ethyl)-malonamic
acid (HCl salt) and 50 ml toluol were combined in a round-bottomed
flask under N.sub.2 atmosphere. Then 13.9 ml thionyl chloride were
added and the solution was refluxed for 3 hours. The solvent was
evaporated and 150 ml chloroform were added to the residue. After
the addition of 14.7 g cholesterol and 0.023 g
4-Dimethylaminopyridine the mixture was stirred at room temperature
and 15 min later 10.7 ml triethylamine were added. The reaction was
allowed to stir at room temperature for 1.5 days. Then the solvent
was removed at a rotovap and the crude product was dissolved in 100
ml acetic acid ethyl ester and subsequent purified by column
chromatography on silica gel (eluent: acetic acid ethyl
ester/methanol 9:1) and recrystallization from ether. The final
product was characterized by .sup.1H-NMR and HPLC.
Synthesis of C3Mo3
[0437] A. Synthesis of Malonic Acid Monochloride
[0438] Malonic acid monochloride was synthesized as described in
Wilson, et al., J. Org. Chem 39 (1974) 3170. Briefly, 150 g malonic
acid and 600 ml ether were added under N.sub.2 atmosphere in a
round bottom flask. The mixture was stirred and 150.2 ml thionyl
chloride were added dropwise. The suspension was refluxed for 5
hours before the solvent was removed at a rotovap. The residue was
treated three times under sonication and 40.degree. C. with 500 ml
chloroform:hexane 1:2. The three extracts were combined and kept at
-15.degree. C. over night. The mother liquor was decanted and the
yellow crystals were washed with hexane and dried. The mother
liquor was concentrated and again kept at -15.degree. C. resulting
in further product.
[0439] B. Synthesis of Malonic Acid Mono Cholesteryl Ester
[0440] 26 g cholesterol and 12.4 g malonic acid monochloride were
weighed into a round bottom flask under N.sub.2 atmosphere. First
300 ml benzene were added and subsequent 11 ml pyridine dropwise.
The mixture was stirred at room temperature and after one hour 100
ml chloroform were added. 3 hours later the mixture was sonicated
for 40 min and subsequent again 100 ml chloroform were added and
the mixture was further stirred for 0.5 days at room temperature.
Then 250 ml H.sub.2O and 100 ml chloroform were added to the
mixture. The organic phase was dried and the solvent was removed at
a rotovap. The crude product was dissolved in 100 ml
dichloromethane/methanol 9:1 and subsequent purified by column
chromatography on silica gel (eluent: dichloromethane/methanol 9:1)
and recrystallization from ether and petrol ether. The final
product was characterized by 1H-NMR.
[0441] C. Synthesis of C3Mo3
[0442] 6 g Malonic acid mono cholesteryl ester was weighed into a
round bottom flask and under N.sub.2 atmosphere and at room
temperature, 200 ml tetrahydrofurane, 2.2 ml
4-(2-Aminopropyl)-morpholine and 2.8 ml N-Methylmorpholine were
added. The mixture was stirred and cooled down to 0.degree. C.
Then, 8.2 g TBTU were added stepwise and the reaction was allowed
to stir at room temperature for 1 day. The suspension was
pre-purified by filtering the mixture through a frit filled with
silica gel (eluent: acetic acid ethyl ester/methanol 1:1). Finally
the crude product was purified by column chromatography on silica
gel (eluent: chloroform/methanol 9:1) and the resulting product was
characterized by .sup.1H-NMR and LC-MS.
Synthesis of C3Mo2
[0443] 6 g Malonic acid mono cholesteryl ester was weighed into a
round bottom flask and under N.sub.2 atmosphere and at room
temperature, 200 ml Tetrahydrofuran, 2.0 ml
4-(2-Aminoethyl)-morpholine and 2.8 ml N-Methylmorpholine were
added. The mixture was stirred and cooled down to 0.degree. C.
Then, 8.2 g TBTU were added stepwise and the reaction was allowed
to stir at room temperature for 2 days. The suspension was
pre-purified by filtering the mixture through a frit filled with
silica gel (eluent: acetic acid ethyl ester/methanol 1:1). Finally
the crude product was purified by chromatography on silica gel (1.
chromatography.fwdarw.eluent: chloroform/methanol 9:1; 2.
chromatography.fwdarw.eluent: chloroform/0-3% methanol). The
resulting product was characterized by .sup.1H-NMR and LC-MS.
Synthesis of C4Mo4
[0444] A. Synthesis of 4-Morpholin-4-yl-butyronitrile
[0445] 196.9 ml morpholine, 500 ml toluol and 100 ml chloroform
were stirred in a round-bottomed flask under N.sub.2 atmosphere and
at 80.degree. C. After one hour 100 g 4-chlorobutyronitrile was
added dropwise within one hour. The reaction was stirred one day at
80.degree. C. and another day at room temperature. The mixture was
fritted and the residue was washed two times with ether. The
filtrate was concentrated and finally distilled under vacuum. The
product fractions were collected at 95-100.degree. C. and 1.2-0.89
Torr. The purity of the colourless oil was judged by gas
chromatography and .sup.1H-NMR.
[0446] B. Synthesis of 4-Morpholin-4-yl-butylamine
[0447] 14.8 g lithium aluminium hydride were weighed under N.sub.2
atmosphere into a round-bottomed flask. The substance was cooled
down to -55.degree. C. and 150 ml ether was added dropwise. The
suspension was allowed to stir; then 30 g
4-Morpholin-4-yl-butyronitrile were dissolved in 200 ml ether and
the solution was added dropwise to the reaction mixture. Again 200
ml ether were added to the mixture and the reaction was stirred at
room temperature overnight. At the next day the mixture was cooled
down to less than 10.degree. C. and 35 ml H.sub.2O were added
carefully. After five hours the mixture was fritted and the residue
was washed with 300 ml ether. The filtrate was dried, concentrated
and finally distilled under vacuum. The product fractions were
collected at 74-78.degree. C. and 2.1-1.6 Torr. The purity of the
colourless oil was judged by gas chromatography, GC-MS and
.sup.1H-NMR.
[0448] C. Synthesis of C4Mo4
[0449] 9.7 g cholesterol hemisuccinate were dissolved in 100 ml
tetrahydrofurane and stirred under N.sub.2 atmosphere and at
-15.degree. C. Then 3.3 ml N-Methylmorpholine were added dropwise
within 10 min followed by the slow addition of 2.9 ml isobutyl
chloroformate. Then 3.2 g 4-Morpholin-4-yl-butylamine were added
dropwise. The temperature was raised to room temperature and the
reaction was allowed to stir for 2.5 hours. The mixture was fritted
and the residue was washed with 20 ml tetrahydrofurane. The solvent
of the filtrate was evaporated and to the residue 100 ml boiling
acetic acid ethyl ester was added. After a further filtration the
mixture was kept at room temperature for 2.5 days. The solvent was
again removed and the crude product was purified by column
chromatography on silica gel (eluent: dichloromethane/methanol 96:4
and 83:17). The product was characterized by thin layer
chromatography and .sup.1H-NMR.
Synthesis of C5Mo2
[0450] A. Synthesis of Pentanedioic Acid Mono Cholesteryl Ester
[0451] 35 g cholesterol and 15.5 g glutaric anhydride were weighed
into a round-bottomed flask. Under N, atmosphere 500 ml chloroform,
25.4 ml triethylamine and 0.22 g 4-dimethylamino pyridine were
added. The reaction was refluxed for 5 days. Then 250 ml H.sub.2O
were added and the pH was adjusted under stirring to pH 4-5 with 2N
HCl. The organic phase was dried and finally evaporated. To the
residue again 31 g glutaric anhydride were added together with 250
ml toluol, 22.1 ml pyridine and 0.22 g 4-dimethylamino pyridine.
The mixture was allowed to reflux for 1 day. Then the solvent was
evaporated and the residue was dissolved in dichloromethane/acetic
acid ethylester (96:4) and purified by a frit on silica gel
(eluent: dichloromethane/methanol 94:4). After further purification
by column chromatography on silica gel (eluent: product was
characterized by 1H-NMR and thin layer chromatography.
[0452] B. Synthesis of C5Mo2
[0453] Under N2 atmosphere 6 g pentanedioic acid mono cholesteryl
ester were dissolved in 250 ml tetrahydrofurane. 2.8 ml
4-(2-Aminoethyl)morpholin and 2.6 ml N-Methylmorpholin were added
and the mixture was cooled down to 10.degree. C. Finally 7.7 g TBTU
were added stepwise and the reaction was allowed to stir at room
temperature for one day followed by an incubation at 4.degree. C.
for 3 days. Then the solvent was evaporated and the crude product
was purified by column chromatography on silica gel and
characterized by 1H-NMR.
Synthesis of C6Mo2
[0454] A. Synthesis of Oxepane-2,7-dione
[0455] 100 g adipinic acid and 100 ml acetanhydride were refluxed
for 5 hours. The solvent was removed at a rotovap and 100 ml
acetonitrile were added to the residue and the mixture was kept in
a freezer over night. Then the mixture was fritted and the
resulting residue was washed with 50 ml acetonitrile and dried.
[0456] B. Synthesis of Hexanedioic Acid Mono Cholesteryl Ester
[0457] 65 g cholesterol and 33 g oxepane-2,7-dione were weighed
into a round-bottomed flask. Under N.sub.2 atmosphere 300 ml
toluol, 21.2 ml pyridine and 0.21 g 4-dimethylamino pyridine were
added. The reaction was refluxed for 2 days. Then the solvent was
evaporated and the residue was dissolved in dichloromethane/acetic
acid ethylester (96:4) and purified by a frit on silica gel
(eluent: dichloromethane/acetic acid ethyl ester (96:4). The
product was characterized by 1H-NMR and thin layer
chromatography.
[0458] C. Synthesis of C6Mo2
[0459] Under N.sub.2 atmosphere 10 g hexanedioic acid mono
cholesteryl ester were dissolved in 250 ml tetrahydrofurane. 3.1 ml
4-(2-Aminoethyl)morpholin and 3.2 ml N-Methylmorpholin were added
and the mixture was cooled down to 10.degree. C. After the addition
of further 100 ml tetrahydrofurane 9.4 g TBTU were added stepwise
and the reaction was allowed to stir at room temperature overnight.
Then the solvent was evaporated and the crude product was purified
by column chromatography on silica gel (eluent: chloroform/0-5%
methanol) and characterized by .sup.1H-NMR, thin layer
chromatography and LC-MS.
Example 17
Amphoteric Liposomes Encapsulating siRNA
[0460] siRNA-loaded amphoteric liposomes were manufactured using
non-targeting scrambled siRNA. The lipid mixtures A
(DC-Chol:DMGS:Chol, 26:39:35 mol %) or B (DC-Chol:DMGS:Chol,
20:40:40 mol %) were dissolved at a concentration of 30 mM or 60 mM
(final lipid concentration) for both mixtures in ethanol.
Appropriate volumes of siRNA stock were diluted in 20 mM NaAc, 300
mM Sucrose/NaOH pH 4.0. The organic and the aqueous solution were
mixed in a 3:7 ratio and the liposomal suspension was immediately
shifted to pH>7.5 with 136 mM Na.sub.2HPO.sub.4, 100 mM
NaCl.
[0461] The amount of unencapsulated siRNA was determined by using
ultrafiltration with Centrisart (Molecular Weight Cut off 300 kD
(Sartorius, Gottingen, Germany)). The siRNA concentration of the
filtrate was measured spectroscopically (OD260 nm). The amount of
encapsulated oligonucleotide was determined by subtraction of
unencapsulated amount of siRNA from the total amount of siRNA.
[0462] Particle characteristics after manufacturing:
TABLE-US-00045 Size // Initial lipid Polydispersity Encapsulation
Formulation concentration index efficacy A 30 mM 256 nm // 0.319
63% A 60 mM 313 nm // 0.490 64% B 30 mM 184 nm // 0.055 69% B 60 mM
206 nm // 0.135 77%
[0463] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *
References