U.S. patent application number 11/590143 was filed with the patent office on 2007-11-22 for amphoteric liposomes.
Invention is credited to Frank Essler, Stefan Fankhanel, Cornelia Panzner, Steffen Panzner.
Application Number | 20070269504 11/590143 |
Document ID | / |
Family ID | 7675950 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070269504 |
Kind Code |
A1 |
Panzner; Steffen ; et
al. |
November 22, 2007 |
Amphoteric liposomes
Abstract
Amphoteric liposomes are proposed, which comprise positive and
negative membrane-based or membrane-forming charge carriers as well
as the use of these liposomes.
Inventors: |
Panzner; Steffen; (Halle,
DE) ; Fankhanel; Stefan; (Halle, DE) ; Essler;
Frank; (Halle, DE) ; Panzner; Cornelia;
(Halle, DE) |
Correspondence
Address: |
BROWN, RUDNICK, BERLACK & ISRAELS, LLP.
BOX IP, 18TH FLOOR
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
7675950 |
Appl. No.: |
11/590143 |
Filed: |
October 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10081617 |
Feb 21, 2002 |
|
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11590143 |
Oct 31, 2006 |
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Current U.S.
Class: |
424/450 ;
264/4.1; 435/375; 514/44A; 514/5.9; 536/23.1; 554/124 |
Current CPC
Class: |
A61K 31/7105 20130101;
Y10T 428/2984 20150115; A61K 9/1272 20130101; A61P 35/00 20180101;
A61K 31/711 20130101 |
Class at
Publication: |
424/450 ;
264/004.1; 435/375; 514/002; 514/044; 536/023.1; 554/124 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/7105 20060101 A61K031/7105; A61K 31/711
20060101 A61K031/711; C07H 21/02 20060101 C07H021/02; C12N 5/10
20060101 C12N005/10; C07H 21/04 20060101 C07H021/04; B01J 13/02
20060101 B01J013/02; A61K 38/02 20060101 A61K038/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 21, 2001 |
DE |
101 09 897.9 |
Claims
1. A method of loading nucleic acids in amphoteric liposomes having
an isoelectric point of between 4 and 8 comprising the steps of: a.
providing a mixture of at least one amphipatic cationic lipid, at
least one amphipatic anionic lipid and at least one neutral lipid
and nucleic acids and at least one solvent at a pH of between 3-6;
and b. forming liposomes of said lipids and nucleic acids c.
changing the pH of said mixture to pH 7-9.
2. The method of claim 1, wherein said amphoteric liposomes have an
isoelectric point of between 5 and 7.
3. A method of loading nucleic acids in amphoteric liposomes having
an isoelectric point of between 4 and 8 comprising the steps of: a.
providing a mixture of at least one amphipatic lipid with both a
positive and a negative charge, at least one neutral lipid, and
nucleic acids and at least one solvent, wherein said mixture has a
pH between about 3-6; b. forming liposomes in said mixture; and c.
changing the pH of said liposomes to pH 7-9
4. The method of claim 3, wherein said amphoteric liposomes have an
isoelectric point of between 5 and 7.
5. The method of claim 3 and 4, wherein said amphoteric liposomes
further comprise at least one amphipatic lipid with a positive
charge or at least one amphipatic lipid with a negative charge.
6. The method of claim 1 and 3, wherein said method further
comprises the step: d. removing non-encapsulated nucleic acids from
said mixture.
7. The method of claim 2 and 4, wherein said neutral lipid is
selected from the group consisting of phosphatidyl choline,
phosphatidyl ethanolamine, cholesterol, tetraether lipid, ceramide,
sphingolipid, and diacyl glycerol.
8. The method of claim 2, wherein said anionic lipid is a weak
anion, said cationic lipid is a strong cation, and said anionic
lipid is present in excess over said cation lipid.
9. The method of claim 8, wherein said anionic lipid is selected
from the group consisting of cholesterol hemisuccinate (CHEMS),
diacyl glycerol hemisuccinate, fatty acids and
phosphatidylserine
10. The method of claim 8, wherein cationic lipid is selected from
the group consisting of DOTAP, DC-Chol, DORIE, DDAB, TC-Chol,
DOTMA, DOGS, (C18)2Gly+ N,N-dioctadecylamido-glycin, CTAB, CPyC and
DOEPC.
11. The method of claim 2, wherein the anionic lipid is CHEMS or
diacylglycerol hemisuccinate, the cationic lipid is DOTAP or
DC-Chol and the neutral lipid is phosphatidylcholine.
12. The method of claim 2, wherein said amphoteric liposomes
comprise about 30-60 mol. % POPC, about 10-30 mol. % DOTAP and
about 25-40 mol. % CHEMS
13. The method of claim 2, wherein said anionic lipid is a weak
anion and said cationic lipid is a weak cation.
14. The method of claim 13, wherein said anionic lipid is selected
from the group consisting of cholesteryl hemisuccinate (CHEMS),
diacyl glycerol hemisuccinate, fatty acids and phosphatidyl
serine.
15. The method of claim 13, wherein said weakly cationic lipid is
selected from the group consisting of HisChol and MoChol.
16. The method of claim 2, wherein said anionic lipid is CHEMS or
diacylglycerol hemisuccinate, said cationic lipid is HisCHol or
MoChol and said neutral lipid is phosphatidylcholine.
17. The method of claim 2, wherein said amphoteric liposomes
comprise about 55-60 mol. % POPC, about 20-40 mol. % HisChol and
about 5-20 mol. % CHEMS.
18. The method of claim 2, wherein said anionic lipid is a strong
anion and said cationic lipid is a weak cation and said cation
lipid is present in excess over said anionic lipid.
19. The method of claim 18, wherein said anionic lipid is selected
from the group consisting of cholesterol sulphate, cholesterol
phosphate, phosphatidyl glycerol, phosphatidic acid, phosphatidyl
inositol, and cetyl phosphate.
20. The method of claim 18, wherein said cationic lipid is selected
from the group consisting of HisChol and MoChol.
21. The method of claim 2, wherein said anionic lipid is
phosphatidylglycerol, phosphatidic acid or cetyl phosphate and said
cationic lipid is HisChol or MoChol and said neutral lipid is
phosphatidylcholine.
22. The method of claim 2, wherein the amphoteric liposomes
comprise about 47.5 mol. % POPC, about 40 mol. % HisChol and about
12.5 mol. % DPPG.
23. The method of claim 2 and 4, wherein said liposomes have an
average size between 50 and 1000 nm.
24. The method of claim 2 and 4, wherein said liposomes have an
average size between 70 and 250 nm.
25. The method of claim 2 and 4, wherein said liposomes have an
average size between 60 and 130 nm.
26. The method of claim 2 and 4, wherein said nucleic acids are
selected from the group consisting of DNA, RNA, antisense
nucleotides and decoy nucleotides.
27. The method of claim 6, wherein, following step d., at least 80%
of said nucleic acids are encapsulated within said liposomes.
28. The method of claim 1 and 3, wherein said forming step b.
comprises a method selected from the group consisting of the
injection of an ethanolic lipid solution into an aqueous nucleic
acid solution, drying an organic solution of said lipids and
hydrating the dry lipid films with aqueous nucleic acid solution,
or detergent dialysis of said lipids and nucleic acids.
29. The method of claim 1 and 3, wherein said liposomes are sized
using a method selected from the group consisting of high pressure
homogenization and extrusion.
30. A method of loading active ingredients in amphoteric liposomes
having an isoelectric point of between 4 and 8, wherein said
liposomes are permeabilized and closed at a defined pH.
31. A method of claim 30, wherein the active ingredients comprise
proteins, peptides, DNA, RNA, antisense nucleotide and/or decoy
nucleotide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/081,617 filed Feb. 21, 2002, which claims benefit of German
Application 101 09 897.9, filed Feb. 21, 2001.
[0002] This invention relates to amphoteric liposomes, which
simultaneously comprise positive and negative membrane-based or
membrane-forming charge carriers as well as to the use of these
liposomes.
[0003] The concept of lipids comprises three classes of natural
products, which can be isolated from biological membranes:
phospholipids, sphingolipids and cholesterol with its derivatives.
However, it also comprises synthetically produced materials with
similar characteristics. As representatives of these, diacyl
glycerols, dialkyl glycerols, 3-amino-1,2-dihydroxypropane esters
or ethers and also N,N-dialkylamines are mentioned.
[0004] These substances are of technical interest for the
preparation of liposomes. On of the uses of these liposomes is as a
container for active ingredients in pharmaceutical preparations.
For this purpose, an efficient and stable packaging of the cargo,
compatibility with body fluids and a controllable and optionally
site-specific release of the content are desirable.
[0005] It is a disadvantage that it is difficult to combine the two
requirements. The tighter and more stable the packaging, the more
difficult it is to release the enclosed active ingredient once
again. For this reason, liposomes were developed, which change
their properties in reaction to external stimuli. Heat-sensitive
and pH-sensitive liposomes are known. The pH-sensitive liposomes
are of special interest, since this parameter may change under
physiological circumstances, such as during the endocytotic
absorption of a liposome in cells or during passage through the
gastrointestinal tract. According to the state of the art,
pH-sensitive liposomes comprise, in particular, cholesterol
hemisuccinate (CHEMS).
[0006] Cholesterol hemisuccinate is used in admixture with
phosphatidyl ethanolamine for the preparation of pH-sensitive
liposomes (Tachibana et al. (1998); BBRC 251: 538-544, U.S. patent
4,891,208). Such liposomes can be endocytized by many cells and in
this way are able to transport cargo molecules into the interior of
cells, without injuring the integrity of the cellular membrane.
[0007] The anionic character of CHEMS is a significant
disadvantage. The liposomes, prepared with it, have an overall
negative charge and absorbed by cells only with a low efficiency.
Therefore, in spite of the transfer mechanism described above, they
are hardly suitable for transporting macromolecules into cells.
[0008] Cationic liposomes with the highest possible and constant
surface charge are used to transport active ingredients into cells
(transfection). The overall positive charge of such particles leads
to an electrostatic adhesion to cells and, consequently, to an
efficient transport into cells. The use of these compounds and of
the liposomes, produced therewith is, however, limited to in vitro
or ex vitro uses, since such positively charged liposomes form
uncontrolled aggregates with serum components.
[0009] The limitation to very few pK values, generally to that of
the carboxyl group in the cholesterol hemisuccinate (approximately
4.5) is a disadvantage of the pH-sensitive liposomes, which are
available according to the state of the art. A further disadvantage
of the compounds is the limitation to negative charge carriers.
These are not suitable for binding nucleic acids and, frequently
also, proteins efficiently.
[0010] Cationic liposomes show good bonding of nucleic acids and
proteins and are in a position to bring these active ingredients
into cells. It is a disadvantage that they cannot be used for in
vivo applications.
[0011] It was therefore an objective to produce the liposomal
structures, which [0012] i) permit an efficient inclusion of active
in agents, [0013] ii) can transport these active ingredients into
biological cells, [0014] iii) are compatible with use under in vivo
conditions and [0015] iv) can be produced simply and
inexpensively.
[0016] The inventive object is accomplished by amphoteric
liposomes, which comprise at least one positive and at least one
negative charge carrier, which differs from the positive one, the
isoelectric point of the liposomes being between 4 and 8. This
objective is accomplished owing to the fact that liposomes are
prepared with a pH-dependent, changing charge.
[0017] Liposomal structures with the desired properties are formed,
for example, when the amount of membrane-forming or membrane-based
cationic charge carriers exceeds that of the anionic charge
carriers at a low pH and the ratio is reversed at a higher pH. This
is always the case when the ionizable components have a pKa value
between 4 and 9. As the, pH of the medium drops, all cationic
charge carriers are charged more and all anionic charge carriers
lose their charge.
[0018] The following abbreviations are used in connection with the
invention: [0019] CHEMS cholesterol hemisuccinate [0020] PC
phosphatidyl choline [0021] PE phosphatidyl ethanolamine [0022] PS
phosphatidyl serine [0023] PG phosphatidyl glycerol [0024]
Hist-Chol histidinyl cholesterol hemisuccinate
[0025] The membrane-forming or membrane-based charge carriers have
the following general structure of an amphiphile:
[0026] charge group-membrane anchor
[0027] The naturally known systems or their technically modified
forms come into consideration as membrane anchors. These include,
in particular, the diacyl glycerols, diacyl phosphoglycerols
(phospholipids) and sterols, but also the dialkyl glycerols, the
dialkyl- or diacyl-1-amino-2,3-dihydroxypropanes, long-chain alkyls
or acyls with 8 to 25 carbon atoms, sphingolipids, ceramides, etc.
These membrane anchors are known in the art. The charge groups,
which combine with the these anchors, can be divided into the
following 6 groups: [0028] Strongly cationic, pKa>9, net
positive charge: on the basis of their chemical nature, these are,
for example, ammonium, amidinium, guanidium or pyridinium groups or
timely, secondary or tertiary amino functions. [0029] Weakly
cationic, pKa<9, net positive charge: on the basis of their
chemical nature, these are, in particular, nitrogen bases such as
piperazines, imidazoles and morpholines, purines or pyrimidines.
Such molecular fragments, which occur in biological systems,
preferably are, for example, 4-imidazoles (histamine), 2-, 6-, or
9-purines (adenines, guanines, adenosines or guanosines), 1-, 2-or
4-pyrimidines (uraciles, thymines, cytosines, uridines, thymidines,
cytidines) or also pyridine-3-carboxylic acids (nicotinic esters or
amides). [0030] Nitrogen bases with preferred pKa values are also
formed by substituting nitrogen atoms one or more times with low
molecular weight alkane hydroxyls, such as hydroxymethyl or
hydroxyethyl groups. For example, aminodihydroxypropanes,
triethanolamines, tris-(hydroxymethyl)methylamines,
bis-(hydroxymethyl)methylamines, tris-(hydroxyethyl)methylamines,
bis-(hydroxyethyl)methylamines or the corresponding substituted
ethylamines. [0031] Neutral or zwitterionic, at a pH from 4 to 9:
on the basis of their chemical nature, these are neutral groups,
such as hydroxyls, amides, thiols or zwitterinonic groups of a
strongly cationic and a strongly anionic group, such as
phosphocholine or aminocarboxylic acids, aminosulfonic acids,
betaines or other structures. [0032] Weakly anionic, pKa>4, net
negative charge: on the basis of their chemical nature, these are,
in particular, the carboxylic acids. These include the aliphatic,
linear or branched mono-, di- or tricarboxylic acids with up to 12
carbon atoms and 0, 1 or 2 ethylenically unsaturated bonds.
Carboxylic acids of suitable behavior are also found as substitutes
of aromatic systems. [0033] Other anionic groups are hydroxyls or
thiols, which can dissociate and occur in ascorbic acid,
N-substituted alloxane, N-substituted barbituric acid, veronal,
phenol or as a thiol group. [0034] Strongly cationic, pKa<4, net
negative charge: on the basis of their chemical nature, these are
functional groups such as sulfonate or phosphate esters. [0035]
Amphoteric charge carriers, pI between 4.5 and 8.5, net positive
charge below the p1, net negative charge above the pI: on the basis
of their chemical nature, these charge carriers are composed of two
or more fragments of the groups named above. For carrying out the
invention, it is, initially, immaterial whether the charged groups
are on one and the same membrane anchor or if these groups are on
different anchors. Amphoteric charge carriers with a pI between 5
and 7 are particularly preferred for implementing the invention.
[0036] Strongly cationic compounds are, for example: [0037] DC-Chol
3-.beta.-[N-(N',N'-dimethylethane) carbamoyl]cholesterol, [0038]
TC-Chol 3-.beta.-[N-(N',N',N'-trimethylaminoethane) carbamoyl
cholesterol [0039] BGSC bisguanidinium-spermidine-cholesterol
[0040] BGTC bis-guadinium-tren-cholesterol, [0041] DOTAP
(1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride [0042]
DOSPER (1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide [0043]
DOTMA (1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium chloride)
(Lipofectin.RTM.) [0044] DORIE
(1,2-dioleoyloxypropyl)-3-dimethylhydroxyethylammonium bromide
[0045] DOSC (1,2-dioleoyl-3-succinyl-sn-glyceryl choline ester)
[0046] DOGSDSO (1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl
disulfide ormithine) [0047] DDAB dimethyldioctadecylammonium
bromide [0048] DOGS ((C18).sub.2GlySper3.sup.+)
N,N-dioctadecylarnido-glycol-spermin (Transfectam.RTM.)
(C18).sub.2Gly.sup.+ N,N-dioctadecylamido-glycine [0049] CTAB
cetyltrimethylammonium bromide [0050] CpyC cetylpyridinium chloride
[0051] DOEPC 1,2-dioleoly-sn-glycero-3-ethylphosphocholine or other
O-alkyl-phosphatidylcholine or ethanolamines, [0052] amides from
lysine, arginine or omithine and phosphatidyl ethanolaamine.
[0053] Examples of weakly anionic compounds are:
His-Chol-histaminyl-cholesterol hemisuccinate, Mo-Chol
morpholine-N-ethylamino-cholesterol hemisuccinate or
histidinyl-PE.
[0054] Examples of neutral compounds are: cholesterol, ceramides,
phosphatidyl cholines, phosphatidyl ethanolamines, tetraether
lipids or diacyl glycerols.
[0055] Examples of weakly anionic compounds are: CHEMS cholesterol
hemisuccinate, alkyl carboxylic acids with 8 to 25 carbon atoms or
diacyl glycerol hemisuccinate. Additional weakly anionic compounds
are the amides of aspartic acid, or glutamic acid and PE as well as
PS and its amides with glycine, alanine, glutamine, asparagine,
serine, cysteine, threonine, tyrosine, glutamic acid, aspartic acid
or other amino acids or aminodicarboxylic acids. According to the
same principle, the esters of hydroxycarboxylic acids or
hydroxydicarboxylic acids and PS are also weakly anionic
compounds.
[0056] Strongly anionic compounds are, for example: SDS sodium
dodecyl sulfate, cholesterol sulfate, cholesterol phosphate,
cholesteryl phosphocholine, phosphatidyl glycerols, phosphatid
acids, phosphatidyl inositols, diacyl glycerol phosphates, diacyl
glycerol sulfates, cetyl phosphate or lyosophospholipids.
[0057] Amphoteric compounds are, for example, [0058] Hist-Chol
N.alpha.-histidinyl-cholesterol hemisuccinate, [0059] EDTA-Chol
cholesterol ester of ethylenediaamine tetraacetic acid [0060]
Hist-PS N.alpha.-histidinyl-phosphatidylserine or
N-alkylcarnosine.
[0061] The inventive liposomes contain variable amounts of such
membrane-forming or membrane-based amphiphilic materials, so that
they have an amphoteric character. This means that the liposomes
can change the sign of the charge completely. The amount of charge
carrier of a liposome, present at a given pH of the medium, can be
calculated using the following formula:
z=.SIGMA.ni((qi-1)+10.sup.(pK-pH)/(1+10.sup.(pK-pH)) in which
[0062] qi is the absolute charge of the individual ionic groups
below their pK (for example, carboxyl=0, simple nitrogen base=1,
phosphate group of the second dissociation step=-1, etc.) [0063] ni
is the number of these groups in the liposome.
[0064] At the isoelectric point, the net charge of the liposome is
0. Structures with a largely selectable isoelectric point can be
produced by mixing anionic and cationic portions.
[0065] The structures can also be constructed so that, in
particular, as the pH drops, the charge on the molecule as a whole
is actually changed from negative to positive. Such a reversal of
charge is advantageous particularly when the liposomes, produced
with these structures, are to be used in physiological
interrelationships. Only liposomes with an overall negative charge
are compatible with blood and serum components. A positive charge
leads to aggregations. Liposomes with a positive charge are,
however, very good fusogenically and can transport active
ingredients into cells. A pH-dependent reversal of charge therefore
permits compounds to be constructed, which are compatible with
serum because they have a negative charge; however, after their
endocytotic absorption, their charge is reversed and they become
fusogenic only in the cell.
[0066] In a preferred embodiment of an embodiment of the invention,
the amphoteric liposomes have an isoelectric point between 5 and
7.
[0067] The invention also relates to amphoteric liposomes, which
comprise at least one amphoteric charge carrier, the amphoteric
charge carrier having an isoelectric point of between 4 and 8.
[0068] In a preferred variation, the amphoteric charge carrier of
the liposomes has an isoelectric point of between 5 and 7.
[0069] The invention also relates to amphoteric liposomes, the
liposomes comprising at least one amphoteric charge carrier and an
anionic arid/or cationic charge carrier.
[0070] It is appropriate that, in a preferred variation, the
amphoteric liposomes have an isoelectric point between 5 and 7.
[0071] In a special variation of the invention, the inventive
liposomes comprise phosphatidyl choline, phosphatidyl ethanolamine,
diacyl glycerol, cholesterol, tetraether lipid, ceramide,
sphigolipid, and/or diacyl glycerol. However, the preparation of
the liposomes can, of course, be carried out with many lipid
combinations of the inventive teachings. For examples, liposomes
can be synthesized using a large amount of CHEMS (about 40%) and a
smaller amount of DOTAP (about 30%). At the pK of the carboxyl
group of the CHEMS, the negative charge of this component is
already suppressed so far, that the positive charge carrier
predominates overall. An alternative formulation is the mixing of
CHEMS with HisChol the stronger charging of the positive charge
carrier HisChol going along synergistically with the discharging of
the negative CHEMS.
[0072] If Hist-Chol, which in itself is amphoteric, is incorporated
in a neutral membrane of, for example, phosphatidyl choline, an
amphoteric liposome with an isoelectric point, which largely
corresponds to that of Hist-Chol, also results.
[0073] It is known to those skilled in the art how the important
parameters can be adapted by manifold variations of the inventive
teachings: [0074] (i) the charge density of the liposomes at the
end points of the of the charge reversals by the amount and the pKa
values of the charge carriers used, [0075] (ii) the slope of the
charge reversal curve by the ratio of the two charge carriers, by
their absolute amounts and by an optimally synergistic effect of
two complementary pH-sensitive lipids and [0076] (iii) the passing
of the zeta potential through zero due to the ratio of the two
charge carriers or also due to the position of the pK value or
values.
[0077] In a further variation of the invention, the liposomes have
an average size of between 50 and 1000 nm, preferably of between 70
and 250 nm and particularly between 60 and 130 nm. The amphoteric
liposomes are synthesized by methods known in the art, such as the
injection of ethanol into a lipid solution in an aqueous buffer, by
hydrating dry lipid films or by detergent dialysis. The size of the
liposomes can vary, generally between 50 nm and 10,000 nm.
Homogeneous populations can be produced by high-pressure
homogenization or by extrusion.
[0078] In a preferred variation of the invention, the liposomes
comprise an active ingredient.
[0079] Advisably, in a preferred variation, the active ingredient
is a protein, a peptide, a DNA, an RNA, an antisense nucleotide
and/or a decoy nucleotide.
[0080] In a further preferred variation of the invention, at least
80% of the active ingredient in the interior of the liposome.
[0081] The invention also relates to a method for charging a
liposome with active ingredient, a defined pH being used for the
encapsulation and the pH being adjusted to a second value for
separating the unbound material.
[0082] The invention furthermore also relates to a method for
charging a liposome with active ingredient, the liposomes being
permeabilized and closed at a defined pH.
[0083] The invention also relates to the use of the liposomes for
the preparation of nanocapsules by depositing polymers or
polyelectrolytes on the lipid layer. Such substances can be
precipitated once or several times on the surface. With a repeated
deposition, which optionally can be carried out in the absence of
cross-linking agents, liposomal nanocapsules of the type described
in the WO 00/28972 or in the WO01/64330 are formed. It is
advantageous that the electrostatic interaction with the
polyelectrolyte can be interrupted when the substances described
here are used. It is known that the interaction of a
polyelectrolyte with charge carriers of the liposomal membrane can
lead to the de-mixing of membrane components and to the formation
of lipid clusters. In many cases, this de-mixing is associated with
a permeabilization of the liposome. The inventive substances enable
this interaction to be switched off after the coating process. The
liposomes are enclosed only sterically in the nanocapsules if the
pH is increased at this time and there no longer is any interaction
between the membrane and the polyelectrolyte. Cluster formation of
the lipids and the permeabilization of the membrane, associated
therewith, can thus be avoided.
[0084] The invention also relates to the use of the inventive
liposomes for packaging and releasing active ingredients. In this
variation, the liposomes bring about, in particular, the efficient
packaging of active ingredients, such as nucleic acids. Nucleic
acids are incubated with said lipids particularly at a low pH
(about 3 to 6). After the formation of the liposomes, nucleic
acids, adhering to the outside, can be washed off by changing to a
high pH (about 7 to 9).
[0085] An analogous procedure can be used to package proteins.
Advantageously, the pH of the medium is adjusted to a value here,
which lies between the pI of the liposome and that of the protein.
It has proven to be particularly advantageous, if the two pI values
are more than one unit apart.
[0086] In a further variation of the invention, the liposomes are
used to prepare release systems in diagnostics.
[0087] In a further preferred variation of the invention, the
liposomes are used as transfection systems, that is, for bringing
active ingredients into cells.
[0088] In a further variation of the invention, the liposomes are
used for the controlled release of their contents by fusion or
permeabilization of the membrane. For example, liposomes of a
lipid, which by itself is not membrane-forming, can be stabilized
by the incorporation of charge carriers, such as PE. If the charge
carrier is transformed into a neutral, uncharged or zwitterionic
state, the permeability of the membrane is increased. Known
liposomes of the state of the art (PE/CHEMS, Tachibana et al.)
permit such a permeabilization at the low pH values, which are
attained under physiological conditions only in the interior of
endosomes or during passage through the stomach. Amphoteric
liposomes can be produced by the measures listed above in such a
manner, that their neutral point lies at any desirable pH between 4
and 9. Under these conditions, the liposomes are permeable and can
deliver cargo to the medium.
[0089] However, the liposomal formulations can be produced,
processed and stored under conditions of lesser permeability. In a
preferred embodiment of the invention, liposomes are produced so
that they release of their cargo under conditions of a
physiological pH, but enclose their cargo securely at a low pH.
Such liposomes are suitable particularly for the preparation of
formulations with slow release kinetics, the release being
initiated only by contact with body fluids and not during storage
or transport.
[0090] A preferred embodiment of the inventive teaching therefore
consists of the use of such liposomes for therapeutic purposes,
especially for such uses, which employ the specific targeting of
the liposomes. The slight nonspecific binding is a prerequisite
here for transporting the liposomes to the target place. In
contrast to this, a high nonspecific binding would prevent any
transport of the liposomes to the target place. A specific binding
can be attained by further measures of the state of the art, that
is, by selecting the size of the liposomes or also by binding the
ligands to the liposomal surface, which binds to a target receptor
of the cell surface. Ligands may, for example, be antibodies or
their fragments, sugars, hormones, vitamins, peptides, such as
arg-gly-asp (RGD), growth factors, bilirubin or other
components.
[0091] The preferred variation of the inventive teachings relates
to the use of the liposomes for therapeutic or diagnostic
applications under in vivo conditions. Preferably, such liposomes
are ones, which have a slight nonspecific binding and, with that, a
slight tendency to fuse under physiological conditions, but are
combined strongly and with a high fusion competence under changed
conditions. Such liposomes are amphoteric liposomes, which have an
overall anionic particle charge under physiological conditions and
an increasingly cationic charge at a pH below 6.5. Such pH values
occur during the endocytosis of the liposomes into cells. Such pH
values also occur in the interior of tumors and in the external
layers of the skin. Low pH values can also be obtained by perfusing
an organ ex vivo for a certain period of time. A high binding
strength and fusion competence is therefore limited to those
liposomes, which were already taken up by cells or special tissue.
The binding strength and increasing fusion competence support the
fusion of the liposomal membrane with the cell membrane. This event
leads to a direct release of the cargo into the interior of the
cell without releasing components of the lysis of the endosome and,
with that, endangering the cargo or cell components.
[0092] Furthermore, the use of the liposomes as a sustained release
formulation and/or as a circulating depot is appropriate. The
liposomes can also be used advantageously for intravenous or
peritoneal application. In a particularly preferred variation of
the invention, the liposomes are: used as a vector for the in vivo,
in vitro and ex vivo transfection of cells.
[0093] The inventive liposomes have several advantages.
Cationically chargeable liposomes of 40 percent HisChol and PC bind
the nucleic acids, such as DNA, to their membrane even under
conditions of a neutral pH. Surprisingly, this binding is
suppressed completely if the above-mentioned liposomes are produced
using 5 percent of PG in addition and then have amphoteric
properties. However, the binding of nucleic acids to the membrane
can be restored once again by decreasing the pH. The inventive
liposomes are therefore particularly well suited for the
pH-dependent binding of nucleic acids.
[0094] Furthermore, it was surprisingly found that a series of
proteins also behaves in the manner described for nucleic acids.
For example, antibodies bind not at a neutral pH, but under
slightly acidic conditions effectively to the membrane of the
inventive liposomes. Such a behavior cannot be observed in the case
of pH-sensitive liposomes from a neutral lipid and CHEMS nor from
such a liposomes from a neutral lipid and HisChol. It is therefore
a special property of the amphoteric liposomes. Surprisingly, it
was also found that inventive liposomes, contrary to the known,
constitutive, cationic liposomes, are compatible with serum. An
appropriate embodiment of the inventive teachings therefore
consists of the use of such liposomes for therapeutic properties.
It is an advantage of the liposomes that, in comparison to known,
constitutive, cationic liposomes, the nonspecific binding to cells
is significantly less.
[0095] It is, however, also surprising that the fusion competence
of the inventive liposomes depends on the pH of the medium. In
comparison to biological membranes of cells, the fusion competence
is determined by the lipid selected and also by the charging of the
liposomes. Usually, a binding step precedes the actual fusion.
However, strong binding of the liposomes to cell membranes is not
always desirable and should take place, as described above, only
under controlled conditions in particular cells or tissue.
[0096] The liposomes cane therefore by used to construct liposomal
vectors for the transport of active ingredients into cells. All
materials, which do not form micelles, come into consideration as
active ingredients. Water-soluble materials are particularly
suitable as active ingredients. They include many proteins and
peptides, especially antibodies or enzymes or antigens, all nucleic
acids, independently of their molecular weight and their derivation
from RNA or DNA. However, they include also other biological
macromolecules, such as complex sugars, natural products and other
compounds, as well as low molecular weight active ingredients of
synthetic or natural origin, which otherwise cannot penetrate
through the cell membrane as barrier. With the help of vectors,
such materials can then be transported into the interior of cells
and initiate actions, which are not possible without this
transport.
[0097] Accordingly, with the help of the inventive teachings,
liposomes can be prepared, the fusion and binding properties of
which differ at different pH values. Serum-compatible liposomes,
which are laden with a large amount of active ingredients and
transport these into the interior of cells, can therefore be
produced in this way. Someone, skilled in the art, is able to
combine elements of the inventive teachings with one another and,
with that, produce liposomes, which are optimally suitable for a
particular purpose.
[0098] The invention is described in greater detail in the
following by means of examples without being limited to these
examples.
EXAMPLE 1
Preparation and Charge Properties of Amphoteric Liposomes with
Charge Carriers, which can be Charged Positively and are Constantly
Charged Negatively
[0099] His-Chol (5 mg) and 7.8 mg of POPC and 2 mg of DPPG are
dissolved in 4 ml of a 1:1 (v/v) mixture of chloroform and methanol
and dried completely in a rotary evaporator. The lipid film is
hydrated with 4.3 mL of the appropriate buffer (10 mM KAc, 10 mM
HEPES, 150 mM NaCl, pH 7.5, in a lipid concentration of 5 mM by a
five-minute treatment with ultrasound. Subsequently, the suspension
is frozen and, after thawing, extruded several times (Avestin
LiposoFast, polycarbonate filter with a 200 nm pore width). For
measuring the zeta potential, the final concentration of the
liposomes is adjusted to a value of 0.2 mM. For the dilution, the
buffer system, named above, is used at a pH of 7.5 or 4.2. The zeta
potentials measured lie between -18 mV (at pH 7.5) and +35 mV (at
pH 4.2).
EXAMPLE 2
Preparation and Charge Properties compatible of Amphoteric
Liposomes with Constant Positive and Variable Negative Charge
Carriers
[0100] POPC, DOTAP and CHEMS are dissolved in the molar ratios
given below in 4 mL of a 1:1 (v/v) mixture of chloroform and
methanol and evaporated completely in the rotary evaporator. The
lipid film is hydrated with 4.3 mL of the appropriate buffer (10 mM
KAc, 10 mM HEPES, 150 mM NaCl, pH 7.5, in a total lipid
concentration of 5 mM by a five-minute treatment with ultrasound.
Subsequently, the suspension is frozen and, after thawing, excluded
repeatedly (Avestin LiposoFast, polycarbonate filter with a 200 nm
pore width). The Table below shows the zeta potentials as a
function of pH. TABLE-US-00001 Composition of the liposomes in mole
percent liposome 1 POPC 50 DOTAP 40 Chems 10 liposome 2 POPC 50
DOTAP 30 Chems 20 liposome 3 POPC 50 DOTAP 25 Chems 25 liposome 4
POPC 50 DOTAP 20 Chems 30 liposome 5 POPC 50 DOTAP 40 Chems 10
[0101] TABLE-US-00002 TABLE 1 Zeta Potentials in mV Liposome
Liposome Liposome Liposome Liposome pH 1 2 3 4 5 4 44.2 38.4 34.7
31.7 16.2 5 39.9 25.6 27.2 22.1 3.3 6 37 21.4 16.4 2.5 -7.3 7.5
29.2 1.8 -7.9 -18.9 -34.6
[0102] The height of the zeta potential and its slope can be
selected within why limits by means of a suitable composition.
EXAMPLE 3
[0103] Preparation and Charge Properties of Amphoteric Liposomes
with Complete Switchability in One Compound
[0104] His-Chol (5 mg) and 9.8 mg of POPC are dissolved in 4 ml of
a 1:1 (v/v) mixture of chloroform and methanol and dried completely
in a rotary evaporator. The lipid film is hydrated with 4.3 mL of
the appropriate buffer (10 mM KAc, 10 mM HEPES, 150 mM NaCl, pH
7.5, in a lipid concentration of 5 mM by a five-minute treatment
with ultrasound. Subsequently, the suspension is frozen and, after
thawing, extruded several times (Avestin LiposoFast, polycarbonate
filter with a 200 nm pore width). The course of the zeta potential
at different pH values and ionic strengths is shown in the table
below (Table 2). TABLE-US-00003 TABLE 2 pH Without Salt 100 mM of
NaCl 4 45.6 20.2 5 26.9 2.2 6 -4.1 -5.2 7 -31.4 -15.3 8 -45.7
-25.4
EXAMPLE 4
Serum Aggregation
[0105] Lipid films are prepared as in Example 1. A lipid mixture,
which did not contain any DPPG, was used as comparison sample. The
lipid films were hydrated in buffer (10 mM of phosphate, 150 mM of
sodium chloride, pH of 7.4) and extruded as above. Human serum is
diluted with an equal amount of buffer (10 mm of phosphate, 150 mM
of sodium chloride, pH of 7.4), particular components and fat being
removed by centrifuging (20 minutes, 13,000 rpm, 4.degree. C.); the
clear serum is filtered sterile with a filter having a pore width
of 0.2 .mu.m.
[0106] The liposomes, prepared above are added to the serum in
concentration of 1 mM and incubated for 15 minutes at 37.degree. C.
After the incubation, the suspension of the DPPG-containing
liposomes is uniformly cloudy; however, flocculation cannot be
observed. The diameter of the liposomes is determined by means of
dynamic light scattering and is changed by less than 10% from that
of the starting sample. The suspension of the DPPG-free liposomes
clearly shows flocculation.
EXAMPLE 5
Serum Stability of the Membrane
[0107] Aside from serum aggregation, the precipitation of an active
ingredient (carboxyfluorescein, CF) in the presence of human serum
was also investigated. For this purpose, POPC/DOTAP/CHEMS liposomes
of different decomposition were prepared by the method of Example
2: POPC 100% (as control), POPC/DOTAP/CHEMS 60:30:10, 60:20:20 and
60:10:30 (in mole %). Any CF, which is not enclosed, was removed by
gel filtration. For the measurement, the liposomes were diluted to
0.1 mM in serum and incubated at 37.degree. C. A 30 .mu.l sample
was removed at certain times and diluted to 300 .mu.L with 100 mM
of tris buffer, having a pH of 8.2 and the fluorescence was
measured. The 100% values were obtained by dissolving the liposomes
with 10 .mu.l of Triton X-100 (10% in water). The enclosed CF as a
function of time is shown in the Table below.
[0108] The liposomes lose only a little CF into the serum during
the 4-hour period of measurement. POPC/DOTAP/CHEMS 60:30:10 and
60:20:20 still contain about 75%, POPC and POPC/DOTAP/CHEMS
60:10:30 even 100% of their original CF content (see Table 3).
TABLE-US-00004 TABLE 3 POPC/DOTAP/ POPC/DOTAP/ POPC/DOTAP/ Time in
CHEMS CHEMS CHEMS Min. POPC 60:30:10 60:20:10 60:10:30 0 100% 100%
100% 100% 15 91% 84% 95% 107% 60 94% 81% 87% 110% 120 96% 80% 76%
105% 240 96% 80% 77% 107%
EXAMPLE 6
Binding DNA
[0109] Liposomes of the following compositions (in mole %) are
prepared as in Example 1 (all data is in mole %). TABLE-US-00005 A:
60 POPC 40 HisChol B: 55 POPC 40 HisChol 5 CHEMS C: 60 POPC 20
HisChol 20 CHEMS
[0110] The liposomes are suspended in a concentration of 0.2 mM in
buffer (10 mM of potassium acetate, 10 mM of HEPES, pH 4.2 or 7.5).
A DNA solution (45 .mu.L, 1 mg of DNA (Hering sperm, SIGMA D3 159)
in 1 mL of water) are added in each case to 1 mL of the different
liposomes samples and mixed quickly. After an incubation period of
15 minutes, the sample is filled up with 6 mL of the appropriate
buffer and the zeta potential of the liposomes is measured (Table
4). TABLE-US-00006 TABLE 4 pH 4.2 pH 7.5 Lipid -DNA +DNA -DNA +DNA
A +47.6 -32.0 +2.4 -44.4 B +47.8 -28.1 +0.1 -38.4 C +34.0 -28.6
-10.1 -24.7
[0111] Under the conditions of an excess of cationic charges (pH
4:2), there is a strong reversal of the charge of the particles. At
a neutral pH of 7.5, the CHEMS in high concentration (liposome C)
can overcompensate the charge of the HisChol and the particles have
a negative zeta potential. Only slight amounts of DNA bind to such
particles.
Example 7
Binding and Detaching DNA
[0112] Liposomes having the compositions POPC/DOTAP/CHEMS in the
ratio of 60:15:25 and POPC/DCChol/CHEMS in the ratio of 60:15:25
(in mole %), were prepared by the method of Example 2. The binding
of the DNA was carried out at a pH of 4.2 by the method of the
above example and the zeta potentials were determined.
Subsequently, the pH of the samples was adjusted to a value of 7.5
and the zeta potential was measured once again. TABLE-US-00007
Mixture Zeta (mV) a) POPC/DCChol/CHEMS 60:15:25 (pH 4.2)
(aggregate) -43.5 b) POPC/DOTAP/CHEMS -43.7 c) POPC/DCChol/CHEMS
-18.5 d) POPC/DOTAP/CHEMS -14.5
[0113] In the presence of DNA, a negative zeta potential is
measured at a low pH; however, the original particles were charged
positively. After the change to the neutral pH, this charge, which
is due to the DNA, is decreased. The zeta potentials approach that
of the untreated liposomes (-11 mV at a pH of 7.5).
EXAMPLE 8
DNA Inclusion and Detachment of Material not Encapsulated
[0114] Two liposome formulations, having compositions of
POPC60/DOTAP15/CHEMS25 and POPC85/DOTAP15 respectively, are
prepared as dry lipid films as described above. In each case, the
total amount of lipid was. 4 .mu.moles. For hydration, Herings DNA
was dissolved in 10 mM of potassium acetate, 10 mM of HEPES and 100
mM of sodium chloride at a pH of 4.0. The DNA (4 mg) was added
directly to the lipid films. The resulting liposomes were frozen
and thawed repeatedly and subsequently extruded through a 200 nm
filter.
[0115] Each 500 .mu.L of particles was mixed with 2.5 mL of a
sucrose solution (0.8M sucrose in the above buffer, at a pH of 4.0
or 7.5). Over this, 1.5 mL of a 0.5 M sucrose solution and 0.5 mL
of the buffer were placed.
[0116] Liposomes were then separated by flotation from unbound DNA.
After the flotation, the liposomes were removed from the buffer/0.5
M sucrose interface. The amount of bound DNA was determined by
intercalation of propidium iodide. The Stewart assay was used to
determine the amount of lipid. Only the PC used responds in the
Stewart assay. The other lipids were not calculated by means of
this value. The results are shown in the Table below (Table 5).
TABLE-US-00008 TABLE 5 Liposome pH 4.0 pH 7.5 POPC/DOTA/ .sup. 2
.mu.g DNA/.mu.g DOTAP 1.2 .mu.g DNA/.mu.g DOTAP CHEMS 60/15/25
POPC/DOTAP 2.3 .mu.g DNA/.mu.g DOTAP 2.3 .mu.g DNA/.mu.g DOTAP
85/15
[0117] With the amphoteric liposomes, only about half of the bound
DNA floats up after the change in pH to 7.5. This material is the
actually enclosed material. Similar results are obtained by
digesting with DNAse
[0118] DNA cannot be detached once again from constitutively
cationic liposomes by changing the pH or by additionally increasing
the ionic strength and always remains on the outside.
EXAMPLE 9
Fusion Properties
[0119] Liposomes with the following compositions are prepared as in
Example 1 (all data in mole %): TABLE-US-00009 A) POPC 60 HisChol
40 B) POPC 55 HisChol 40 CHEMS 5 X) POPC 100 Y) POPC 60 DPPG 40
[0120] The facultative cationic liposomes A or B are incubated with
the neutral liposomes X or the anionic liposomes Y in the buffer
(10 mM HEPES, 10 mM potassium acetate, pH 4.2 or 7.5). The possible
fusion of liposomes is analyzed by size measurement by means of
dynamic light scattering (Table 6). TABLE-US-00010 TABLE 6 Liposome
1 X X Y Y Liposome 2 A B A B pH 4.2 161.6 nm 191.9 nm 1689.3 nm
2373.2 nm pH 7.5 191.8 nm 202.4 nm 250.0 nm 206.3 nm
[0121] The starting sizes of the liposomes were 161.8 nm at pH 4.2
and 165.9 nm at pH 7.5 [0122] A) 183.2 nm [0123] X) 195.2 nm [0124]
Y) 183.2 nm
[0125] The size of the pairs with the complementary charge (YA and
YB) differs clearly from the size of the mixed suspensions with the
neutral liposome X. The extent of the interaction is determined by
the magnitude of the charge of the facultative cationic liposomes.
The extent of the fusion to larger units does not depend on the
fusogenic lipid PE.
EXAMPLE 10
Permeability to Macromolecules
[0126] DOPE (13.75 .mu.moles), 2.5 Emotes of CHEMS and 10 .mu.moles
of HisChol are dissolved in isopropanol and the solvent is drawn
off under a vacuum. A solution (2.5 mL) of proteinase K in buffer
(1 mg/mL of proteinase K, 10 mM of potassium acetate, 10 mM HEPES,
150 mM of sodium chloride, pH 4.2) is added to the dried lipid
film. After the film is hydrated, the liposomes formed are extruded
through a 400 nm membrane. Proteinase, which is not enclosed, is
removed by floatation of the liposome in the sucrose gradient. The
liposomes, so produced, are incubated with 7.5 mL of buffer at a pH
of 4.2 and 7.2 (buffer as above, starting pH 4.2 and 8.0). After
the combination, the proteinase K released is removed using a 0.1
.mu.m membrane. The liposomes, remaining in the filter, are then
treated with 7.5 mL of a solution of Triton X-100 in buffer (as
above, pH 8.0).
[0127] All filtrates are tested for the presence of proteinase K.
For this purpose, a solution of azocasein (6 mg/mL of azocasein in
1 M urea, 200 mM tris sulfate, pH 8.5) is used. This solution (500
.mu.L) is mixed with 100 .mu.L of filtrate or buffer and incubated
for 30 minutes at 37.degree. C. The reaction is terminated by the
addition of 10% trichloroacetic acid. Precipitated proteins are
removed by centrifuging. The coloration is measured at 390 nm
(Table 7). TABLE-US-00011 TABLE 7 Absorption at 390 nm pH of
Incubation Triton X-100 Blank 4.2 - 0.0192 4.2 + 0.2345 7.2 -
0.2210 7.2 + 0.0307
[0128] If the incubation of the liposomes is carried out at a pH of
about 4.2, very little if any proteinase K is released. Only the
dissolution of the liposomes with Triton X-100 leads to the release
of the enzyme. If the liposomes are incubated at a pH of 7.2, the
bulk of the enzyme is released already without the addition of the
Triton and is found in the first filtrate. Hardly any additional
enzyme is then dissolved from the liposomes by the addition of
Triton.
EXAMPLE 11
Protein Binding
[0129] Liposomes, having the composition POPC50/DOTAP10/CHEMS40
(all data in mole %) are prepared as in the preceding examples. A
solution of 0.26 mg/mL of lysozyme in buffer (10 mM MES of pH 5.0
or pH 6.0 or 10 mM of HEPES of pH 7.0 or pH 8.0) is used to hydrate
the lipid film. After the hydration, all samples were frozen and
thawed repeatedly. Subsequently the liposomes are homogenized by
ultrasound and extruded through a 200 nm filter.
[0130] The liposome suspension, so prepared, is adjusted to a pH of
4.0 by the addition of acetic acid. Subsequently the liposomes are
separated by flotation from protein, which has not been
incorporated. The proportion of enclosed protein is given in the
Table below (Table 8). TABLE-US-00012 TABLE 8 pH during Inclusion %
of Material Enclosed 5.0 4 6.0 21 7.0 75 8.0 80
[0131] Liposomes of the composition used show a pI of 5; the
lysozyme is a basic protein with a pI of 11.5. The two partners
therefore have opposite charges at a pH between 6 and 8. An
efficient inclusion in the liposomes is brought about by
electrostatic attraction. Protein, not encapsulated, was removed at
a pH of 4. The interaction between the partners is cancelled at
this pH.
EXAMPLE 12
Transfection Into Cells
[0132] HeLa cells or CHO cells (3.times.10.sup.5) were plated into
each cavity of a 6-well titer plate and cultured for three days.
Liposomes (POPC/DOTAP/CHEMS 60/30/10) were prepared in the presence
of fluorescence-labeled dextran (TRITC dextran 10 mg/mL in the
hydration buffer). TRITC dextran, which had not been incorporated,
was removed by gel filtration. The liposomes, so prepared, were
added to the cells and incubated for 6 hours at 37.degree. C.
Subsequently, the cells were washed twice with buffer. The
absorption of dextran was followed in the microscopic image. The
results are shown in FIG. 1.
EXAMPLE 13
Ligand Binding and Transfection
[0133] Liposomes, having the composition of
POPC/DOTAP/Chems/N-glutaryl-DPPE (50:10:30:10 (mole %)) are
prepared as in Example 2. At the same time, they are hydrated with
a solution of 3 mg/mL of TRITC-Dextran (having a molecular weight
of about 4,400) in HEPES 10 mM and 150 mM of sodium chloride at a
pH of 7.5. TRITC-Dextran, which is not enclosed, is removed by gel
filtration through a Sephadex G-75 column. Activation of the
N-glutaryl DEPPs with EDC (1-ethyl-3-(3-dimethylaminopropyl
carbodiimide) (3.5 mg of EDC per 400 .mu.L of liposome suspension)
and subsequent stirring in the dark for 5 hours brings about the
binding of the cyclic peptide RCDCRGDCFC to the liposomal surface.
The RGD peptide (250 .mu.g in 150 .mu.L of buffer) was then added
and stirring was continued overnight. The liposomes were separated
by gel filtration from the peptide, which had not been bound.
[0134] Human endothelium cells (HUVEC) were cultured in a special
medium. The liposomes, modified with ligand, and control liposomes
without RGD ligand were added as a 0.5 mM suspension to the cells.
After 2 hours, the liposomes are removed and the cell chambers
rinsed 3 times with PBS buffer and viewed under the fluorescence
microscope. The TRITC fluorescence of cells, which had been treated
with RDG liposomes, is distinctly more red than that of the control
liposomes.
EXAMPLE 14
Pharmacokinetics (Blood Level and Organ Distribution of
pH-Switchable Liposomes)
[0135] Liposomes of POPC/Chol (60:40), POPC/Hist-Chol/Chol
(60:20:20) and POPC/DOTAP/Chems (60:10:30) (500 .mu.L) were
injected into the tail vein of male Wistar rats.
[0136] Liposome suspensions (50 mM) were prepared by hydrating a
lipid film of the corresponding formulation (addition of 0.03 moles
of [14]C-DPPC) with 2 mL of a solution of 1 mg [3]H-insulin in
HEPES 10 mM, sodium-chloride 150 nm at a pH of 7.5). After 3
freezing and thawing cycles, the suspensions were extruded
repeatedly through a 400 nm membrane (LiposoFast, Avestin).
[3]H-Insulin which had not been enclosed, was removed by gel
filtration though a G-75 Sephadex-column and subsequent
concentration over CENTRIPREP (Millipore) centrifuging units.
[0137] Liposome suspension (0.5 mL) was administered to 4
experimental animals per formulation and blood samples were taken
after 5 minutes, 15 minutes, 60 minutes, 3 hours, 12 hours and 24
hours. The radioactivity of the membrane fraction and of the
soluble cargo was measured by scintillation and gave the following
values: TABLE-US-00013 Elimination half-life times from the blood
POPC/Chol greater than 120 minutes POPC/DOTAP/Chems greater than
120 minutes POPC/Hist-Chol greater than 120 minutes
[0138] With their relatively long half-life in the blood, the
inventive liposomes fulfill the basic prerequisites for a vector
system. They are not acutely toxic and not absorbed immediately by
their reticuloendothelial system. Up to the end of the experiment,
the ratio of the 3[H] to the 14[C] radioactivity of the blood
samples was constant. Release of the cargo by complement lysis
therefore does not take place in any of the cases.
* * * * *