Note: Descriptions are shown in the official language in which they were submitted.
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Periodic structures comprising lipids, polyelectrolytes, and structure-
inducing
soluble oligovalent linkers, and biological use thereof
The invention relates to a method for preparing pharmaceutically usable
compositions which comprise periodic structures consisting of polyelectrolytes
1 o sandwiched between lipid aggregates having at least one charged component,
and
the use thereof; it further relates to a kit comprising, in a bottled or
otherwise
packaged form, at least one dose of said pharmaceutically usable composition.
Assemblies of lipid membranes and DNA have attracted large interest as
artificial
15 carriers of genetic material, being suitable for the use in gene therapy
and DNA
vaccination. For the purpose, various complexes of DNA and cationic lipids
(CL)
were tested without that the details of CL-DNA were completely understood or
perfectly controlled to date.
2o The advantages of CL-DNA complexes over natural, viral gene vectors include
the absence of a viral DNA and a greatly reduced immunogenicity. The main
disadvantages are the poor reproducibility of CL-DNA complex formation and
relatively low transfection efficiency in comparison with the viral vectors.
In
order to overcome said deficiencies, it is essential to master better the
factors
25 governing complex formation and also to maximise the payload of carriers.
When DNA is interspersed with a suspension of multilamellar lipid vesicles,
multilamellar DNA-CL complexes also arise spontaneously. They comprise
stacks of lipid bilayers alternating with monolayers of densely packed
parallel
3o DNA helices; often, more than 10 lamellae are counted in one complex.
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CL-DNA complexes for practical applications are usually prepared by mixing
DNA solution with an aqueous suspension of liposomes containing CLs. As a
result, globular lipid-DNA particles often form, sometimes with a structure
similar
to that described in preceding paragraph. However, the size and morphology of
the CL-DNA complexes, that stem from unilamellar vesicles, varies widely
between the preparations; variability is a problem even within the same
preparation. As any variability is prone to influence the transfection
efficiency
substantially, greater control over the key parameters of
transfection/vaccination
vehicle is desirable. This can only be based on a clear understanding of the
process of (multilamellar) DNA-CL complex formation and on a good rationale
for its modulation.
Based on a close examination of cryo electron-micrographs of multilamellar
complexes formed from a suspension of unilamellar vesicles we previously
suggested the following model for multilamellae formation:
Starting with a single template membrane, multilamellar structures emerge.
This
involves repeated, alternating adsorption of DNA and lipid layers: first, a
DNA
monolayer adsorbs to the template CL bilayer; then, a vesicle from the bulk
adsorbs to the bulk side of the DNA monolayer, ruptures, and rolls its bilayer
over
2o the complex; the bulk side of this bilayer is now available for further DNA
adsorption, and so on. In the systems containing lipid multilamellae, DNA can
intercalate between the multilayers under the influence of mixing or osmotic
stress, e.g. at the sites of maximum membrane curvature, which can easily lead
to
local bilayer rupture and DNA translocation. It was believed to date that
salts
play a role in this, but ruled by simple laws of electrostatics. These laws
suggest
that electrostatic interactions between the CL and DNA, as well as inter-DNA
repulsion, will be screened progressively (in a square-root like fashion). A
decreased DNA solubility and a lower affinity of oppositely charged (counter-
ionic) surfaces to bind DNA polymer from the solution result from this.
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Scientific publications reveal no consistent picture of the effect of EDTA, or
other
chelators, on lipid-DNA interaction or its biological effects. Some authors
report
positive while others observe negative or no effect of said additive.
An example for the former effect are asialofetuin-liposomes (AF-lps),
developed
as a vector for gene transfer to hepatocytes (tiara, T., Aramaki, Y., Takada,
S.,
Koike, K., Tsuchiya, S., 1995, "Receptor-mediated transfer of pSV2CAT DNA to
a human hepatoblastoma cell line HepG2 using asialofetuin-labelled cationic
liposomes" in Gene 159: 167-174). Plasmid pSV2CAT DNA was associated with
such liposomes (AF-lps-pSV2CAT). AF-lps was found to bind to HepG2 cells
through specific interaction with asialoglycoprotein receptors (AGPR);
internalisation follows by the receptor-mediated endocytotic pathway.
Transfection of HepG2 cells with AF-lps-pSV2CAT was much stronger than the
effects of either pSV2CAT associated with non-derivatised control lps (N-lps-
pSV2CAT) or else a mixture of pSV2CAT and empty AF-lps. Pretreatment with
EDTA- encapsulated AF-lps increased the transfection efficiency of AF-lps-
pSV2CAT.
2o A negative example is given in the article of Watanabe, Y., Nomoto, H.,
Takezawa, R., Miyoshi, N., Akaike, T. entitled "Highly efficient transfection
into
primary cultured mouse hepatocytes by use of canon-liposomes: an application
for
immunization" J. Biochem. 116: 1220-1226, 1994. Specifically, four
conventional artificial transfection vectors were examined. Amongst the four -
DEAE-dextran, calcium phosphate, canon-multilamellar liposomes, and cation-
liposomes (lipofection)- only the latter were highly efficient in primary
cultured
mouse hepatocytes, but less so in three other commonly used cell types (CHO-
K1,
COS-1, 3T3-L1). The transfection efficiency was strongly decreased (inhibited)
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by EDTA, free low density lipoprotein (LDL), and an endocytosis inhibitor,
cytochalasin B.
In a different study, cellular uptake and gene expression of plasmid DNA and
its
cationic liposome complexes were studied using primary cultures of bovine
brain
microvessel endothelial cells (BMEC). An avid association of naked plasmid
DNA with the BMEC monolayer was observed at 37 °C, which is
comparable to
that of the DNA/liposome complex. The binding at 4 °C was saturable and
significantly inhibited by polyanions involving polyinosinic acid and dextran
1 o sulfate; EDTA or polycytidylic acid had no effect (Nakamura et al., 1998).
We found unexpectedl~that EDTA, a common constituent of many buffers with
the charges of similar sign as DNA (a co-ion), drastically and reproducibly
affects
the morphology of CL-DNA complexes. We also observed that EDTA can
promote efficiently the unilamellar-to-multilamellar structures transition. We
consequently infer that oligovalent co-ions, including but not limited to
EDTA,
can beneficially affect the morphology of CL-DNA complexes for practical
applications and should influence the efficacy of transfection in vitro and in
vivo.
Why does the presence of EDTA in suspension so drastically alter the
morphology
of CL-DNA complexes? One possible, but as yet speculative, answer, by which
the applicants wish not to be bound, is the mechanism by which assemblies
form.
It seems that the DNA chains, in the process of complex formation, function as
'molecular glue'. The application-relevant polyelectrolyte therefore can keep
the
substrate and adsorbed vesicles close together. Catalysed by such ' glue'
action,
the ruptured vesicles roll their bilayers over the substrate and form an
adsorbed
bilayer, which then improves/grows over time. Further layers develop
similarly.
EDTA can be involved in the process by either affecting the strength / range
of
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electrostatic interactions or else by more intimate (e.g. H-bond mediated)
interaction with the polymer.
The presence of EDTA is not a prerequisite for DNA adsorption to the lipid
layer,
however. Rather, EDTA seems to influence, in a subtle fashion, the
electrostatic
interplay in interactions between the complex constituents. This may rely on
the
formation of inter-molecular hydrogen bonds, on very efficient electrostatic
screening, which increases rapidly with ion valency or simply on modified Van
der Waals interactions ('correlation forces'). It therefore stands to reason
that
1o molecules with force field similar to that of EDTA, most notably di- and
oligovalent ions, can play a pivotal role in the creation of vectors suitable
for of
gene-therapy and / or DNA vaccination.
W092/0666 discloses multilayer liposomes for thermal water encapsulation. The
systems described therein generally form multilayer (periodical) structures
spontaneously. This prior art comprises no teaching, how perdiodical
structures
can be built from monolayer (non-periodical) vesicles. Systems as disclosed in
W092/06666 are e.g. discussed in
Koltover, L, Salditt, T., Radler, J.O. Safinya C.R. (1998). An Inverted
Hexagonal
2o Phase of Cationic liposomes/DNA Complexes Related to DNA Release and
Delivery. Science 281:78-81 and
Radler, J.O., Koltover I, Salditt, T., Safinya, C.R. (1997), Structure of DNA
cationic liposome complexes: DNA intercallation in multilamellar membranes in
distinct interhelical packing regimes Science 275:810-814.
Definitions:
An adsorbent, or less generally a carrier, means an aggregate, independent of
its
composition and/or the nature and /or the source of its generation, capable of
associating with one or more charged macromolecules (adsorbates) suitable for
a
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practical purpose, such as application on or the delivery into mammalian body.
The substrate for the positively charged adsorbates is typically negatively
charged,
while for the positively charged adsorbates negative adsorbents are
preferable.
The terms "adsorbate", "adsorbing (macro)molecule", "binding (macro)molecule",
"associating (charged) (macro)molecule", etc., are therefore used
interchangeably
in this application to describe one of the participants in association between
molecules which do not form an extended surface under the conditions chosen
and
the "adsorbent" or a "binding surface", etc., in above mentioned sense.
1o An associate, by the definition used in this application, is any complex
between an
adsorbate and a carrier (adsorbent). Such an associate typically comprises two
or
more different kind of molecules, at least one of which forms aggregates with
one
or several well defined surface(s), independent of the reason for the complex
formation but excluding covalent binding. Adsorption of polyelectrolyte
15 molecules onto an aggregate surface driven by electrostatic interactions
between
the differently charged system components is the most frequent type of
association, and is also the main topic of this application. Binding of
anionic
DNA onto an aggregate surface containing cationic molecular ' anchors' is most
prominent and practically relevant example for this.
An oligovalent linker is any molecule with two or more groups capable of
binding,
non-covalently, to other groups on different molecules. Very often, the
binding is
of electrostatic nature, such as between chelators and oppositely charged
groups
on various molecules / ions, but other types of binding, e.g. via hydrogen
bonds
or directed dispersion forces, are also possible.
A chelator, within the framework of this disclosure, denotes any molecule
capable
of bringing and/or keeping together two charged entities whether dissolved or
(partially) aggregated, independent of whether or not the underlying force is
of
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electrostatic or some other origin. (Further possible interactions include H-
bonds
or other non-covalent forces.)
An extended stable lipid aggregate typically contains more than a few hundred
molecules. Most often, and advantageously, it takes the form of a macroscopic
monolayer or a bilayer, depending on the purpose of a formulation.
A lipid, in the sense of this invention, is any substance with characteristics
similar
to those of fats or fatty materials. In a rule, molecules of this type possess
an
1 o extended apolar region (chain, X); often they also have a water-soluble,
polar,
hydrophilic group, so-called head-group (Y). Basic structural formula 1 for
lipids
therefore reads
Xm - Yn (1)
n being greater than or equal to zero and m typically exceeding the value of 3
(and
more often of 6 and in most cases of 10). Lipids with n = 0 are called apolar
lipids; substances with n >_ 1 are polar. In this context, all amphiphiles are
called
simply lipids. For an explicit list and definitions previous patents and
patent
2o applications by one of the authors, such as German Patents DE 41 07 152.2
and
DE 44 47 287.0, and International Patent Applications PCT/EP91/01596,
PCT/EP96/04526, PCT/EP98/05539, PCT/EP98/06750, PCT/EP98/08421 and
PCT/EP99/04659 should be considered, which are incorporated herein by
reference.
In this description, all implicitly and explicitly mentioned lipids are well
known in
the art. Many lipids and phospholipids which form stable aggregates, most
often
in the form of bilayer vesicles are described in above mentioned patents and
patent
applications and are surveyed in 'Phospholipids Handbook' (Cevc, G., ed.,
Marcel
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Dekker, New York, 1993); 'An Introduction to the Chemistry and Biochemistry
of Fatty acids and Their Glycerides' (Gunstone, F.D., ed.) and 'Lipids' (D. M.
Small, ed., Plenum Press, London). A survey of commercial surfactants, some of
which may be suitable for the purposes of this application, is given in
Handbook
of Industrial Surfactants, M. Ash & I. Ash, eds., Gower).
A cationic amphiphile, with the basic formula similar to (1) except in that Y -
-~
Y+, which can act as an anchor for polyelectrolytes in the associate, is a
substance
that remains an integral part of the adsorbent during polyelectrolyte-
substrate
(carrier) association process. Monoamines, that advantageously can take the
role
of Y+, include ethanolamine, methylamine, dimethylamine and trimethylamine,
ethylamine, diethylamine and triethylamine, n-propylamine, n-butylamine, etc.,
furthermore methoxyamine, 2-methoxyethylamine, and 2-ethoxyethylamine;
diamines, such as ethylenediamine, 1,3-diaminopropane, 1,3-diaminobutane,
etc.,
hydrazine, putrescine, and cadaverine; polyamines, spermine and spermidine;
amides, such as acetamide, propionamide, and isonicotinic acid hydrazide, or
semicarbazide, etc.. For a comprehensive list, Handbook of Cationic
Surfactants
should be considered; specific examples used for the purpose of transgene
delivery and an extensive list of cationic (amphiphilic) anchors is given in
2o International Patent Application W096-18372, US-Patent No. 5,910,487 and US-
Patent No. 5,650,096, as well as "Cationic amphiphiles and plasmids for
intracellular delivery of therapeutic molecules", Genzyme Corp. (USA), which
are all incorporated herein by reference.
An anionic anchor differs from the corresponding cationic anchor functionally
in
the sign of its charges (Y -~ Y-). In principle, any group with a pK below the
pH
value of formulation containing periodic structures can take the role of Y-. A
representative list of such groups can be found in CRC Handbook of Chemistry
and Physics, for example; a comprehensive list of corresponding anchors is
given
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in Handbook of Cationic Surfactants should be considered; both documents are
incorporated herein by reference.
A polyelectrolyte (a charged macromolecule) is any straight or branched chain
molecule with charges, which are more often than not concentrated in/along
molecular segments, along the chain. This includes poly-cations as well as
poly-
anions, mixed forms being also possible.
The group of poly-anions encompasses, amongst others, oligo or
polynucleotides,
1o such as homo- or hetero-chains of desoxyribonucleic- (DNA) or ribonucleic
acid
(RNA), especially the genomic DNA, cDNA and mRNA that encode for
therapeutical) their chemical, biological, or molecular biological (genetic)
modifications or derivatives, etc., with at least 4 charges per chain. The
group of
nucleotides includes adenine, adenosine, adenosine-3',5'-cyclic monophosphate,
15 n6,o2'dibutyryl, adenosine-3',5'-cyclic monophosphate, n6,o2'-dioctanoyl,
adenosine, n6-cyclohexyl, salts of adenosine-5'-diphosphate, adenosine-5'-
monophosphoric acid, adenosine-5'-o-(3-thiotriphosphate), salts of adenosine-
5'-
triphosphate, 9-beta-D-arabinoturanosyladenine, 1-beta-D-
arabinoturanosylcytosine, 9-beta-D-arabinoturanosylguanine, 9-beta-D-
20 arabinoturanosylguanin 5'-triphosphate, 1-beta-Darabinoturanosylthymine, 5-
azacytidine, 8-azaguanine, 3'-azido-3'-deoxythymidine, 6-beniylaminopurine,
cytidine phosphoramidite, beta-cyanoethyl diisopropyl, cytidine-5'-
triphosphate,
2'-deoxyadenosine, 2'-deoxyadenosine 5'-triphosphate, 2'-deoxycytidine, 2'-
deoxycytidine 5'-triphosphate, 2'-deoxyguanosin, 2'-deoxyguanosine 5'-
25 triphosphate, 2',3'-dideoxyadenosine, 2',3'-dideoxyadenosine 5'-
triphosphate, 2',3'-
dideoxycytidine, 2',3'-dideoxycytidine 5'-triphosphate, 2',3'-
dideoxyguanosine,
2',3'-dideoxyguanosine 5'triphosphate, 2',3'-dideoxyinosine, 2',3'-
dideoxythymidine, 2',3'-dideoxythymidine 5'-triphosphate, 2',3'dideoxyuridin,
n6-
dimethylallyladenine, 5-fluoro-2'deoxyuridin, S-fluorouracil, 5-fluorouridin,
5-
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fluorouridin 5'-monophosphate, formycin a 5'-triphosphate, formycin b,
guanosin-
3'-5'-cyclic monophosphate, guanosin-5'-diphosphate-3'-diphosphate, guanosin-
5'-
o-(2thiotriphosphate), guanosin-5'-o-(3'-thiotriphosphate), guanosine 5'-
triphosphate, 5'-guanylyl-imidodiphosphate, inosine, 5-iodo-2'-deoxyuridine,
nicotinamide-adenine dinucleotides, nicotinamide-adenine dinucleotides,
nicotinamide-adenine dinucleotide phosphate, oligodeoxythymidylic acid,
(p(DT)10), oligodeoiythymidylic acid (p(DT)12-18), polyadenyl acid (poly A),
polyadenyl acid-oligodeoxythymidynic acid, polycytidyl acid, poly(deoxyadenyl-
deoxiythymidylic acid, polydeoxyadenylic-acid-oligodeoxythymidynic acid,
polydeoxythymidylin acid, polyinosin acid-polycytidyl acid, polyuridynic acid,
ribonuclein acid, tetrahydrouridin, thymidine, thymidin-3',5'-diphosphate,
thymidine phosphoramidite, beta-cyanoethyl diisopropyl, thymidine 5'-
triphosphate, thymin, thymine riboside, uracil, uridine, uridine-5'-
diphosphoglucose, uridine 5'triphosphate, xanthine, zeatine, transeatine
riboside,
etc. Further suitable polymers are: poly(DA) ss, poly(A) ss, poly(C) ss,
poly(G)
ss, poly(U) ss, poly(DA)-(DT) ds, complementary homopolymers, poly (D(A-T))
ds, copolymers, poly(DG)~(DC) ds, complementary homopolymers, poly (d(GC))
ds copolymers, poly (d(L-C)) ds copolymers, poly(I)-poly(C) ds, etc..
Especially
advantageous is the genomic DNA, cDNA and mRNA that encodes for
2o therapeutically useful proteins as are known in the art, ribosomal RNA;
further
antisense polynucleotides, whether RNA or DNA, that are useful to inactivate
transcription products of genes, and which are useful e.g. as therapies to
regulate
the growth of cells in diseased mammals; or ribozymes.
The group of poly-canons includes certain poly-amino acids, such as poly-
lysine.
More complete list can be found in pertinent scientific literature, primary
sequence
databanks, etc.. The chain molecules quoted in such lists, which can be
formulated most advantageously following the prescriptions of this
application,
excel in biological activity and are typically either agonists or antagonists
of
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biological action, such as protein synthesis. Words poly-nucleic acids with
sense
or antisense have their common meaning, as used in term anti-sense DNA, for
example.
The group of poly-cations includes certain poly-amino acids, such as poly-
lysine.
More complete list can be found in pertinent scientific literature, primary
sequence
databanks, etc.
The word biocide describes any ingredient added with the purpose of improving
biological stability of the formulation. An exemplary list is given in
International
Patent Application by the same first applicant (cf. PCT/EP98/08421 ), which is
incorporated herein by reference.
In order to solve the above-mentioned problems the inventions describes a
method
15 for preparing pharmaceutically usable compositions comprising periodic
structures consisting of polyelectrolytes sandwiched between lipid aggregates
having at least one charged component is described which is characterised in
that
- a suspension of non-periodic, preferably mono- or bilayer like, lipid
aggregates,
2o - a solution of polyelectrolyte molecules,
- a solution of oligovalent linkers
are separately made and then mixed to form said periodic structures, the
simultaneous presence of said components catalysing the formation of said
periodic structures comprising at least one layer of lipid component
associated
25 with a layer of polyelectrolyte molecules.
It is a preferred feature of said method that the formation of periodic
structures
does not take place or proceeds at least 10 time less rapidly if any of said
components is left out.
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Said lipid aggregates preferably have the original form of multilamellar, more
preferably of unilamellar lipid vesicles or of freely suspended or supported
lipid
monolayers.
Accordingly, the polyelectrolytes are selected from the classes of poly-
deoxyribonucleic acids, poly-ribonucleic acids, or derivatives thereof.
It is another preferred feature of the invention that said oligovalent linkers
belong
1 o to the class of chelators. Another preference is to use polar lipids for
forming
lipid aggregates.
It is preferred that a suspension of lipid aggregates and a polyelectrolyte
solution
are mixed, to form a relatively stable suspension in a solution, and
oligovalent
15 linkers, preferably in a solution, are then added to start or to control
otherwise the
formation of said periodic structures. It then is advantageous that said
periodic
structures are suspended or remain suspended in the supporting solution after
their
formation.
2o According to an advantageous embodiment of the invention, the average size
of
plain lipid aggregates is between 30 nm and 5000 nm, preferably is between 20
nm and 1000 nm, more preferably is between 30 nm and 500 nm and most
preferably is between 450 nm and 100 nm.
25 It is preferred that the concentration of at least one of the above-listed
system
components and / or the respective relative concentrations are used to control
the
speed of formation and / or the final size and / or the degree of periodicity
for the
structures generated in the system.
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Accordingly, it may be advantageous to select the final size, which for
spherical
structures corresponds to diameter, of suspended periodic structures is
between 10
nm and 10 Vim, preferably is between 20 nm and 2.5 Vim, even more
advantageously is between 30 nm and 600 nm or even better between is 40 nm
and 350 nm, and most preferably is between 50 nm and 200 nm.
Another important feature is the choice of final periodicity of said
structures,
which preferably should be between 2 and 100, more advantageously is between 4
and 50 and even more advantageously is between 8 and 25.
According to a preferred feature of the invention the chelator is selected
amongst
EDTA, EGTA, EDDA, EDDS (ethylenediamine-N,N'-disuccinic acid),
iminodiacetic acid, or their salts, DMPS (2,3-dimercaptopropane-1-sulfonic-
acid),
8-hydroxyquinoline, lipoic acid (thioctic acid), deferoxamine mesilate,
polycarboxylate, 2-furildioxime, N-2-hydroxypropyl sulphonic acid aspartic
acid,
N-carboxymethyl N-2 hydroxypropyl 3 sulphonic acid, (3-alanine N,N diacetic
acid aspartic acid, N,N diacetic acid aspartic acid N-monoacetic acid,
iminodisuccinic acid, is an amino acid based chelating agent, such as
isoserine
diacetic acid (ISDA), 2-phosphonobutane-1,2-4-tricarboxylic acid, GADS, alkyl
2o iminodiacetic acid; dipicolinic acid; hydroxy-1,1-ethylidene diphosphonic
acid
(HEDP) or a derivative thereof, or is some other oligo- or poly-anions and
cations,
or any other molecules with several polar, polarisable, or otherwise
associable
groups, which often have hydrogen bond donors and/or acceptors on them.
It is also preferred to use lipids (or lipoids) from biological sources or
made
synthetically, directly or by modifying the former lipids, advantageously
comprising a glyceride, glycerophospholipid, isoprenoidlipid, sphingolipid,
steroid, sterine or sterol, a sulphur- or carbohydrate-containing lipid, or
else, any
other lipid which forms bilayers, in particular a half protonated fluid fatty
acid,
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and very frequently a phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylinositol, a phosphatidic acid, a
phosphatidylserine, a sphingomyelin or sphingophospholipid, glycosphingolipid
(e.g. cerebroside, ceramidpolyhexoside, sulphatide, sphingoplasmalogene), a
ganglioside or any other glycolipid or a synthetic lipid, in particular with
oleoyl-,
linoleyl-, linolenyl-, linolenoyl-, arachidoyl-, lauroyl-, myristoyl-,
palmitoyl-,
stearoyl chains, which can also be attached to the corresponding sphingosine
base,
is a glycolipid or any other diacyl-, dialkenoyl, dialkyl-lipid or branched
aliphatic
chain-lipid with two identical or mixed chains.
to
Cationic anchors which can be used particularly advantageously belong to the
class of lipids with one or several aliphatic chains or other siutable apolar
residues, if appropriate branched or derivatised, and a headgroup with one or
several positive charges; the latter most often reside on a quaternary or
ternary
15 amine, which in case of monoamines includes ethanolamine, methylamine,
dimethylamine and trimethylamine, ethylamine, diethylamine and triethylamine,
n-propylamine, n-butylamine, etc., furthermore methoxyamine, 2-
methoxyethylamine, and 2-ethoxyethylamine; diamines, such as ethylenediamine,
1,3-diaminopropane, 1,3-diaminobutane, etc., hydrazine, putrescine, and
2o cadaverine; polyamines, spermine and spermidine; when an amide can be e.g.
acetamide, propionamide, and isonicotinic acid hydrazide, or semicarbazide,
etc.;
an alkylamine or a polyalkylamine is also particularly useful. Preferable
choices
include N-[1-(2,3-diacyl)-, N-[1-(2,3-dialkyl)- or N-[1-(2,3-
dialkenoyl)propyl]-
N,N,N-trialkylammonium, -N,N-dialkylammonium or -N-alkylammonium salt,
25 such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium bromide
(DOTMA), 1,2-diacyloxypropyl-N,N-dialkyl-hydroxyalkyl ammonium salt, 1,2-
dialkenoyloxypropyl-N,N,N-dialkyl-hydroxyalkyl ammonium salt, or -N,N-alkyl-
hydroxyalkyl, or N,N,N-alkyl-dihydroxyalkyl, such as 1,2-dimyristyloxypropyl-
N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE), [N-(N', N'-
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dialkylaminoethane) carbamyol] cholesterol, such as [N-(N', N'-
dimethylaminoethane) carbamyol] cholesterol (DC-Chol), or [N-(N'-
alkylaminoethane) carbamyol] cholesterol, dialkylamidoglycyl spermine or
spermidine, such as dioctadecylamidoglycyl spermidine (DOGS), diacyl,
dialkenoyl or dialkyl diacylammonium or acylammonium salt, such as dimethyl
dioctadecylammonium bromide (DDAB), 2,3-diacyl-, 2,3-dialkenoyl- or 2,3-
dialkyl-N-[2(sperminecarbozamide-0-ethyl]-N,N-dialkyl- or N-alkyl-1-
propanaminium trifluoroacetate, such as 2,3-dioleoyloxyl-N-
[2(sperminecarbozamide-0-ethyl]-N,N-dimethyl-1-propanaminium
1o trifluoroacetate (DOSPA), a I-[2-(alkenoyloxy)-ethyl]-2-alkenoyl-3-(2-
hydroxyalkyl) imidazolinium salt, such as 1--[2-(oleoyloxy)-ethyl]-2-oleyl-3-
(2-
hydroxyethyl) imidazolinium chloride (DOTIM), 1,2-dialkenoyloxy-3-
(trialkylammonio)- or (dialkylammonio)- or alkylammonio-propane, such as 1,2-
dioleoyloxy-3-(trimethylammonio) propane (DOTAP), 1,2-diacyl-3-
trimethylammonium propane (TAP), 1,2-diacyl-3-dimethylammonium propane
(DAP) or 1,2-diacyl-3-methylammonium propane (MAP), and fatty acid salts of
quaternary amines.
Anionic anchors which are selected most often, or with an advantage, carries a
2o carboxylate, succinate, sulfosuccinate, sulphate, sulphonate, ether
sulphate,
phosphate, phosphonate or amine oxide, or other anionic substances which also
appear in anionic linkers, with some preference for long-chain fatty acid
derivatives, alkylsulphate-, phosphate or phosphonate salts, cholate-,
deoxycholate-, glycodeoxycholate-, taurodeoxycholate-salts, dodecyl- dimethyl-
aminoxides, especially lauroyl- or oleoylsulphate-salts, sodium deoxycholate,
sodium glycodeoxycholate, sodium oleate, sodium elaidate, sodium linoleate,
sodium laurate or sodium myristate.
It is advantageous if the concentration of charged anchors used in the mixing
process, relatively to the concentration of the lipids that form basic
aggregates, is
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in the range 1-80 mol-%, more preferably is 10-60 mol%, and most preferably is
20-50 mol-%, the specific chosen value also depending on the selected
polyelectrolyte concentration; higher concentrations of latter ingredient
typically
require a relatively high concentration of charged anchor molecules.
It is furthermore advantageous if the total lipid concentration, including
charged
anchors and basic lipids in the aggregates is 0.0005-30 w-%, more preferably
is
0.001-20 w-%, even more preferably is 0.01-15 w-%, and most preferably is 0.05-
w-%.
It may be advantageous as well that the bulk polyelectrolyte concentration is
selected to be in the range 0.0005-30 w-%, more preferably is 0.001-20 w-%,
even
more preferably is 0.01-15 w-%, and most preferably is 0.05-10 w-%.
Preferably, the specific total lipid concentration and polyelectrolyte
concentration
values are chosen so as to ensure that the resulting periodic structures carry
less
than 50 % of the original charge density and more preferably less than 25 % of
residual charge.
Furthermore, it is preferred that the concentration and the composition of
2o background electrolyte is chosen so as to maximise the positive effect of
charge-
charge interactions on the association and normally is below I = 1, more
preferably
below 0.5 and even more preferably is between 0.01 and 0.3.
According to a preferred feature of the invention the formation of (mixed)
lipid
suspension is induced by substance addition into the fluid phase, evaporation
from
a reverse phase, by using an injection- or a dialysis procedure, with the aid
of
mechanical stress, such as shaking, stirring, vibrating, homogenisation,
ultrasonication, shear, freezing and thawing, or filtration using convenient
driving
pressure.
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It is another preferred feature of the invention that the lipids) and charged
anchor
molecules are separately mixed, if required in an organic solution (which in
case is
eliminated in due time), and the resulting suspension is combined with the
solution of polyelectrolytes and the chosen linkers solution under the action
of
mechanical energy.
It may be advantageous to generate the starting suspension of lipid aggregates
or
to achieve the final mixing by filtration, suitably elevated pressure or
velocity
homogenisation, shaking, stirring, mixing, or by means of any other controlled
mechanical fragmentation.
Preferably, the formation of aggregates with the desired size is ensured by
filtration, the filtering material having pores sizes between 0.02 ~m and 0.8
Vim,
very frequently between 0.05 ~m and 0.4 Vim, and most frequently between 0.08
~m and 0.2 Vim, several filters being potentially used in a row or
sequentially.
According to another preferred feature, the composition of periodic structures
is
prepared just before the application, if convenient, from a suitable
concentrate or a
lyophilisate.
It is another characteristic feature of the invention that the composition
comprising
periodic structures, prepared according to the above-described method, is used
to
manipulate cells, their metabolism, reproduction or survival. It then may be
of an
advantage if the composition is used in or on the mammalian body, preferably
as
drug, drug depot, or some other kind of device with a desirable medical or
biological action.
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It may then be advantageous if said structures contain oligo- or poly-nucleic
acids
that are either sense or antisense or else comprise an expressible form of a
transgene, and are used to deliver said nucleic acids into cells. Preferably,
said
composition is used for gene delivery, gene therapy or any other kind of
modulation of genetic action or in bioengineering; it may furthermore be
advantageous for said transgene to encode a protein, which preferably is
selected
from the group consisting of a ligand, a receptor, an agonist of a ligand, an
agonist
of a receptor, an antigonist of a ligand, and an antigonist of a receptor. It
then also
is preferred that said protein is a soluble protein.
to
It is a preferred feature of said inventive to use transgene which expresses
antisense RNA.
Another preferred use of said composition refers to selection of above-
mentioned
15 cells in a mammal with a disorder or a potential disorder, said use then
being for
treating the disorder or for preventing the potential disorder, as in the case
of
vaccination. Said disorder preferably is an inflammatory disease, dermatosis,
kidney or liver failure, adrenal insufficiency, aspiration syndrome, Behcet
syndrome, blood disorder, such as cold-haemagglutinin disease, haemolytic
2o anemia, hypereosinophilia, hypoplastic anemia, macroglobulinaemia,
trombocytopenic purpura, a bone disorder, cerebral oedema, Cogan's syndrome,
congenital adrenal hyperplasia, connective tissue disorder, such as lichen,
lupus
erythematosus, polymyalgia rheumatica, polymyositis and dermatomyositis,
epilepsy, an eye disorder, such as cataracts, Graves' ophthalmopathy,
25 haemangioma, herpes infection, neuropathy, retinal vasculitis, scleritis, a
gastro-
intestinal disorder, such as inflammatory bowel disease, nausea and
oesophageal
damage, hypercalcaemia, an infection, e.g. of the eye (as in infections
mononucleosis), Kawasaki disease, myasthenia gravis, one of pain syndromes,
such as postherpetic neuralgia, polyneuropathy, pancreatitis, respiratory
disorder,
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such as asthma, rheumatoid disease or osteoarthritis, rhinitis, sarcoidosis,
skin
disease, such as alopecia, eczema, erythema multiforme, lichen, pemphigus and
pemphigoid, psoriasis, pyoderma gangrenosum, urticaria, a thyroid or vascular
disorder.
Another advantageous piece of the invention is a kit comprising, in a bottled
or
otherwise packaged form, at least one dose of the pharmaceutically usable
composition prepared according to the above-described method designed to be
used in or on a mammal for prophylactic purposes, e.g. in the course of
vaccination, or for therapy.
The following two examples (cf. comparative panels 1 and panel 2) illustrate
the
determining influence of chelator (linking agent) EDTA on the formation of
lipid
multilayers from a suspension of originally unilamellar / monolayer systems.
(Electron density profiles given in both examples, pertaining to the
aggregates at
the air-water interface, were determined with a laboratory built X-ray
reflectometer and are shown in Figure 1.)
For the preparation of comparative example 1 (cationic vesicles / liposomes in
2o absence of EDTA) and of example 2 (cationic vesicles / liposomes in
presence of
EDTA), the following general experimental conditions were used: a solution of
linear DNA fragments with an average length of 6 000 by was prepared by
digesting calf thymus DNA (Sigma Chemical Co., USA) with the restriction
endonuclease EcoRV (Stratagene, USA). The fragments were purified by
repeated phenol/chloroform extraction according to standard procedures and
exhaustive dialysis against 25 mM triethanolamine buffer (pH adjusted to 7.4
with
HCl) through a membrane with a molecular weight cut-off of 3 kDa.
The lipid mixture used in this study consisted of the cationic lipid 3 (3 [N-
(N ~,N ~-
dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol, Bachem Biochemica,
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Heidelberg, Germany) and the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC, Avanti Polar Lipids, Alabaster, AL, USA) at a molar
ratio of 2:3. Part of the mixture was dissolved in chloroform to a total lipid
concentration of 1 g/L. The other part of the mixture was used to prepare
small
unilamellar vesicles by repeated sequential extrusion through 400 nm to 50 nm
polycarbonate filters in the buffer described above. Quasi-elastic light
scattering
revealed final vesicle diameters of 63 (~22) nm.
To measure X-ray reflectivity, as shown in Figure 1, a Langmuir trough was
filled
with DNA solution to which vesicle suspension was added. To this, 0.54 mM
EDTA was added to induce multilayer formation. The final DNA concentration
in the solution was 6 mg/L, total vesicles concentration was 100 mg/L. A
Langmuir film at the air water interface was prepared by spreading said
mixture
from a chloroform solution onto the surface of the suspension of vesicles and
DNA solution. To suppress capillary waves, a film of only 300 ~m thickness was
used. The temperature was kept constant at
25(~0.1)°C.
The X-ray reflectometer was laboratory made. A sealed 3 kW Molybdenum
anode serves as X-ray source. A Germanium solid state detector (Silena, Milan,
2o Italy, E.U.) with an energy resolution of about 1 % was used to analyse the
specularly reflected beam. The source as well as the detector were mounted on
goniometers. The Langmuir trough was positioned on a lifting jack between the
two goniometers and enclosed in a gas-tight box to avoid water evaporation. In
this study, the reflectometer was operated in the energy dispersive mode with
angles fixed at a = 8.61 mrad. In the energy dispersive mode, the fact was
exploited that the wave vector transfer q is a function of X-ray energy and
incident
angle a. The energy spectrum of the specularly reflected beam was multiplied
with a previously recorded calibration function, to correct for the energy
distribution of the source, detector sensitivity and secondary effects, such
as
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scattering at the water vapour in the sample chamber. From the calibrated
energy
spectrum the reflectivity R(q) was obtained. A more detailed description of
the
method is given in Vierl, U., Cevc, G., Metzger, H. 1995 "Energy-Dispersive X-
ray Reflectivity Study of the Model Membranes at the Air/Water Interface" in
Biochim. Biophys. Acta. 1234: 139-143.
The upper panel of Figure 1 gives the electron density profile of a lipid
monolayer
at the air-water interface in contact with the suspension of corresponding
vesicles
and DNA without chelators (linkers) added (example 1 ). Only a mixed
(cationic)
lipid monolayer is observed at all times. In contrast to this, the electron
density
profile illustrated in lower panel of Figure 1 (example 2) pertains to the air-
water
interface covered with a mixed (cationic) lipid monolayer in contact with a
suspension of corresponding vesicles and a solution of DNA with EDTA added,
after 100 hours of incubation. The underlying molecular structure is shown for
better reflectogram understanding. The multilayers apparent in the lower panel
consist of lipid bilayers alternating with monolayers of DNA. Low electron
densities correspond to the hydrocarbon tail region of lipid bilayers. High
electron
densities correspond to the lipid headgroup regions and intercalated DNA
layers.
The vertical grid corresponds to the repeat distance of surface adsorbed CL-
DNA
multilayers. As the only difference between examples 1 and 2 is the presence
of
EDTA the importance of latter is evident.
The validity of general conclusions is not restricted to the narrowness of
illustrated examples, described here in detail, nor is the use of resulting
associates
only useful in the field of human or veterinary medicine.
