EP2451441A2

EP2451441A2 - Mixture of amphipathic molecules and method for modifying cell membranes by means of fusion

Info

Publication number: EP2451441A2
Application number: EP10747151A
Authority: EP
Inventors: Bernd Hoffmann; Agnes CSISZÁR; Nils Hersch; Rudolf Merkel
Original assignee: Forschungszentrum Juelich GmbH
Current assignee: Forschungszentrum Juelich GmbH
Priority date: 2009-07-09
Filing date: 2010-07-08
Publication date: 2012-05-16
Also published as: JP5775073B2; WO2011003406A3; DE102009032658A1; WO2011003406A2; US20120100077A1; JP2012532151A

Abstract

The invention relates to a mixture of amphipathic molecules and to a method for modifying cells in vivo by means of membrane fusion using said molecules.

Description

Mixture of Amphipathic Molecules and Methods for

Cell membrane modification by fusion

The invention relates to a mixture of amphipathic molecules and a method for cell membrane modification by fusion with these molecules.

State of the art

From the publication by Simberg et al. (D. Simberg, Weisman S., Talmon, Y, Barenholz (2004) DOTAP (and Other Cationic Lipids): Chemistry Biophysics, and Transfection. Crit. Reviews in Therap. Drug Carr. Systems, 2004, 21, 257- 317.) it is known that cationic lipids, such as. B. DOTAP mixed with other phospholipids, such as. B. DOPE (SW Hui, M. Langner, Y. Zhao, P. Ross, E. Hurley, and K. Chan (1996) The Roel of Helper Lipids in Cationic Liposome-Mediated Gene Transfer, Biophysical Journal, 1996, 71, 590-599.) Can be used for the introduction of DNA into cells (transfection). The way these particles are taken up by the cells is endocytosis, as shown by Weijun et al. (Weijun Li and Francis C. Szoka Jr. 2007. Lipid-based Nanoparticles for Nucleic Acid Delivery., Pharmaceutical Research, 24, 438-449.). This pathway is particularly inefficient because only 1 to 10% of the liposome-entrapped DNA is taken up by the cells. A modification of these mixtures with other proteins, such. B. Transferrin is from Sakaguchi et al. Sakaguchi, Ch. Kojima, A. Harada, K. Koiwai and K. Kono, 2008. The correlation between fusion capability and transfection activity in hybrid complexws of lipoplexes and pH-sensitive liposomes., Biomaterials 29, 4029-4036). ,

A further modification of a fusion mixture is effected by the binding of viral components on the surface of the carrier particles. A disadvantage of this process is the complexity and cost with which the complexes must be prepared. Another fusion system is known from Gopalakrishnan et al. (G. Gopalakrishnan, C. Daneion, P. Izewska, M. Prummer, P.-Y. Bolinger, I. Geissbühler, D. Demurtas, J. Dubochet, and H. Vogel (2006) Multifunctional Lipid / Quantum Dot Hybrid Nanocontainers for Controlled Targeting of Live Cells, Angew Chem. Int. Ed. 2006, 45, 5478-5483.). The authors describe the fusion of a living cell membrane with a vesicle structure of DMPE, DOTAP, DPPE-PEG2000, and quantum nanodots. The disadvantage of the method is that non-biodegradable particles (quantum nanodots) are used. As a result, further medical or pharmaceutical uses are excluded.

Thus, the prior art methods are disadvantageously inefficient or associated with high stress strains. In addition, they must be optimized for each cell type and are therefore labor-intensive and cost-intensive.

Task and solution of the invention

The object of the invention is therefore to provide a molecule mixture which leads to an efficient fusion with cell membranes also in vivo. It is a further object of the invention to provide a method for efficient in vivo cell membrane modification by fusion with a molecular mixture. Another object of the invention is to demonstrate uses for the molecular mixture in the modification of cells.

The object is achieved by a mixture according to claim 1 and by the method for in vivo Zellmembran-) modification and by the cells provided in this way according to subsidiary claims. The method is based on membrane fusion between any cell membrane with a mixture of amphipathic molecules according to the invention. As a result, biologically or pharmaceutically relevant molecules can be incorporated into the cell interior and / or into the cell membrane by means of fusion and / or also coupled to the surface thereof. The mixture can be used on individual cells as well as advantageously on tissues or tissue sections.

The fusion mixture

The mixture of amphipathic molecules has an amphipathic type of molecule A, which carries a positive total charge in the hydrophilic region, and an amphipathic molecule. Lekülsorte B, which forms a delocalized electron system in the hydrophilic and / or hydrophobic region.

A type of molecule B with a delocalized electron system therefore comprises according to the invention at least one (or even more) cyclic structural motif, which are formed by conjugated double bonds and / or lone pairs of electrons and / or unoccupied p orbitals. These follow the Hückel rule.

Covalently bonded neighboring atoms of the molecule type B in the delocalized electron system particularly advantageously have an electronegativity difference (Δχ) of greater than or equal to 0.4. A type of molecule B that complies with these rules increases the fusion efficiency. Furthermore, it is advantageous that in the molecule type B at the hydrophobic or hydrophilic region a group R is bound, which forms the delocalized electron system.

In one embodiment of the invention, the mixture comprises l, 2-dioleoyl-3-trimethylammonium propane (DOTAP) as molecule type A.

In a further embodiment of the invention, the molecule type B is formed by a phospholipid to which a fluorescence dye with delocalized electron system is bound. But it can also be another dye, for. An azo dye, to be bound to a phospholipid.

As phospholipid of the molecule type B, in particular l-hexadecanoyl-2-dodecanoyl-stt-glycero-S-phosphocholine or 1,2-dihexadecanoyl-1-glycero-3-phosphoethanolamine, or 1,2-dioleoyl-57-ol Glycero-3-phosphoethanolamine provided.

The delocalized electron system in the molecule type B is advantageously formed by a group R, as indicated below: 2- (4,4-difluoro-5-methyl-4-bora-3a, 4a-diaza- 5-indacene-3-dodecanoyl) or iV- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-5'-indacene-3-propionyl) or Lissamine Rhodamine B sulfonyl or (5- (chlorosulfonyl) -2- (IH, 2H, 3H, 5H, 6H, 7H, 1H, 12H, 13H, 15H, 16H, 17H-pyrido [3, 2, 1-ij] quinolizino [1 ', 9 ': 6, 7, 8] chromeno [2, 3-f] quinolin-4-ium-9-yl) benzenesulfonate or 7-nitro-2-l, 3-benzoxadiazol-4-yl or carboxyfluorescein or 1-pyrensulfonyl ,

The specified basic components of the molecule type B, that is z. As the phospholipids and said groups R are freely combinable with each other, that is, that each of the dyes can be bound to a phospholipid.

The fusion mixture has far-reaching advantages over the previously known fusion mixtures. Thus, the mixture in all (mammalian) cell types, and thus ubiquitously applicable. The mixture is highly efficient (> 50%), and there are no damage to the cells due to the fusion. The cells are therefore fully chargeable after the merger. The mixture is also applicable to tissue sections. Further advantages are achieved by the functionalization of the cell membrane with specific molecules, as described below, for. B. in the way 3. indicated.

In the mixture according to the invention, a further amphipathic type of molecule C can be provided as helper molecule.

In particular, 1,2-dioleoyl-5-glycero-3-phosphoethanolamine (DOPE) can be present as molecule type C for this purpose.

The preferred ratio between the types of molecules A, B and C is about 1: 0.05-0.2: 1 wt / wt.

The mixture comprises up to 99% wt amphipathic molecule type A, 40% wt amphipathic type of molecule B (with group R), and up to 70% wt amphipathic type of molecule C.

For the process according to the invention, any mixtures are dissolved in an organic solvent and homogenized well (eg vortex, ultrasound). The solvent is removed, for. In vacuo and the dried mixture of molecular species dissolved in an aqueous buffer. This mixture is durable while cooling. Particularly preferably, the molecule types A, B and C are present as lipids. These then form a liposome upon uptake in an aqueous buffer. Liposomes are preferred because in an excellent manner, further types of molecules can be arranged in and on the cell membrane, and also molecules incorporated in the liposome can be introduced into the cell interior during the fusion of the liposome with the cell membrane.

Most preferably, therefore, the mixture is present as a liposome. The liposome according to the invention has a particularly advantageous effect in that further molecules can be arranged in it, which can lead to preferred modifications of the cells.

However, it is conceivable to specify polymers instead of the lipids as molecule type A and / or B and / or C.

For the inventive method for in vivo cell modification, the selected cell with its cell membrane with a preferably buffered mixture, or liposome, in

Brought in contact. The process of contacting takes advantageously only a few minutes, z. 1 to 120, preferably 10 to 30 minutes, during the fusion. Single cells are advantageously fused in a very short time after contact with the mixture, or the liposome, hereby. Cell tissues may take a little longer, that is, up to about 120 minutes. From the fusion, the endocytosis of the liposome is strictly distinguishable.

The method has a high proportion of cells fused to the mixture or liposome compared to the prior art and for the shortness of contact time. The efficiency is therefore advantageously over 50%.

During the process, various other molecules can be added to the mixture according to the invention. The method according to the invention proves to be versatile and robust in this respect. By this is meant that a variety of different functional molecule types can be mixed individually or simultaneously and thus lead to the desired modifications of the cell and not only the membrane. Because it is a vivo system, the cell remains functional. By way of example, an amphipathic type of molecule D having a functional group in the hydrophilic region, in particular having a chelate group as functional group, can be supplied to the mixture according to the invention having molecular types A, B and optionally C. Then the mixture comprises z. As the molecule types A, B, D and optionally C. The functional group is placed during the fusion of the mixture, or the liposome, with the cell membrane of the cell on the surface of the cell membrane.

With this approach, the surface of the cell membrane can be specifically modified and functionalized. For this purpose, the functional group protrudes outwards from the surface of the cell and can be selectively modified by further chemical groups. This can result in a particularly advantageous embodiment of the invention for successful cancer therapy.

A mixture according to the invention therefore comprises for this purpose z. B. l, 2-Dioleoyl-3-trimethylamine-propane as the molecular species A and N- (4,4-difluoro-5,7-dimethyl-4-boron-Sa-a-diaza-A-Indacene-S-Propiony ^ -l ^ -Dihexadecanoyl- ^ n-glycero-S-

Phosphoethanolamines as molecule type B and triethylammonium salt / 1, 2-dioleoyl-s «- glycero-3-phosphoethanolamine as molecule type C and l, 2-dioleoyl-s-glycero-3 - [(N- (5-amino-1-yl) Carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) as molecule type D, e.g. In a mixing ratio of 1: 0.1: 1: 0.002 wt / wt. The said, further cell modification is then carried out by adding tumor necrosis factor (TNF) α, bound to a όxHistidin repeat. This leads to the successful therapy of cancer, as indicated under way 3 (see below).

An amphipathic type of molecule E can be added to the mixture and, during the fusion, arranged in the membrane of the cell, in particular bacteriorhodopsin or glycophorin A or integrin as molecule type E.

Furthermore, a non-amphipathic hydrophilic type of molecule F in a buffered solution can be added to a pre-dried mixture and released into the lumen of the cell during the fusion. With molecule type F, in particular RNA, DNA, Ca ²⁺ , peptide, protein or a medicament, such as acetylsalicylic acid, is included. A non-amphipathic hydrophobic type of molecule G can also be simultaneously added to the mixture and placed in the membrane of the cell during fusion. These include in particular cholesterol, vitamin A, D, E and K or drugs such as cortisone.

All types of molecules A, B, C, D, optionally G can be dissolved in an organic solvent and homogenized. They can then be dried and dissolved in an aqueous buffer and stored. In the solution in an aqueous buffer, the mentioned types of molecules E and / F can be added. Depending on the type of molecule, liposomes from the molecule types A, B, C, D, E, F, G can advantageously be formed.

The following molecules for preparing a fusion mixture for cell membrane modification by fusion with the membrane can be used. The following list is not exhaustive or restrictive.

Exemplary embodiments of molecule type A:

The criteria for the molecule A are that (a) the molecule has a hydrophilic region with at least one or more positive charges so that the total charge of the hydrophilic part of the molecule is positive. The object of this molecule is to bring the fusion mixture by electrostatic forces in the vicinity of the negatively charged cell membrane, (b) It also has a hydrophobic region, preferably C ₁₀ -C _{30 -content} with or without double bonds. Double bonds advantageously have the effect that the membrane of the liposome formed becomes elastic, so that fusion of the liposome with the cell membrane is facilitated. (C) The proportion of molecule type A in the mixture according to the invention can be up to 99 wt / wt%.

Suitable are molecules such. L, 2-dioleoyl-3-trimethylammonium-propanes (DOTAP), N- (2,3-dioleyloxypropyl) -N, N, N-trimethylammonium chloride (DOTMA), dimetyl-dioctadecyl ammonium bromide (DDAB), or ( 1- [2- (oleoyloxy) ethyl] -2-oleyl-3- (2-hydroxyethyl) imidazolinium chlorides (DOTIM). As a first example, DOTAP (1,2-dioleoyl-3-trimethylammonium propane (Chloride SaIz)) is called. Exemplary embodiments of molecule type B:

The criteria for the amphipathic type of molecule B as fusion-inducing molecules according to the invention are as follows: (a) it must have a hydrophilic area with or preferably no charge, (b) and it must have a hydrophobic area, preferably C ₁₀ -C ₃₀ content with or have no double bonds. For the function of the double bonds, see variety A. (c) The molecule has a group (R), either hydrophobic and / or hydrophilic

Area, which has a delocalized electron system. This means a cyclic structural motif of conjugated double bonds and / or lone pairs of electrons and / or unoccupied p orbitals.

Particularly preferred in group R is a large difference in electronegativity between covalently connected neighboring atoms. This can be particularly advantageous at least 0.4 (Δχ> 0.4). This serves to increase polarizability and positively affect fusion, (d) The amount of fusion-inducing type B molecule in the mixture can be adjusted up to 40 wt / wt%.

The table below summarizes some relevant groups R for the molecules of variety B.

Table 1: Summary of the properties of fusion inducing and non-fusion inducing groups R bound to amphipatic molecules B (eg: phospholipids, such as DHPE or DOPE).

a 1-very good, 2-good, 3-satisfying, 4-sufficient, 5-hardly works, 6-does not work

First example of molecule type B (P-BODIPY-C _I2 -HPC):

2- (4,4-Difluoro-5-methyl-4-bora-3a, 4a-diaza-, ϊ-indacene-3-dodecanoyl) is β-BODIPY and represents the group R. This group R is bound to l ^{-hexadecanoyl-2-dodecanoyl-1} Λ «- glycero-3-phosphocholines (C ₁₂ -HPC). Both together give the following structural formula: Hg) ₃

Second example of molecule type B (BODIPY FL DHPE):

N- (4,4-difluoro-5,7-dimethyl-4-boro-3a, 4a-diaza - ^ -indacene-3-propionyl) is BODIPY-FL. This group R is bound to DHPE (1,2-dihexadecanoyl, S77-glycero-3-phosphoethanolamine (triethylammonium salt)). Both together give the following structural formula:

(CH ₃ CH ₂ J ₃ NH

Third example of molecule type B ( ¹ DiO '):

DiOCi ₈ (3) 3,3'-dioctadecyloxacarbocyanine perchlorate. This molecule is not a lipid-bonded dye as the first two molecules B, but an amphiphatic molecule per se of the structural formula:

Fourth example of molecule type B (LR-DOPE):

Lissamine Rhodamine B sulfonyl is group R. This is bound to 1, 2-dioleoyl-s "- glycero-3-phosphoethanolamine (DOPE) as an amphiphatic molecule. Both give the structural formula:

Fifth Exemplary Embodiment for Molecule B (Texas Red®-DHPE):

Texas Red is (5- (chlorosulfonyl) -2- (IH, 2H, 3H, 5H, 6H, 7H, 1H, 12H, 13H, 15H, 16H, 17H-pyrido [3, 2, 1-ij] quinolizino [ 1 ', 9': 6, 7, 8] chromeno [2, 3-f] quinolin-4-ium-9-yl) benzenesulfonates, ie group R. This is bonded to 1,2-dihexadecanoyl-s-glycero 3-phosphoethanolamines and together give the structural formula:

Sixth example of molecule type B (NBD-DOPE):

NBD is (7-nitro-2-l, 3-benzoxadiazol-4-yl) also group R. This is bound to 1,2-dioleoyl-5'-glycero-3-phosphoethanolamines having a structural formula:

Seventh example of molecule type B (fluorescein-DOPE):

Carboxyfluorescein is group R and attached to l, 2-dioleoyl-5'-glycero-3-phosphoethanolamines having the structural formula:

Eighth example of molecule type B (Pyrene - DOPE)

1 -pyrenesulfonyl is group R and attached to l, 2-dioleoyl-.SM-glycero-3-phosphoethanolamines having the structural formula:

Ninth example of molecule type B (ß-py-C ₁₀ -HPC):

Pyrenedecanoyl group R is attached to l-hexadecanoyl-2-5-glycero-3-phosphocholines having the structural formula:

All groups R have a delocalized electronic structure in the sense of the invention.

Due to the delocalization, the group R in molecule type B is often present as a dye.

It is conceivable that the group R itself is formed by a hydrophobic part of an amphipathic molecule and by delocalized double bonds.

In addition, as shown in Table 1, the greater the difference between the neighboring atoms in terms of their electronegativity, the better the fusion quality.

Do not work, however, succeeding molecules, such as. CapBio-DOPE: cap biotinyl would be group R bound to 1-dioloyl-sn-glycero-S-phosphoethanolamines having the structural formula:

Also does not work PI: L-α-phosphatidylinositol (sodium salt)

and

Methoxy (Polyethylene glycol) -2000 would be group R bound to 1,2-dioleoyl-5Η-glycero-3-phosphoethanolamine (PEG2000-DOPE) having the structural formula:

The last three molecules lack delocalization of the electronic structure. This shows that the mixture according to the invention must have these.

As described, it is conceivable to combine the positive properties of the type of molecule A with respect to the positive charge and that of the type of molecule B with respect to the delocalized electron system, or with respect to the differences in the electronegativity in a chemically synthesized molecule. This, the inventors hitherto unknown molecule, therefore, should also be the subject of the invention.

Optionally, and for further advantageous embodiment of the mixture, the following further molecules are possible for producing a fusion mixture or for cell membrane modification.

Exemplary embodiments of molecule type C as helper molecule

The criteria for helper molecule / carrier molecule C are: (a) The molecule must have a hydrophilic region and (b) a hydrophobic region (in particular Ci ₀ -C ₃₀ ) with or without double bonds. For the function of the double bonds, see variety A and / or B. (c) Both ranges should have a neutral character in order to avoid the large La density and the repulsive forces between positively charged molecules of molecule type A to neutralize. This effect leads to the stabilization of the system, (d) The proportion of the carrier molecule can be set to a maximum of 70% wt / wt.

Suitable are molecules such. B. phosphatidylethanolamines, phosphatidylcholines (1,2-dioleoyl - ^ - glycero-3-phosphoethanolamines, 1,2-dipalmitoyl-5-glycero-3

Phosphoethanolamines, 1,1-Dimiristoyl-SM-glycero-S-phosphoethanolamines, 1,2-Dielaidoxy® -glycero-3-phosphoethanolamines, 1,2-diphytano-L-S-glycero-3-phosphoethanolamines, 1-dilinoleoyl-glycero-S- Phosphoethanolamines or 1,2-dioleoyl 5'-glycero-3-phosphatidylcholine).

As a first example, 1,2-dioleoyl-s-glycero-3-phosphoethanolamine (DOPE) is named.

Advantageously, it is possible to form the molecule types A, B and C as lipids or as polymers. In the case of amphipathic lipids, liposomes are formed in the preparation of the ready-to-use mixture.

The weight-mixing ratio (wt / wt) is advantageously 1: 0.05-0.2: 1 between the molecule types A, B and C.

Molecule Type D (amphipathic with functional group)

The criteria for the amphiphatic molecule type D are: (a) The molecule must have a hydrophilic region and (b) a hydrophobic region (in particular with Ci _O -C ₃₀ ) with or without double bonds. For the function of the double bonds, see species A, B, or C. (c) The molecule must have a functional group in the hydrophilic region that allows further chemical bonding on the cell surface after fusion.

Suitable are molecules such. Long-chain fatty acids and alcohols, chelate complex modified lipids, eg. For example, 1, 2-dioleoyl, 5-glycero-3 - [(N- (5-amino-1-ol)

Carboxypentyl) iminodiacetic acid) succinyl] (nickel salt), biotin modified lipids (1,2- Dioleoyl-sw-glycero-S-phosphoethanolamine-N-cap-biotin), or PEG-modified lipids (1,2-dioleoyl-5-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt ) or pharmaceuticals.

A first exemplary embodiment of this is (iminodiacetic acid) succinyl (nickel salt) as a functional group, here in the form of a chelate group, attached to 1,2-dioleoyl-H-glycero-5-amino-1-carboxypentyl. This gives the structural formula:

Type of molecule E (amphipatic):

The criteria for molecule type E are further: (a) they are amphiphatic. (b) These may include membrane proteins, peptides, surface receptors, such as. B. Bacteriorhodopsin, integrin, glycophorin A and many others, (c) type of molecule E, like the other components A, B, C, D are artificially synthesized or derived from cells, (d) modification of these molecules, such as. For example, fluorescence or radioactive label may be present. (E) Molecule Type E may be added to the mixture up to 20% wt / wt. The amphiphatic molecule types E is incorporated into the liposome membrane when using lipids as grade A-D. Non-amphiphatic molecule types E are incorporated on the liposome membrane when lipids are used as grade A-D.

Type of molecule F (water-soluble or hydrophilic):

The criteria for molecule type F are: (a) The molecules should be water-soluble. The following examples are given: ions, peptides, proteins, drugs, DNA or RNA.

Type of molecule F is incorporated into the lumen of the liposome when using lipids as molecular species. Type of molecule G (not water-soluble or hydrophobic):

The criteria for molecule type G are: (a) The molecules have hydrophobic character and are readily soluble in organic solvents. The following examples are given: cholesterol, vitamin B. Together with the molecule types A, B, C, and D, these are mixed in an organic solvent.

The merger procedure

The constituents of the mixture according to the invention with the molecule types A and B are together with the optionally further, mixed molecules C, optionally D and optionally G simultaneously in an organic solvent, preferably in chloroform, methanol, ethanol, propanol, hexane, heptane or a mixture thereof, added in the desired weight ratio. After homogenization in the organic solvent, the organic solvent should be removed.

The dried mixture is taken up in an aqueous buffer solution, preferably at a pH around 7, and homogenized again. Then, advantageously, a liposome is formed which comprises the added types of molecules. In this case, molecules of the molecule type E and F can also be added. In this form, the mixtures are durable, for example at 4 ⁰ C for at least one month. These steps serve to prepare the process according to the invention.

The actual method provides that the fusion mixture with a cell medium z. B. 1: 100 (mixture: cell medium v / v) diluted, homogenized again and added to the cells.

By adding the fusion mixture according to the invention to live cells, the fusion is preferably generated in vivo. For this it is sufficient that the cells with the mixture for about 1 to 60 minutes, in tissue samples z. B. 60 to 120 minutes are brought into contact. A washing step of the cells should then be inserted. The method is advantageously characterized by a particularly high fusion efficiency of at least 50%, preferably more than 70%, in particular more than 90%.

Membrane fusion is particularly not limited to single cell types, but provides an almost universal mechanism as set forth in Table 2. Even dense tissue samples can be successfully treated by prolonged incubation. The method is therefore particularly advantageous very versatile.

When the mixture according to the invention is fused with a cell membrane, the vesicular volumes in the form of type of molecule F are introduced into the cell, which can be advantageously used to introduce soluble molecules, ions, proteins or DNA into the cytoplasm of the cell. Molecule type F is therefore in the true sense not a component of the mixture of amphipathic types of molecules.

During the fusion process, the biologically or pharmaceutically relevant molecule types D, E, or G can be incorporated into intact cell membranes. The introduced in the cell membrane reactive groups of the molecule type D can, for. B. be used for coupling reactions. Additional membrane E-type molecules can be incorporated into the cell membrane, such as peptides or proteins.

The mixture of the invention and the method of the present invention provide ways to perform analyzes in a variety of basic research fields, such as the diffusion analysis of molecules incorporated into intact cell membranes, the characterization of lipid micro and nanodomains, also known in the literature as lipid rafts, or the investigation of the regulation and transport pathways of membrane molecules in the cell.

At the same time, the system, for example, the medically highly relevant, targeted marking of certain cell types z. For example, for the labeling of cancer cells, to support wound closure by induction of cell migration, for the formation of cellular cell contacts, of cell spurs or of proliferation induction or cell-cell fusion. Certain inventive amphiphatic mixtures of the molecule types A, B, (C), D with TNFα can therefore be used as medicaments and at the same time in the targeted labeling and treatment of cancer cells.

An overview of some cell membrane modification procedures is shown in Table 2. Table 2: Summary of cell modification experiments using the fusion mixture.

^a Type of molecule C / type of molecule A Type of molecule B = 1/1 / 0.1 wt / wt in each experiment carried out. For the exact composition, see route 2 below. ^c for the exact composition see way 3. below. ^d DOPE / DOTAP / β-BODIPY C ₁₂ -HPC / PI = 1/1 / 0.1 / 0.1 Abbreviations to Table 2:

Method 1: cell membrane staining; Method 2: Cell Surface Modification; Method 3: cell lumen modification; Method 4: Protein Reconstruction in the Cell Membrane To Molecule C: DOPE = l, 2-Dioleoyl-5'-glycero-3-phosphoethanolamine To type of molecule A: DOTAP = l, 2-dioleoyl-sn-glycero- (trimethylammonium-propane (chloride salt)

To type of molecule B: β-BODIPY C ^ -HPC ^^^ - difluoro-S-methyl ^ -Bora-Sa ^ a-Diaza - ^ -indacene-S-dodecanoyl) -1-hexadecanoyl-sH-glycero-S-phosphocholine ß-Pyrene C ₁₀ -HPC: 1 -hexadecanoyl-2- (1-pyrenedecanoyl) -5-glycero-3-phosphocholine

BODIPY FL DHPE: JV- (4,4-difluoro-5,7-dimethyl-4-borora-3a, 4a-diaza-s-indacene-3-propionyl) -1,3-dihexadecanoyl-sw-glycero-S-phosphoethanolamine , (Triethylammonium salt) DiO: DiOC ₁₈ (3) 3,3'-dioctadecyloxacarbocyanine perchlorate

Fluorescein-DHPE: 1, 2-Dioleoyl-.sn-glycero-3-phosphoethanolamine-N- (carboxyfluorescein) (ammonium salt)

LR-DHPE: lissamine rhodamine B 1 ^ -dihexadecanoyl-stt-glycero-S-phosphoethanolamine, (triethylammonium salt) LR-DOPE: lissamine rhodamine B l, 2-diolenoyl-5-glycero-3-phosphoethanolamine, (triethylammonium salt)

NBD-DOPE: N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) -l, 2-diolenoyl-1-glycero-3-phosphoethanolamine (triethylammonium salt)

Pyrene-DOPE: 1, 2-Dioleoyl-5-glycero-3-phosphoethanolamine-N- (1-pyrenesulfonyl) (ammonium salt)

Texas Red-DHPE: Texas Red 1, 2-Dihexadecanoyl-i-glycero-3-phosphoethanolamine (triethylammonium salt) To molecular types DG:

BR-TRITC: bacteriorhodopsin from Halobacterium salinarum gelabeled with tetramethylrhodoin isothiocyanate mixed isomers as molecule type E

DOGS-NTA: 1, 2-Dioleoyl-5'-glycero-3 - [(N- (5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) as molecular species D. capBioDOPE: 1-dioloyl-SM -Glycero ^ -phosphoethanolamine-N-cap-biotin as molecule type D

B FL-SM: N- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-dodecanoyl) sphingosyl phosphocholine as the molecular species D GPA-TRITC: glycophorin A (MNS blood group) labeled with tetramethylrhodamine isothiocyanate mixed isomers as molecule type E.

PI: L-α-phosphatidyl inositol (sodium salt) as molecule type E.

CaCl ₂ XH ₂ O as molecule type F.

Cell types and cell lines: BSMC: Bronchial smooth muscle cells

ESMC: Embryonic smooth muscle cells

HEK: Human Embryonic Kidney, HEK293 h. Fibroblasts: human fibroblasts

Keratinocytes: human keratinocytes Macrophages: human macrophages Myofibroblasts: cardiac fibroblasts of rat neurons: embryonic cortical neurons of rat R37: breast cancer cell line pericardium: rat embryonic heart bag Miscellaneous to Table 2:

TNF: TNFα-His tag: tumor necrosis factor linked with όxhistidine repeat

Furthermore, the invention will be explained in more detail with reference to embodiments and the accompanying figures, without this being intended to limit the invention.

1: Schematic representation of three possible uses of the fusion reagent.

2: Corresponds to line 11 in Table 2. Microscopic images of HEK293 cells preincubated in Fluo-4 AM after addition of the fusion mixture (DOPE (type of molecule C) / DOTAP (type of molecule A) / LR-DHPE (type of molecule B) = 1 / l / 0, l wt / wt) filled with 1 mM CaCl ₂ (molecular grade F) solution. The red-labeled lipid mixture (molecule types AC) (LissRhod channel) was incorporated into the cell membrane while the green Ca ²⁺ indicator (fluo-4 channel) indicates an increased Ca ²⁺ concentration (molecule type F) in the cell interior. (Scale = 20 μm).

Fig. 3: Corresponds to line 25 in Table 2. Microscopic images of fibroblasts 10 minutes after the addition of the fusion mixture (DOPE (molecule type C) / DOTAP (type of molecule A) / β-Bodipy-C, ₂ HPC (molecule type B) / BR- TRITC (molecule type E) = 1/1/0, 1 / 0.0005 wt / wt). The green-labeled lipid mixture (type of molecule AC) (β-Bodipy channel) and the red-labeled channel protein (molecule type E) (TRITC channel) were incorporated into the cell membrane. (Scale = 20 μm) 4: Corresponds to line 25 in Table 2. Microscopic images of fibroblasts 4 hours after washing off of the fusion mixture (DOPE (molecule type C) / DOTAP (molecule type A) / β-Bodipy-C ₁₂ HPC (molecule type B) / BR- TRITC (type of molecule

E) = 1/1/0, 1 / 0.0005 wt / wt). The green-labeled lipid mixture (molecule type A-C) (β-Bodipy channel), as well as the red-labeled channel protein (molecule type E) (TRITC channel) were incorporated into the cell membrane. The separation of the two dyes as a function of time suggests a different transport process of the membrane lipids and proteins from the cell membrane into the cell interior. (Scale = 20 μm).

Fig. 5: Corresponds to line 16 in Table 2. Microscopic images of HEK293 cells 10 minutes after addition of the fusion mixture (DOPE (type of molecule Q / DOTAP (type of molecule A) / Bodipy FL-DHPE (type of molecule B) / DOGS-NTA (type of molecule D ) = 1/1 / 0.1 / 0.002 wt / wt) (scale = 50 μm)

FIG. 6: Corresponds to line 16 in Table 2. Microscopic uptake of TNFα-His-tag treated HEK293 cells with incorporated DOGS-NTA (molecule type D) in the membrane and macrophages and B: Microscopic image of TNFα-His-tag treated HEK293 cells without DOGS-NTA (molecule type D) in the cell membrane and macrophages. (Scale = 50 μm)

FIG. 7: corresponds to row 23 in table 2. Microscopic images of fibroblasts after washing off of the fusion mixture (DOPE (type of molecule C) / DOTAP (type of molecule A) / TexasRedDHPCC (type of molecule B) / capBioDOPE (molecule type D) = 1/1 / 0.1 / 0.01 wt / wt). The red-labeled lipid mixture (molecule type A-C) (TexasRed channel), as well as the green-labeled surface protein (AlexaFluor488 channel) show a clear colocalization of phospholipid and cell surface bound proteins. (Scale = 50 μm)

Figure 8: Corresponds to row 24 in Table 2. Microscopic images of fibroblasts after washing off the fusion mixture (DOPE (molecule type C) / DOTAP (molecule type A) / TexasRedDHPC (molecule type B) / B FL-SM (molecule type D) = 1 / l / 0, l / 0.01 wt / wt). The images indicate a successful incorporation of the phospholipids (TexasRed channel) as well as the sphingolipids (BFL channel). (Scale = 20 μm) Figure 9: Corresponds to row 47 in Table 2. Micrograph of primary pericardium from embryonic rats (day 19) after one hour of treatment with the fusion mixture (DOPE (molecule type C) / DOTAP (molecule type A) / Bodipy FL) DHPE (type of molecule B) = 1/1/0, 1 wt / wt). The image clearly shows that the fusion mixture of molecular types AC has penetrated deep into the tissue up to about 3-4 cell layers and has induced membrane fusion. (Scale = 70 μm).

Fig. 1 shows three ways of cell membrane modification according to the invention. Each path is executed in another embodiment.

Path 1 from FIG. 1: cell membrane modification by mixing the varieties AC and introducing Ca ²⁺ ions as type F into the lumen of cells.

Human Embryonic Kidney (HEK293) (DSMZ Germany) cells were grown in DMEM medium (Sigma Aldrich, St. Luis, Mo., USA) at a cell density of 30,000 cells per cell culture dish (0-3.5 cm) in Fluo-4 AM-Ca ^{2 +} Indicator (Invitrogen, Eugene, OR, USA) in (lμg / ml) for 30 minutes. After incubation, the cells were washed with DMEM medium and incubated with a fusion mixture according to the invention for 30 minutes.

The fusion mixture according to the invention consists of a lipid mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE: molecular grade C), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP: molecular grade A) (both Avanti Polar Lipids Inc., Alabama, AL, USA), Lissamine Rhodamine B1, 2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (LR-DHPE: Type B molecule with group R) (Invitrogen) in the weight ratio of DOPE / DOTAP / LR-DHPE (1/1 / 0.1 wt / wt).

The lipid components were first homogeneously mixed in chloroform (VWR, Darmstadt, Germany). Subsequently, the solution was dried in vacuo for 30 to 60 minutes at room temperature. The lipid molecules were resuspended in a buffer solution of 20 mM (2- [4- (2-hydroxyethyl) -l-piperazinyl] -ethanesulfonic acid) (HEPES buffer) (VWR) and 1 mM CaCl ₂ as the molecular species F (VWR) in a 2 mg lipid / ml buffer and homogenized in an ultrasonic bath (80-100 W) for 20 minutes at room temperature. The mixture is thus present as a liposome. This mixture is stable for about 1 month in the refrigerator at 4 ° C and reusable after a repeated Homogenisierungsschritt.

5 μl of this fusion mixture are diluted 100 times with DMEM medium and, before being added to a cell culture dish with HEK293 cells incubated with Ca ²⁺ indicator Fluo-4 AM (Invitrogen), again homogenized for 5 to 10 minutes by means of ultrasound.

The cells are incubated in the inventive fusion process for 10 to 30 minutes with the inventive mixture at 37 ° C and 5% CO ₂ . Thereafter, the cells were washed with DMEM medium and the fusion of the reagent with the cell membrane was checked on a fluorescence microscope (LSM 710 from Carl Zeiss Microimaging GmbH, Jena) (FIG. 2).

After successful membrane fusion, the red fluorescent lipid molecules of molecule type B are incorporated into the cell membrane of HEK293 cells. As a result, the cell membrane under a fluorescent microscope well visible (see Fig. 2 - LissRhod channel).

Evidence of the delivery of the vesicle lumen into the cell interior, and thereby also the type of molecule F, provides the enhanced green fluorescence intensity of the Ca ²⁺ indicator Fluo4 AM, which was caused by the increased Ca ²⁺ concentration in the cell volume after membrane fusion (FIG 2 - Fluo-4 channel).

The images and associated counts show a fusion efficiency above 80%, ie more than 80% of all cells fused with the mixture / liposome according to the invention.

As a control, HEK293 cells were treated identically except that the vesicle lumen was filled with 20 mM HEPES instead of 1 mM CaCl ₂ buffer. In this case, the Ca ²⁺ indicator showed a significantly lower Ca ²⁺ concentration in the cells (not shown).

Route 2 from FIG. 1: Cell membrane modification by mixing the varieties AC and introducing bacteriorhodopsin (molecule type E) into the cell membrane: Cardiac fibroblasts were isolated from embryonic (day 19) rat hearts and in FlO urinary medium (Sigma Aldrich, St. Luis, MO, USA) at a density of 20,000 cells per cell culture dish (0 3.5 cm) at 37 ° C and 5% CO ₂ incubated. Over a period of 6 days, the cardiac fibroblasts differentiated into myofibroblasts and were used in the subsequent method according to the invention.

The myofibroblasts were prepared in a fusion mixture of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE: molecular grade C), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP: molecular grade A) (both from Avanti Polar Lipids Inc. Alabama, AL, USA) and 2- (4,4-difluoro-5-methyl-4-bora-3a, 4a-diaza-5'-indacene-3-dodecanoyl) -1-hexadecanoyl-SH-glycero. 3-phosphocholines (β-BODIPY C ₁₂ -HPC: Type B of B vitamins from Invitrogen (Eugene, OR, USA) in the weight ratio 1/1 / 0.1 in a final concentration of 20 μg lipid / ml medium and fluorescence-labeled channel protein (bacteriorhodopsin of Halobacterium Sainarium (Sigma Aldrich) -Tetramethylrhodamine isothiocyanate (Sigma Aldrich)) (BR-TRITC: molecule type E) 0.4 ng / ml). The mixture according to the invention was prepared in the following manner. The lipid components (type of molecule AC) were homogeneously mixed in chloroform (VWR). After mixing, the solution was dried in vacuo for 30-60 minutes. The lipid molecules were resuspended in a buffer solution of 20 mM HEPES (2- [4- (2-hydroxyethyl) -l-piperazinyl] -ethanesulfonic acid (VWR)) to a final concentration of 2 mg lipid / ml buffer and sonicated (80- 100 W) homogenized for 20 minutes. All steps were carried out at room temperature. The mixture is thus present as a liposome.

5 μl of this fusion mixture was incubated with 0.7 μl of a BR-TRITC solution (molecule type E) (0.06 mg / ml HEPES) for 60 minutes, while being gently stirred. The channel proteins E are taken up in the lipid bilayers of the liposome. The solution was diluted 100-fold with FlO Ham's medium (Sigma Aldrich) and sonicated (80-100W) once more for 1 to 2 minutes prior to addition to a cell culture dish with myofibroblasts. The cells were incubated with the fusion mixture according to the invention for 10 to 20 minutes, then washed with FlO Ham's medium and analyzed.

After washing, about 80 to 100% of the cells cytoplasmic membranes of the cells could be detected in the fluorescence microscope (green for lipids, red for proteins) (LSM 710 from Carl Zeiss Micrimaging GmbH, Jena), resulting in successful membrane incorporation of both the lipid mixture (type of molecule AC) as well as the protein molecules (type of molecule E) (Figures 3 and 4).

Path 3 from FIG. 1: cell membrane modification by mixing the varieties A-D for protein binding on the cell membrane surface, principles of a novel tumor treatment and a medicament therefor.

Human Embryonic Kidney (HEK 293) (DSMZ, Germany) cells were grown in RPMI medium (Sigma Aldrich, St. Luis, Mo., USA) at a cell density of 40,000 cells per cell culture dish (0 3.5 cm) at 37 ° C and 5 % CO ₂ incubated and used at a confluence density of about 90% in the subsequent steps. HEK293 cells were directly in a fusion mixture comprising l, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE: molecular grade C), l, 2-dioleoyl-3-trimethylammonium-propane (DOTAP: molecular grade A), (both from Avanti Polar Lipids Inc. Alabama, AL, USA), JV- (4,4-difluoro-5,7-dimethyl-4-bora- 3 a, 4a-diaza-5-indacene-3-propionyl) - 1, 2-Dihexadecanoyl-5-glycero-3-phosphoethanolamine, triethylammonium salt (Bodipy FL-DHPE: molecular grade B) (In Vitrogen, Eugene, OR, USA) and 1,2-dioleoyl-sn-glycero-3 [(N- (5-Amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) (DOGS-NTA: Molecule D Avanti Polar Lipids Inc.) in the weight ratio of DOPE / DOTAP / Bodipy FL-DHPE / DOGS-NTA (1/1 / 0.1 / 0.002 wt / wt) in a final concentration of 20 μg lipid / ml RPMI medium (Sigma Aldrich) for 10 to 20 minutes at 37 ° C and 5% CO ₂ incubated. The fusion mixture was prepared in the following manner. The lipid components (molecule types A to D) were homogeneously mixed first in chloroform (VWR, Darmstadt, Germany). Subsequently, the solution was dried in vacuo for 30 to 60 minutes. The lipid molecules were resuspended in a buffer solution of 20 mM (2- [4- (2-hydroxyethyl) -1- piperazinyl] -ethanesulfonic acid) (HEPES buffer) (VWR) in a final concentration of 2 mg lipid / ml buffer and homogenized in an ultrasonic bath (80-100 W) for 20 minutes.

The mixture is thus present as a liposome. This mixture is stable for about 1 month in the refrigerator at 4 ° C and after a repeated Homogenisierungsschritt reusable.

5 μl of this fusion mixture was diluted 100 times with RPMI medium (Sigma Aldrich) and homogenized again with ultrasound (80-100 W) for 5 to 10 minutes before addition to a cell culture dish with HEK293 cells. All preparation steps were carried out at room temperature. Subsequently, the cells were washed with RPMI medium (Sigma Aldrich) and the fusion of the reagent with the cell membrane on the fluorescence microscope (LSM 710 from Carl Zeiss Microimaging GmbH, Jena) checked.

Fig. 5 shows such a check and indicates a fusion efficiency above 80%. This efficiency was determined by counting the microscopic images.

In order to allow binding between the reactive head group of the DOGS-NTA used as molecule type D with a reaction partner, the cells were bound in a solution of Tumor Necrosis Factor-α to a όxhistidine repeat (TNFa- His-tag) (ProSpec, Rehovot , Israel) at the concentration of 1 to 5 ng / ml of RPMI medium for about 20 minutes, incubated at 37 ^° C. and 5% CO ₂ .

Residues of the protein were removed from the suspension by a three-time wash with RPMI medium. TNFα functions as a macrophage activating factor in the body and is the major factor in the immune system's recognition and control of cancer cells. To test the functionality of the system, differentiated macrophages were used. Human monocytes were obtained from blood by Leukoseptsystem (Greiner bio-one, Kremsmuenster, AU) and Biocoll separation medium (Biochrom, Cambridge, UK). After centrifugation at 1000 g, 10 minutes were monocytes harvested from the interphase formed and subsequently washed with phosphate buffer (PBS from Sigma Aldrich). Monocytes were picked up in RPMI medium (Sigma Aldrich) and incubated at 37 ° C and 5% CO ₂ . After 2 days, 0.1 ng / ml granulocyte macrophage colony stimulation factor (G-MCF from Sigma Aldrich) was added to the monocytes. After another 3 days, inactive macrophages differentiated from monocytes.

The macrophages thus obtained were then plated out on a HEK 293 cell lawn, which should carry the recognition signal for macrophages by means of the fusion and subsequent incubation with TNFα-His-tag. Upon detection, the cellular immune response in macrophages should be induced and HEK 293 cells lysed. As a control, HEK cells were treated identically, except that the amphipathic molecule with reactive head group of molecule type D (DOGS-NTA) was released from the fusion mixture. Both batches were incubated for 24 hours and subsequently analyzed by light microscopy (LSM 710 from Carl Zeiss Microimaging GmbH, Jena). Figure 6 shows the detection and lysis of HEK293 cells (formation of plaques) only on the cells incubated with DOGS-NTA (molecule type D) in the fusion mixture. This example shows that by means of membrane fusion targeted cell types can be marked and made visible to the immune system by way of example.

Another exemplary embodiment of cell membrane modification by mixing varieties A-D for protein binding on the cell membrane surface by biotin-avidin / streptavidin binding:

Cardiac fibroblasts were isolated from embryonic (day 19) rat hearts and in FlO urinary medium (Sigma Aldrich, St. Luis, MO, USA) at a density of 20,000 cells per cell culture dish (0 3.5 cm) at 37 ° C and 5% CO ₂ incubated. Over a period of 6 days, the cardiac fibroblasts differentiated into myofibroblasts and were used in the subsequent method according to the invention.

The myofibroblasts were directly incubated in a fusion mixture comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE: molecular grade C), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP: molecular grade A), both by Avanti Polar Lipids Inc. Alabama, AL, USA), Texas Red l, -diihexadecanoyl-n-glycero-S-phosphoethanolamine (triethylammonium salt) (Texas Red DHPE: Molecule B) (Invitrogen, Eugene, OR, USA) and 1,2-dioleoyl-s.srt Glycero-3-phosphoethanolamine-N-cap-biotin (capBioDOPE: molecule type D Avanti Polar Lipids Inc.) in the weight ratio of DOPE / DOTAP / Texas Red DHPE / capBioDOPE (1/1 / 0.1 / 0.1 wt / wt ) in a final concentration of 20 μg lipid / ml FlO urinary medium medium (Sigma Aldrich) for 10 to 20 minutes at 37 ° C and 5% CO ₂ .

The fusion mixture was prepared in the following manner. The lipid components (molecule types A to D) were first homogeneously mixed in chloroform (VWR, Darmstadt, Germany). Subsequently, the solution was dried in vacuo for 30 to 60 minutes. The lipid molecules were resuspended in a buffer solution of 20 mM (2- [4- (2-hydroxyethyl) -l-piperazinyl-1-ethanesulfonic acid) (HEPES buffer) (VWR) to a final concentration of 2 mg lipid / ml buffer and sonicated (80-100 W) homogenized for 20 minutes.

The mixture is thus present as a liposome. This mixture is stable for about 1 month in the refrigerator at 4 ° C and reusable after a repeated homogenization step.

5 μl of this fusion mixture was diluted 100 times with FlO Ham's medium medium (Sigma Aldrich) and homogenized again for 5 to 10 minutes by means of ultrasound (80-100 W) before addition to a cell culture dish with myofibroblasts. All preparation steps were carried out at room temperature.

Subsequently, the cells were washed with FlO Ham's medium medium (Sigma Aldrich) and the fusion of the reagent with the cell membrane on the fluorescence microscope (LSM 710 from Carl Zeiss Microimaging GmbH, Jena) checked.

Fig. 7 shows such a check and indicates a fusion efficiency above 80%. This efficiency was determined by counting with the help of microscopic images. In order to allow binding between the reactive head group of capBioDOPE used as molecule type D with a reaction partner, the cells in a solution of streptavidin / streptavidin-AlexaFluoro488 (both from Invitrogen, Eugene, OR, USA) 100/1 mol / mol% in the total protein concentration of 1 mg / ml FlO urinary medium for about 20 minutes, incubated at 37 ° C and 5% CO ₂ .

Residues of the protein were removed from the suspension by a three-time wash with FlO Ham's medium.

Figure 7 shows a clear colocalization of phospholipid (red) and cell surface bound proteins (green). Another embodiment for cell membrane modification by mixing the grades A-D to modify the cell membrane composition:

Cardiac fibroblasts were isolated from embryonic (day 19) rat hearts and resuspended in FlO Ham's medium (Sigma Aldrich, St. Luis, Mo., USA) at a density of 20,000 cells per cell culture dish (0-3.5 cm) at 37 ° C and 5%. CO ₂ incubated. Over a period of 6 days, the cardiac fibroblasts differentiated into myofibroblasts and were used in the subsequent method according to the invention.

The myofibroblasts were directly incubated in a fusion mixture comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE: molecular grade C), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP: molecular grade A), both by Avanti Polar Lipids Inc. Alabama, AL), Texas Red 1, 2-dihexadecanoyl-5'n-glycero-3-phosphoethanolamine (triethylammonium salt) (TexasRed-DHPE: molecular grade B) and N- (4, 4-difluoro-5,7-dimethyl-4-bora- 3a, 4a-diaza-s-indacene-3-dodecanoyl) sphingosyl phosphocholines (B FL-SM: molecular type D) (both from Invitrogen, Eugene, OR, USA) in the weight ratio of DOPE / DOTAP / Texas Red-DHPE / BFL-SM (1/1 / 0.1 / 0.01 wt / wt) in a final concentration of 20 μg lipid / ml FlO Ham's medium medium (Sigma Aldrich) for 10 minutes incubated at 37 ° C and 5% CO ₂ . The fusion mixture was prepared in the following manner. The lipid components (molecule types A to D) were homogeneously mixed first in chloroform (VWR, Darmstadt, Germany). Subsequently, the solution was dried in vacuo for 30 to 60 minutes. The lipid molecules were resuspended in a buffer solution of 20 mM (2- [4- (2-hydroxyethyl) -l-piperazinyl] ethanesulfonic acid) (HEPES buffer) (VWR) to a final concentration of 2 mg lipid / ml buffer and incubated in Ultrasonic bath (80-100 W) homogenized for 20 minutes.

The mixture is thus present as a liposome. This mixture is stable for about 1 month in the refrigerator at 4 ° C and reusable after a repeated Homogenisierungsschritt. 5 μl of this fusion mixture was diluted 100 times with FlO Ham's medium medium (Sigma Aldrich) and homogenized again for 5 to 10 minutes by means of ultrasound (80-100 W) before addition to a cell culture dish with myofibroblasts. All preparation steps were carried out at room temperature.

Subsequently, the cells were washed with FlO Ham's medium medium (Sigma Aldrich) and the fusion of the reagent with the cell membrane was checked on the fluorescence microscope (LSM 710 from Carl Zeiss Microimaging GmbH, Jena).

Fig. 8 shows such a check in the context of the embodiment and the incorporation of the phospholipid molecules (Texas Red-DHPE) and the sphingolipids (BFL-SM) in the plasma membrane of a myofibroblast. Route 4: Cell membrane modification by mixing varieties A-C on tissue samples:

Not only individual mammalian cells but also cells in the tissue and in tissue sections can be brought into fusion with the fusion mixture (see FIG. 9). Primary pericardium from embryonic rats (day 19) was isolated and transplanted into FlO Ham's medium (Sigma Aldrich, St. Luis, Mo., USA) on a 3.5 cm cell culture dish with a fusion mixture of 1,2-dioleoyl-sn Glycero-3-phosphoethanolamine (DOPE: molecular grade C), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP: molecular grade A), (both from Avanti Polar Lipids Inc. Alabama, AL, USA) and N- (4 , 4-Difluoro-5,7-dimethyl-4-boron-Sa-a-diaza-s-indacene-S-propionyl-1-dihexadecanoyl-.src-glycero-S- Phosphoethanolamine, triethylammonium salt (Bodipy FL-DHPE: molecule type B) (In Vitrogen, Eugene, OR, USA) in a weight ratio of 1/1 / 0.1 wt / wt, incubated at 37 ° C and 5% CO2 for 1 hour. (Molecule C and B were obtained from Avanti Polar Lipids Inc., Alabama, AL, USA; B, type molecule from Invitrogen, Eugen, OR, USA). The fusion mixture was prepared in the following way:

The lipid components (molecule type A to C) were homogeneously mixed first in chloroform (VWR, Darmstadt, Germany). Subsequently, the solution was dried in vacuo for 30 to 60 minutes. The lipid molecules were resuspended in a buffer solution of 20 mM (2- [4- (2-hydroxyethyl) -l-piperazinyl] ethanesulfonic acid) (HEPES buffer) (VWR, Darmstadt, Germany) to a final concentration of 2 mg lipid / ml Buffer recorded and homogenized in an ultrasonic bath (80-100 W) for 20 minutes. The mixture is again present as a liposome stable at 4 ° C.

5 μl of this fusion mixture was diluted 100 times with FlO Ham's medium (Sigma Aldrich) and again homogenized for 5-10 minutes by means of ultrasound (80-100 W) before addition to the tissue sample. All preparation steps were carried out at room temperature.

Subsequently, the fused tissue was analyzed by fluorescence microscopy (LSM 710 from Carl Zeiss Microimaging GmbH, Jena). Figure 9 shows that both cells of the outer tissue layer and cells in deeper cell planes were successfully fused. About 3-4 cell layers of connective tissue cells are clearly visible. Eventual use in gene therapy, cancer treatment and wound healing is conceivable.

Other embodiments can be specified. These are shown in Table 2. The processes for the preparation of the mixtures are to be taken from the four example protocols given here. The cell membrane fusion follows those of the above-mentioned methods. Within the meaning of the invention, all method steps in the exemplary embodiments are to be regarded as of a non-restrictive nature. In particular, the fusion mixtures and their use should be widely interpreted. This means that the paths and fusion mixtures shown in exemplary embodiments can be combined with one another in order to arrive at new mixtures and designs. The presently described in detail four ways are based on liposomes, since after chloroform addition and the inclusion in buffer, the amphiphatic components to form liposomes.

In the patent application, reference is made to colored figures 1 to 9 which accompany this patent application. Reproductions of these figures in black and white are attached to the patent application on the basis of the disclosure rules as Figures 10-18. 11 corresponds to FIG. 2. FIG. 12 corresponds to FIG. 3. FIG. 13 corresponds to FIG. 4. FIG. 14 corresponds to FIG. 5. FIG. 15 corresponds to FIG. FIG. 16 corresponds to FIG. 7. FIG. 17 corresponds to FIG. 8. FIG. 18 corresponds to FIG. 9.

Claims

Patent claims

1. A mixture of amphipathic molecules having an amphipathic type of molecule A, which has a positive total charge in the hydrophilic region, and an amphipathic type of molecule B, which forms a delocalized electron system in the hydrophilic and / or hydrophobic region.

2. Mixture according to one of the preceding claims,

marked by

a type of molecule B with a delocalized electron system comprising at least one cyclic structural motif, which are formed by conjugated double bonds and / or lone pairs of electrons and / or unoccupied p orbitals.

3. Mixture according to one of the preceding claims,

characterized in that

covalently bonded neighboring atoms of the molecule type B in the delocalized electron system have an electronegativity difference (Δχ) of greater than or equal to 0.4.

4. Mixture according to one of the preceding claims,

marked by

a type of molecule B comprising a group R which delocalized

Electron system trains.

5. Mixture according to one of the preceding claims,

marked by

l, 2-Dioleoyl-3-trimethylammonium propane (DOTAP) as molecule type A.

6. Mixture according to one of the preceding claims,

marked by

a phospholipid with a delocalized fluorescent dye

Electron system is bound as a group R, as molecule type B.

7. Mixture according to the preceding claim,

marked by

1 -hexadecanoyl-2-dodecanoyl-5-glycero-3-phosphocholine or 1,2-dihexadecanoyl-5-glycero-3-phosphoethanolamine, or 1,2-dioleoyl-5-glycero-3-phosphoethanolamine as phosholipid the molecule type B.

8. Mixture according to one of the preceding claims,

marked by

2- (4,4-difluoro-5-methyl-4-boro-3a, 4a-diaza-5'-indacene-3-dodecanoyl) or N- (4,4-difluoro-5,7-dimethyl-4- Bora-3a, 4a-diaza-s-indacene-3-propionyl) or Lissamine rhodamine B sulfonyl or (5- (chlorosulfonyl) -2- (IH, 2H, 3H, 5H, 6H, 7H, 1HH,

12H, 13H, 15H, 16H, 17H-pyrido [3, 2, 1-ij] quinolizino [1 ', 9': 6, 7, 8] chromeno [2, 3-f] quinolin-4-ium-9- yl) benzenesulfonate or 7-nitro-2-l, 3-benzoxadiazol-4-yl or carboxyfluorescein or 1-pyrensulfonyl, which forms the delocalized electron system in the molecule type B.

9. Mixture according to one of the preceding claims,

marked by

another amphipathic type of molecule C as a helper molecule.

10. Mixture according to one of the preceding claims,

marked by

1, 2-dioloylo-an-Glycolo-S-phosphoethanolamine (DOPE) as molecule type C.

11. Mixture according to one of the preceding claims,

marked by

a ratio of 1: 0.05-0.2: 1 wt / wt between the molecule types A, B and C.

12. Mixture according to one of the preceding claims,

up to

99% wt amphipathic type of molecule A.

13. Mixture according to one of the preceding claims,

up to

40% wt amphipathic type of molecule B.

14. Mixture according to one of the preceding claims,

up to

70% wt amphipathic type C.

15. Mixture according to one of the preceding claims,

characterized in that

this is dissolved in an aqueous buffer.

16. Mixture according to one of the preceding claims,

marked by

Lipids as molecule type A and / or B and / or C.

17. Liposome comprising

a mixture according to the preceding claims 15 and 16.

18. Mixture according to one of the preceding claims,

marked by

Polymers as molecule type A and / or B and / or C.

19. Method for in vivo cell modification,

marked by

contacting and fusion of the cell membrane with a mixture or a liposome according to any one of the preceding claims.

20. Method according to the preceding claim,

marked by

no more than 1 to 120 minutes, preferably 10 to 30 minutes, of contact between the cell membrane of a cell and the mixture during fusion.

21. The method according to claims 19 to 20,

marked by

a fraction of fused cells with the mixture or liposome (efficiency) of over 50%.

22. The method according to any one of the preceding claims 19 to 21,

characterized by

a functional group in the hydrophilic region of an amphipathic molecule type D, in particular a chelate group as functional group, is added to the mixture and placed on the surface of the cell during the fusion.

23. Method according to the previous claim,

characterized by

a mixture comprising 1,2-dioleoyl-3-trimethylammonium propane as the molecule type A and N- (4,4-difluoro-5,7-dimethyl-4-borora-3a, 4a-diaza-s-indacene-3 Propionyl) -l, 2-dihexadecanoyl-5'-glycero-3-phosphoethanolamine as type of molecule B and triethylammonium salt / 1, 2-dioleoyl-srt-glycero-3-phosphoethanolamine as molecule type C and l, 2-dioleoyl-5 - glycero-3 - [(N- (5-amino-l-

Carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) as molecule type D z. B. in a mixing ratio of 1: 0, 1: 1: 0.002 wt / wt is used for fusion.

24. Method according to the preceding claim 23,

characterized in that

a further cell modification by adding Rumor necrosis factor (TNF) α, bound to a όxHistidin repeat occurs.

25. The method according to any one of the preceding claims 19 to 24,

characterized in that

an amphipathic type of molecule E is added to the mixture and placed during the fusion in the membrane of the cell, in particular bacteriorhodopsin or glycophorin A or integrin as molecule type E.

26. The method according to any one of the preceding claims 19 to 25,

characterized in that

a non-amphipathic hydrophilic type of molecule F is added to a dried mixture in a buffered solution and released into the lumen of the cell during the fusion, in particular with RNA, DNA, Ca ²⁺ , peptide, protein or a drug such as acetylsalicylic acid as molecule type F ,

27. The method according to any one of the preceding claims 19 to 26,

characterized in that

a non-amphipathic, hydrophobic molecule G is added to the mixture and placed during the fusion in the membrane of the cell, in particular cholesterol, vitamin A, D, E and K or drugs such as cortisone as molecule type G.

28. Mixture according to one of the preceding claims 1 to 18,

marked by

1, 2-Dioleoyl-3-trimethyl-ammonium-propane as molecule type A and iV- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-5-indacene-3-propionyl) - 1 , 2-Dihexadecanoyl-s "-

Glycero-3-phosphoethanolamine as type of molecule B and triethylammonium salt / 1,2-dioleoyl-SH-glycero-3-phosphoethanolamine as molecule type C and 1,2-dioleoyl-5Η-glycero-3 - [(N- (5-amino-) 1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt) as molecule type D, in particular in a mixing ratio of 1: 0.1: 1: 0.002 wt / wt.

29 cell fused in vivo with a mixture or a liposome according to any one of the preceding claims.