RU2482837C2 - Improved liposomes and their application - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of molecular medicine and pharmacology and describes liposome, which contains, at last, one lipid bi-layer surrounding water compartment, in which said lipid bi-layer contains, at least, one amphiphilic synthetic pyridinium derivative, in which total amount of amphiphilic pyridinium derivative constitutes from 2 to 25 mol % of total content of lipids in liposome. Liposomes are suitable for delivery of medicinal compounds.

EFFECT: liposome is stable and makes it possible to improve intracellular release of medication.

18 cl, 1 ex, 9 dwg

Description

The invention relates to the field of molecular medicine and pharmacology. More specifically, it relates to liposomes and their use as a delivery vehicle for drug compounds. Also provided is a method for producing liposomes.

The therapeutic benefits of many compounds are limited by the low absorption of the compound by target cells. In general, for the maximum therapeutic effect, delivery of the compound to the cytoplasmic or nuclear compartment of a cell in which gene transcription and translation of mRNA into protein takes place is necessary. Lipophilic compounds, including many small, uncharged compounds, can penetrate the cell membrane and enable relatively efficient uptake by the cell. However, for various larger and / or charged compounds, such as proteins, nucleic acids and water-soluble charged organic compounds, passive absorption by penetration through the cell membrane is limited.

Several methods have been proposed for improving the uptake of these compounds by cells. For example, a drug can be administered in a modified form or in the form of a prodrug to be transported into cells and then enzymatically converted to an active form within the cell. However, it will be clear that obtaining a suitable form of prodrug is not possible for all drug compounds without exception.

Alternatively, cell processes of phagocytosis or endocytosis can be used in which particles containing the drug are captured by target cells. In the case of the delivery of particles of most modern marketed drugs, this approach, however, is limited to several types of cells, for example, phagocytosis is limited in cells of monocytic origin and in some other myeloid cells, such as neutrophils, while endocytosis is limited in several other cells, among other cells of mesenchymal origin, such as endothelial cells, epithelial cells and fibroblasts. Therefore, unlike macrophages and other cells that specialize in removing and processing particles from the blood, there are many cells that are not or little adapted to the processing of particles containing a drug substance.

Another approach to improving drug uptake by cells involves the use of fusogenic particles designed to fuse with the membrane surface of the target cell, releasing the contents of the particles into the cytoplasmic compartment of the cell. For this purpose, inactivated and reconstructed viral particles are proposed, in particular in gene therapy, when large chains of nucleic acids are introduced into the cells. Virus-like particles are composed of fusion-promoting viral proteins embedded in an artificial lipid double layer, representing another example. However, the safety and cost problems associated with the cultivation, isolation and deactivation of viral components limit the widespread use of these approaches.

Endothelial cells that line the vascular wall of all blood vessels are examples of cells that interfere with the movement of particles containing the drug, for example, drug-loaded liposomes. Endothelial cells play a major role in the homeostasis of the whole organism and the pathology (physiology) of important diseases, including (chronic) inflammation and cancer. They are also attractive target cells / target tissues for drug delivery, as they are fully accessible to any blood-borne compound. In addition, endothelial heterogeneity in terms of type and function allows drug delivery to a particular tissue and in a particular disease. Endothelial cells are a promising target for cancer treatment, since angiogenesis is vital for supplying solid tumors with oxygen and nutrients. In addition, in inflammatory diseases, they direct the movement of leukocytes from the blood into tissues, and thus are necessary for the development of the disease. The authors of the present invention experimentally found that liposomes targeting E-selectin or VCAM-1 expressed on TNFα-activated endothelial cells easily undergo endocytosis in amounts that are comparable to macrophage endocytosis ability. However, unlike macrophages, endothelial cells do not intensively process or destroy liposomes and, thus, accumulate a drug-loaded carrier inside the vesicles. The lack of destruction of liposomes and / or destabilization causes the preservation of the encapsulated drug in the liposome, respectively, resulting in low pharmacological efficacy.

Therefore, it was an object of the present invention to provide a lipid-based drug delivery vehicle that improves the intracellular release of the drug in the target cells compared to known delivery vehicles. Another objective is to provide a lipid-based drug delivery vehicle for efficient intracellular drug release in target cells that are not specialized in the removal and / or processing of lipid-based particles such as endothelial and epithelial cells, muscle cells, brain cells, nerve cells cells, skin cells, hair cells, subpopulations of undifferentiated progenitor cells and pericytes, and all other cells not specialized in the processing of carriers for delivery.

Using a method that distinguishes between a) liposome uptake by a target cell and b) drug release from a liposome in a target cell, the inventors of the present invention unexpectedly found that incorporating an artificial amphiphilic pyridinium derivative into a bilayer of “classical” or “traditional” liposome ( for example, an aqueous compartment surrounded by a lipid bilayer) provides a lipid-based delivery vehicle that provides efficient intracellular drug delivery in cells that cat rye do not specialize in the processing of particles, based on lipid. Only liposomes comprising a bilayer containing one or more artificial amphiphilic pyridinium derivatives in an amount of from 2 to 25% mol, based on the total lipid content, show sufficient stability. Accordingly, the inventors provide a liposome containing at least one lipid bilayer, comprising an aqueous internal compartment, wherein said lipid bilayer contains at least one artificial amphiphilic pyridinium derivative in an amount of from 2 to 25% mol, based on the total lipid content in the liposome.

The term "liposome", as known in the art, refers to aqueous compartments surrounded by a phospholipid bilayer membrane. Molecules of phospholipids consist of an elongated non-polar (hydrophobic) structure with a polar (hydrophilic) structure at one end. When dispersed in water, they spontaneously form a two-layer membrane, also called lamella, which consists of two single-layer layers of lipid molecules with non-polar (hydrophobic) surfaces facing each other and polar (hydrophilic) surfaces facing the aqueous medium.

An amphiphilic substance is a compound consisting of molecules having a polar water-soluble group attached to a water-insoluble hydrocarbon chain. Pyridinium refers to the cationogenic form of pyridine. This form may result from protonation of the nitrogen ring or the addition of a substituent to the nitrogen ring, usually by alkylation. The lone pair of electrons on the nitrogen atom of pyridine is not delocalized, and thus, pyridine can be easily protonated. The expression “pyridinium derivative”, as used herein, refers to any amphiphilic substance having a pyridinium molecule in its polar group.

Artificial amphiphilic pyridinium derivatives are known in the art. Of particular interest for use in the present invention is a group of artificial amphiphilic pyridinium derivatives provided by Synvolux, SAINT (Synthetic, Amphiphilic, INTeractive) technology. See, for example, European Patent EPO 755924-B1. In one embodiment of the present invention, at least one artificial amphiphilic pyridinium derivative is selected from the group of SAINT compounds with the general formula (I):

wherein

R ₁ represents (C ₁ -C ₅ ) alkyl, ap (alkyl) or an alkyl group with a cationogenic functional group, such as

or R ₁ represents (C ₁ -C ₅ alkylene) R ₅ in which R ₅ represents a structure with the general formula I, excluding R ₁ ;

X is a halide ion selected from Cl ^- , I ^- , Br ^- ;

R ₃ is hydrogen and R ₂ and R _{4 are} identical or different, and are selected from the group consisting of branched or linear (C ₁₀ -C ₂₀ ) alkyl, mono- or polyunsaturated (C ₁₀ -C ₂₀ ) alkenyl, O = C- O-alkyl

алкил

alkyl

or ar (alkyl),

or R ₂ and R ₄ are hydrogen and R ₃ is —CH (R ₅ ) ₂ with R ₅ containing (C ₁₀ -C ₂₀ ) alkyl, mono- or polyunsaturated (C ₁₀ -C ₂₀ ) alkenyl, O = C-O-alkyl

алкил

alkyl

or aralkyl.

In an additional embodiment, the composition includes a SAINT molecule, as indicated above, which does not include compounds of general formula I, in which R ₁ represents CH ₃ , R ₂ and R ₄ represent hydrogen, R ₃ represents (C ₁₆ H ₃₃ ) ₂ CH and X are all designated counterions and do not include compounds in which R ₁ is CH ₃ , R ₂ and R ₄ are C ₁₆ H ₃₃ —O — C (O), R ₃ is hydrogen and X represents all designated counterions.

The liposomes of the present invention containing the SAINT molecule are also referred to as “SAINT-o-soms”. Particular embodiments of the present invention use SAINT-18 (1-methyl-4- (cis-9-dioleyl) methylpyridinium chloride) and / or SAINT-12 (1-methyl-4- (pentacosan-13-yl) pyridinium chloride).

The incorporation of artificial amphiphilic pyridinium derivatives, such as SAINT, into traditional liposomes has not been described or is proposed in the art. However, SAINT molecules have so far been used in non-liposomal delivery vehicles (called "lipoplexes") to deliver macromolecules such as nucleic acids or proteins to mammalian cells. EP-A-0755924, for example, discloses a combination of a macromolecule with SAINT, optionally in a 1: 1 mixture with an auxiliary lipid, to form a lipoplex particle up to several microns in diameter. In the lipoplex, only non-covalent interactions are formed, represented between SAINT and the macromolecule. Cationic amphiphilic substances on the particle surface have a high affinity for a negatively charged cell surface. Since the introductory part of EP-A-0755924 refers, in brief, to “liposomes that consist of a double layer of phospholipids”, it must be emphasized that the invention disclosed in EP-A-0755924 does not exactly apply to these liposomes. Rather, it refers to lipoplexes, the structural organization of which has been studied in detail by Oberle et al., Using atomic amplification microscopy (2000, Biophysical Journal 79 (3), 1447-1454). Fig. 7 Oberle et al. illustrates that lipoplexes containing SAINT, disclosed, for example, in EP-A-0755924, consist of DNA wrapped in several layers of amphiphilic substances that are connected in series into a large complex. It is noteworthy that some DNA "stick out" of the lipoplex and are not protected from the environment, as will be the case when a liposome containing SAINT is included in the internal compartment, as disclosed in the present invention. Rejman et al. (Biochimica et Biophysica Acte 1660 (2004) 41-52), Zuhorn et al. (Biochimica et Biophysica Acta 1560 (2002) 25-36), Van de Woude et al. (Proc. Natl. Acad. Sci. USA, Volume 94, pp. 1160-1165, 1997) and WO2006 / 043809 also disclose lipoplexes rather than liposomes containing an aqueous internal compartment.

In contrast to the foregoing, the present invention relates to liposomes containing an aqueous interior surrounded by a double layer of lipids, which include one or more artificial amphiphilic pyridinium derivatives. Without being limited by any theory, the cationogenic pyridinium group of the amphiphilic substance makes the lipid bilayer of the liposome less rigid and more likely to mix (e.g., fuse) with intracellular, endosomal membrane lipids, thereby promoting intracellular release of the encapsulated compound. As demonstrated further in the present description, this effect is not observed for any cationogenic amphiphilic substance, since liposomes with the same amount of DOTAP (propane N- [1- (2,3-dioleoyloxy)] - N, N, N-trimethylammonium ), a well-studied cationic liposome transfer reagent, exhibit low efficacy.

The liposomes of the invention have one or more amphiphilic pyridinium derivatives. The exact composition of the liposomes will depend on the individual cases for which they are used. One of ordinary skill in the art will routinely determine the appropriate lipid composition. General information on liposomes can, for example, be found in Lasic, D.D. “Liposomes: from physics to application”, Elsevier Science B.V. Amsterdam, 1993 (ISBN 0444895485).

The relative amount of the amphiphilic pyridinium derivative in the liposome according to the invention may vary, but is usually up to about 5-25% mol, based on the total amount of other lipid components in the liposome. This circumstance ensures that the liposome is stable outside the target cell (for example, in the blood stream), while at the same time it allows efficient intracellular release of their encapsulated contents by mixing or draining with the membrane of the target cell. A very good result is obtained if the liposomes contain 5-20% mol, preferably 10-20% mol, for example 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% mol, of the amphiphilic pyridinium derivative relative to other used lipids. Particular embodiments include liposomes containing 10-20% mol of SAINT molecules, for example 10, 11, 12, 14, 15, 17, 19 or 20% mol of SAINT-18 or SAINT-12.

In one embodiment, the liposomes of the present invention consist mainly of one type of lipid in addition to at least one amphiphilic pyridinium derivative. In a preferred embodiment, the liposomes contain a mixture of two or more lipids, preferably selected from the group consisting of glycerophospholipids, sphingolipids and cholesterol. Natural and / or synthetic lipids may be used. Essentially, any type of lipid forming a liposome or molecule can be used in combination with at least one amphiphilic pyridinium derivative to produce the liposome of the invention. The lipid constituting the liposomes of the present invention includes phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, gangliosides, glycolipids, phosphatidylglycerols and cholesterol. Phosphatidylcholines preferably include dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine (POPC) and distearoylphosphatidylcholine. Phosphatidylethanolamines preferably include dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylethanolamine. Phosphatidic acids preferably include dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid and dicetylphosphoric acid. Gangliosides preferably include ganglioside GM1, ganglioside GD1a and ganglioside GT1b. Glycolipids preferably include galactosylceramide, glucosylceramide, lactosylceramide, phosphatide and globoside. Phosphatidylglycerols preferably include dimyristoylphosphatidylglycerol, di palmitoylphosphatidylglycerol and distearoylphosphatidylglycerol.

In vivo studies have shown that conventional liposomes are rapidly removed from the circulation by the cells of the reticuloendothelial system, i.e. phagocytes constantly present in tissues, present in a number of organs, in particular in the liver and spleen. This interaction is reduced when sterically stable liposomes are used, which can be obtained by incorporating monosialoganglioside lipid derivatives (GM1) or poly (ethylene glycol) (PEG) into the lipid bilayer of liposomes. These liposomes coated with inert polymers show a significant increase in the half-life in the bloodstream (in humans up to the order of days, as opposed to minutes for conventional liposomes).

Sterically stable immunoliposomes with specific cellular recognition properties are often obtained by combining antibodies with the distal ends of PEG chains. Using the PEG chain as a linker between the liposome and the antibody leads to improved recognition of antigen by the antibody and binding, since the antibody is not protected by the steric barrier activity of PEG. Several covalent binding methods have been improved for attaching (derivatives) of antibodies to the end of PEG. They use functionalized PEG lipids with a chemically active terminal group such as hydrazide, N- (3 '- (pyridyldithio) propionate, maleimide, succinyl, para-nitrophenylcarbonyl or cyanuric chloride. Therefore, in one embodiment of the invention, a derivative (eg, PEGylated a) the phospholipid can be used in vivo to increase the circulation time of the liposome composition, and / or allow the protein to bind to the surface of the liposome. In a particular aspect, the liposome of the present invention contains a phosphorus mixture thidylcholine and cholesterol in relative amounts from 1: 1 to 2: 1 (% mol), optionally in admixture with one or more natural or artificial lipids, for example, PEGylated phospholipid.

The liposome itself can be obtained by any conventional method, including a thin-film method, a phase reversal evaporation method, an ethanol injection method, and a dehydration / rehydration method. Accordingly, the invention provides a method for producing a liposome according to the invention, comprising mixing conventional lipids with a suitable amount of at least one synthetic amphiphilic pyridinium derivative, and preparing the liposome according to conventional methods. Also provided are liposomes that can be obtained by the method of the invention.

For example, a mixture of the above lipids, from which solvents have been removed, can be emulsified using a homogenizer, lyophilized and melted to form multilayer liposomes. Alternatively, single-layer liposomes can be obtained by a phase reversal evaporation method (Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA 75: 4194-4198). Single-layer vesicles can also be obtained by sonication or extrusion. Ultrasonic treatment is generally performed with a bath type ultrasonic disintegrator or a Branson ultrasonic disintegrator with a tip at a controlled temperature, as determined by the melting point of the lipid.

Following the preparation of liposomes, liposomes that were not sized to the desired size during formation can be adjusted to the desired size and relatively limited distribution of liposome size by extrusion. The particle size of liposomes can be controlled by ultrasonic radiation, extrusion, French press, homogenization, or any other conventional method.

Extrusion may be performed by a biomembrane extruder such as a Lipex Biomembrane Extruder (Northern Lipids Inc., Vancouver, BC, Canada). A certain pore size in extrusion filters can lead to the formation of single-layer liposomal vesicles of a certain size. A size range of approximately 200-400 nm will allow sterilization of the liposome suspension by filtration through a conventional filter (e.g., 0.22 micron filter). The filter sterilization method can be performed with high performance. Liposomes can also be obtained by extrusion through an asymmetric ceramic filter, such as a Ceraflow Microfilter (sold from Norton Company, Worcester, Mass.).

For therapeutic use, it is preferred that the liposome of the invention has a size (i.e., diameter) of up to about 300 nm. In one embodiment, the liposome size range is from about 20 to 250 nm, preferably 30-200 nm, such as 50, 60, 75, 100, 120, 135, 150, or 180 nm. A wide range of liposomes (including oligo or multilayer vesicles) can be used in the invention, but small unilamellar liposomes (SUVs) having an outer diameter of from about 30 to about 300 nanometers (nm), most preferably from 50 to 150 nm, are preferred.

The liposome according to the invention may contain one or more other suitable components, for example, means for targeting the liposome to a specific cell. Site-specific drug delivery to diseased cells can lead to an increase in therapeutic effects and a significant reduction in toxicity. Targeting a drug with antibody conjugated liposomes, or immunoliposomes, is a technology that is used to target the drug to specific sites, such as the brain, lungs, cancer cells, HIV-infected cells, or cells of the immune system. Liposomal targeting agents, or targeting ligands, are known in the art and include proteins, peptides, such as antibodies or fragments thereof, having specificity for the surface antigen of a single target cell or for a single intracellular compartment, such as mitochondria. Liposomes, on the entire surface of which antibodies against the target cell are represented, are immunoliposomes in the art. Site-specific targeting is often mediated by a high affinity for binding of monoclonal antibodies to their specific antigens. US patent No. 5258499 discloses compositions of delivery vehicles containing active substances encapsulated in liposomal vesicles to which protein hormones (ligands), such as interlekins-2, are attached. Ligands capable of exhibiting affinity for specific cellular receptors provide delivery of the encapsulated active agent to the target cells, making it possible to deliver active drug substances to a specific cell population in the treatment of conditions such as immune system disorders. In a preferred embodiment, the liposome carries a targeting molecule, for example, a ligand endocytosed by the target cell. In yet another embodiment, the targeting molecule is a ligand that specifically interacts with a tyrosine kinase receptor, such as, for example, EGFR, HER2, HER3, HER4, PD-GFR, VEGFR, bFGFR or IGFR receptors. In yet another embodiment, the targeting molecule specifically interacts with a growth factor receptor, an angiogenesis factor receptor, a transferrin receptor, a phagocytic receptor, a cell adhesion molecule, or a vitamin receptor. Exemplary cell-specific ligands include RGD peptide, NGR peptide, ATWLPPR peptide, APRPG peptide, SMSIARL peptide, TAASGVRSMH peptide, LTLRWVGLMS peptide, CDSDSDITWDQLWDLMK peptide, GPLPLR peptide, H peptide, V peptide, and monoclonal antibodies, bispecific antibodies and single chain fragments against, for example, E-selectin, VCAM-1, endoglin, MHCII, VEGF complex: VEGFR, α _v β ₃ , moc-31, cd-90 and other target cellular epitopes. In addition, a receptor-specific ligand such as transferrin, apolipoprotein E, lactoferrin, modified albumin, etc.

In one embodiment, the liposome contains at least one targeting ligand that is linked via a linker molecule to a SAINT molecule included in the lipid bilayer of the liposomes. See, for example, SAINT-linker-ligand molecules WO 2006/043811. Preferably, the liposome of the invention comprises an unmodified (eg, no targeting agent) SAINT molecule, as well as a SAINT molecule to which at least one cell or organ-specific ligand is attached via a linker.

Due to steric mismatch, the relative amount of modified SAINT molecules relative to unmodified SAINT molecules is preferably less than 5%, more preferably less than 1%, such as about 0.1%. For example, liposomes containing as components of a bilayer 35.8% mol of POPC, 40% mol of cholesterol, 4% mol of PEG-DSPE, 20% mol of SAINT C18 and 0.2% mol of SAINT C18 linker were obtained.

As indicated above, the liposome according to the invention includes an internal space that can be used to deliver one or more molecules of interest, for example, a biologically active compound, to the target cell. For the purposes of the present invention, the term "biologically active compound" is intended to mean all naturally occurring or artificial substances that can cause a biological reaction or have an effect, or beneficial or damaging, including cytotoxic, on biological systems, in particular on tissues, cells and cellular organelles . The compounds listed are intended to include all kinds of drugs, including antibiotics, antibacterial, antiviral, antifungal, anti-inflammatory, antiproliferative and antitumor drugs, and any intracellular signal transmission inhibitor designed as a therapeutic agent, such as a MAPK or SAPK inhibitor; hormones, including peptide hormones and steroid hormones; genes, recombinant nucleic acids, oligonucleotides or other nucleic acids encoding all or part of the mammalian genes, including specialized small interfering RNA (also known as short interfering RNA or siRNA) and specially designed short RNA molecules that can effectively suppress the action of microRNAs in the regulation of expression genes such as antagomirs, viral genes or genes from microorganisms; antigens; enzymes; nutrients; and in the most special way, any biologically active compound that is unable to be absorbed by passive penetration through the cell membrane of the target cell of interest. In one aspect, the liposome contains in the internal compartment a biologically active compound having a neutral or positive net charge, for example, a protein substance.

An additional aspect of the present invention relates to compositions containing the liposome of the invention. In general, the liposome composition of the present invention is sufficiently stable during storage, as measured, for example, by determining the percentage of trapped material outside the liposomes or the substance still stored inside the liposomes from the initially loaded substance inside the liposomes of the present invention after a period of time. For example, the liposome composition according to the present invention is stable at 4 ° C for at least 6 months, less than 10% of the captured substance is released 6 months after the initial loading of the substance. In one embodiment of the invention, the liposome composition according to the present invention is stable at 4 ° C for at least 2 years, less than 20% of the captured substance is released 2 years after the initial loading of the substance.

In accordance with another embodiment of the present invention, the liposome composition of the present invention can be provided in the form of a pharmaceutical composition comprising a liposome composition according to the present invention and a carrier, for example, a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers are saline, isotonic dextrose, isotonic sucrose, Ringer's solution, and Hanks's solution. A buffering agent may be added to provide a pH optimum for storage stability. For example, a pH of from about 6.0 to about 7.5, more preferably a pH of about 6.5, is optimal for the stability of lipids in the liposome membrane and provides excellent retention of encapsulated substances. Histidine, hydroxyethyl piperazine ethyl sulfonate (HEPES), morpholipo ethyl sulfonate (MES), succinate, tartrate and citrate, typically at a concentration of 2-20 mM, are typical buffering agents. Other typical carriers include, for example, water, a buffered aqueous solution, 0.4% NaCl, 0.3% glycine, and the like. Protein, carbohydrate or polymer stabilizers and tonicity regulators, for example, gelatin, albumin, dextran or polyvinylpyrrolidone, may be added. The tonicity of the composition can be adjusted to a physiological level of 0.25-0.35 mol / kg glucose or a more inert compound such as lactose, sucrose, mannitol or dextrin. These compositions may be sterilized by conventional, well-known sterilization methods, for example, by filtration. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized composition is combined with a sterile aqueous medium before administration.

Pharmaceutical liposome compositions may also contain other pharmaceutical excipients, in accordance with approximate physiological conditions, such as pH regulators and buffers, tonicity agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the like Additionally, the liposome suspension may contain lipid protecting agents that protect the lipids from being damaged by free radicals and lipopyroxides during storage. Lipophilic free radical quenchers such as alpha tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

The concentration of liposomes of the present invention in liquid pharmaceutical compositions can vary greatly, i.e. from less than about 0.05% usually or at least about 2-10% up to from 30 to 50% of the mass. and will be determined primarily by the volume of liquid, viscosities, and the like, in accordance with the selected selected route of administration. For example, the concentration may be increased in order to reduce the fluid content associated with the treatment. This may be especially necessary in patients with congestive heart failure associated with atherosclerosis or severe hypertension. On the other hand, liposome pharmaceutical compositions consisting of irritating lipids can be diluted to low concentrations to reduce inflammation at the injection site.

The amount of liposome pharmaceutical composition administered will depend on several factors, for example, the particular drug contained in the liposomes, the disease state being treated, the type of liposome used and / or the decision of the clinician. In general, the amount of the administered liposome pharmaceutical composition will be sufficient to deliver a single drug substance in a therapeutically effective dose.

The amount of liposomal pharmaceutical composition required to deliver a therapeutically effective dose can be determined by in vivo and in vitro methods common in the field of drug research. See, for example, D.B. Budman, A.H. Calvert, E.K. Rowinsky (editors). Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages for various drugs are well known to those skilled in the art; and, in accordance with the present invention, the drug delivered using the pharmaceutical liposome composition according to the present invention provides at least a 2-, 4- or 10-fold increase in activity compared with activity obtained by administering the same amount of drug agents in a conventional liposomal composition not containing at least one amphiphilic pyridinium derivative. Typically, the dosages for the liposomal pharmaceutical composition of the present invention are from about 0.005 to about 500 mg of the drug per kilogram of body weight, most often from about 0.1 to about 100 mg of the drug / kg of body weight or target organ weight.

In one embodiment, the pharmaceutical liposome composition of the present invention is formulated as a topical or injectable composition, or as a liquid solution or suspension. However, solid forms suitable for dissolution or suspension in liquid carriers prior to injection can be prepared. The composition may also be formed into an enteric coated tablet or gel capsule, in accordance with methods known in the art.

The liposomal composition of the present invention may be administered by any medically appropriate method, which may depend on the condition or disease being treated. Possible routes of administration include injections, parenteral routes, such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, epidural, intrathecal, or others, such as oral, nasal, ophthalmic, rectal, vaginal, local or pulmonary, for example, inhalation. For the delivery of drugs enclosed in liposomes prepared according to the invention to tumors of the central nervous system, slow, continuous intracranial infusion of liposomes directly into the tumor (convection enhanced delivery or CED) is particularly successful. See Saito, et al., Cancer Research, vol. 64 pp. 2572-2579, 2004; Mamot et al., J. Neurooncology, Volume 68, pp. 1-9, 2004. The compositions can also be directly applied to the surface of the tissue. The administration of sustained-release, pH-dependent or other-release compositions mediated by a specific chemical or environmental condition is also particularly included in the invention, for example, by methods such as injection of delayed absorption substances or erosion implants.

Preferably, the average particle diameter is 400 nm or less in case of intravenous administration. This is due to the fact that a liposome having a particle size exceeding 400 nm is retained in the liver, spleen and other organs of the endothelial system and the lungs.

Also provided is a method of facilitating the delivery of a biologically active compound to a target cell that is not specialized in removing and processing particles from the blood, comprising intravenously administering the liposome of the invention that contains the biologically active compound. An additional aspect relates to the use of the liposome according to the invention as a means of delivering the target substance into the target cell in vitro and in vivo, which is not specialized in the removal and / or processing of lipid-based particles. Preferably, said target cell is selected from the group consisting of endothelial cells, epithelial cells, muscle cells, brain cells, nerve cells, skin cells, hair cells, a subpopulation of undifferentiated progenitor cells and pericytes. More preferably, the target cell is an endothelial cell. A liposome and the liposome-based delivery method of the invention, for example, facilitates the (targeted) delivery of a biologically active compound to endothelial cells, for example, TNFα-activated endothelial cells. Thus, the liposome disclosed herein is successfully used as a drug delivery vehicle.

Also provided is the use of the liposome of the invention, containing at least one biologically active compound, either in the interior and / or incorporated into the lipid bilayer, and / or attached covalently or otherwise to one of the components of the liposome, to obtain a medicine for treating cancer, chronic inflammation, tissue repair / regeneration, diabetes, or a metabolic disease associated with cell dysfunction.

In one embodiment, the liposome provided by the methods of targeting EPCAM and encapsulating a cytostatic compound is used to prepare a medicament for treating cancer. In another embodiment, the liposome provided by the E-selectin targeting methods and containing the p38MAPK inhibitor is used to prepare a medicament for treating glomerulonephritis or rheumatism.

DRAWINGS FOR DRAWINGS

Figa: Intracellular processing of anti-E-selectin immunoliposomes in HUVEC targeting E-selectin (HES) or VCAM-1 (VCAM) was studied using double conventional isotope-labeled liposomes, i.e. without artificial amphiphilic pyridinium derivative containing cholesteryl [ ¹⁴ C] oleate ester degrading during metabolism in addition to nondestructible ether [ ³ H] cholesteryl oleyl. In the case of undisturbed metabolism, after endocytosis, cholesteryl ether is hydrolyzed and the released [ ¹⁴ C] oleic acid will be released from the cells into the culture medium. The [ ³ H] cholesteryl oleyl ether remains bound and, therefore, the ³ N / ¹⁴ C ratio is a suitable estimate of liposome disruption [5]. As demonstrated in panel A, the ³ H / ¹⁴ C ratio does not change with prolonged incubation, revealing the absence of hydrolysis of cholesteryl [ ¹⁴ C] oleate. Even a 24-hour incubation does not lead to any significant hydrolysis of double conventional immunoliposomes against E-selectin. Panel B compares the destruction of HUVEC immunoliposomes with the destruction of similar liposomes by IC21 cells (peritoneal macrophages). It has been demonstrated that similar liposomes can be efficiently destroyed by specialized cells. Incubations in the presence of inhibitors (chloroquine, NH ₄ Cl) of lysosomal disruption reveal that the disruption is lysosomal (data not shown).

Figure 2: TNFα-activated HUVECs are incubated for 24 hours with anti-E-selectin immunoliposomes (in the absence of an artificial amphiphilic pyridinium derivative), a fluorescently labeled DiI lipid bilayer marker. Liposomes accumulated in certain intracellular vesicles, at the same time, no redistribution of the fluorescent label across cell membranes was detected, i.e. liposomes were not destroyed after endocytosis by endothelial cells.

Figure 3: The liposomes of the invention are prepared as described in the general method and so that they contain the indicated amount of the artificial amphiphilic pyridinium derivative (in the case of SAINT-18) or said cationogenic amphiphilic substance DONAP. Liposomes are stored at 4 ° C in argon for specific periods of time. The particle size of liposomes is measured at a specific time by dynamic light scattering using a Nicomp Model 380 submicron particle analyzer in volumetric weighing mode. Data are presented as a single value for 1, or an average ± SD of 2 to 5 independent liposome compositions.

Figure 4: Liposomes are prepared as described in the general method. Liposomes are stored in a thermostatic bath at 37 ° C or on a laboratory bench at room temperature (20 ° C) in the absence (panel A) or in the presence (panel B) of 10% (v / v) serum. Particle size is analyzed at a specific time by dynamic light scattering using a Nicomp Model 380 submicron particle analyzer in volumetric weighing mode. Data are presented as mean ± SD from 3 or 4 independent liposome compositions.

5: Liposomes are prepared as described with increasing amounts of an amphiphilic pyridinium derivative. The size of liposomes is completely stable if the bilayer is obtained using SAINT C18 0-20% mol. Approximately over 20-25% moles of the amphiphilic pyridinium derivative, liposomes begin to lose size stability.

6: Calcein is released from liposomes depending on pH. The fluorescence of liposomes is monitored during incubation in buffer solutions with a certain pH in 96-well plates in a standard fluorometer. Calcein release is compared with total liposome fluorescence measured after treatment with Triton-X100. Data are presented as relative release compared to liposomes obtained without the use of SAINT or DOTAP (mean ± SD from 3 or 4 independent liposome compositions).

Fig. 7: Calcein release from liposomes prepared using 20% mol of SAINT or DOTAP at pH 7.4 and pH 5. Fluorescence of liposomes was monitored during incubation in buffer solutions at a specific pH in 96-well plates in a standard fluorimeter. Calcein release is compared with total liposome fluorescence measured after treatment with Triton-X100. Data are presented as relative release compared to liposomes obtained without the use of SAINT or DOTAP (mean ± SD from 3 or 4 independent liposome compositions). * p <0.005 compared to pH 7.4; # p <0.05 compared with liposomes containing 20% mol of SAINT.

Fig. 8: Effect of 10% serum (v / v) on the release of calcein from liposomes. Liposome fluorescence is monitored in 96-well plates in a standard fluorometer. Calcein release is calculated after determining the total fluorescence of liposomes measured after treatment with Triton-X100. Data are presented as relative release compared to liposomes obtained without using SAINT or DOTAP (mean ± SD from 3 or 4 independent experiments).

Figure 9: TNFα-activated HUVECs are incubated for 24 hours with calcein-containing immunoliposomes. Panel A: anti-E-selectin immunoliposomes prepared using 20% mol of SAINT. Panel B: anti-E-selectin immunoliposomes not containing SAINT. Panel C: non-targeted liposomes with 20% mol SAINT. Calcein release is most noticeable with targeted liposomes that contain SAINT.

Figure 10: Absorption of DiI (panel A) and calcein (panel B) labeled with HUVEC liposomes. Non-stimulated and stimulated TNFα cells are incubated with these liposomes for 3 hours. Liposome uptake was investigated by FACS. Data are presented for three independent experiments.

11: Effect of E-selectin-targeted liposomes prepared using 20% mol of SAINT containing interleukin-8 (IL-8) siRNA on IL-8 expression in conditionally immortalized human glomerular endothelial cells (ciGEnc). TNF-α activated ciGEnc are incubated with SAINT E-selectin immunoliposomes containing siPHK (either specific for IL-8 or encrypted siRNA) for 48 hours. Inhibition of IL-8 expression is examined by real-time PCR. The data presented are obtained from one experiment.

Fig. 12: Downregulation of the expression of VE-cadherin. Fig. 11: Effect of E-selectin liposomes prepared using 20% SAINT moles and containing siRNA on the mid-T antigen of the H5V polyoma cell line transformed with the endothelioma oncogen. E-selectin SAINT-o-soms containing specific siRNA for VE-cadherin and encoded siRNA are incubated with H5V cells at a siRNA concentration of 1000 pmol / ml for 48 hours. The effects of siRNAs examine real-time PCR. The expression of VE-cadherin in cells treated with liposomes is compared with cells treated with TNF-α (optionally up to 1). Data are presented as mean ± SD.

EXAMPLES

Abbreviations used:

TNFα, tumor necrosis factor α

VCAM-1, vascular cell adhesion molecule 1

HUVEC, human umbilical vein endothelial cells

DiI, 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate

SAINT, N-methyl-4-alkylpyridinium chloride; SATA, (N-succinimidyl-S-acetylthioacetate)

DOTAP, N- [1- (2,3-dioleoyloxy)] - N, N, N-trimethylammonium propane

Liposomal Compositions

Liposomes were prepared in accordance with the following. Lipids from stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (Chol), 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (DSPE-PEG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] maleimide (DSPE-PEG-Mal) in a mixture of chloroform: methanol (9: 1 volume) mixed in a molar ratio of 55: 40: 4: 1, dried under reduced pressure in nitrogen, dissolved in cyclodextran and subjected to freeze-drying. If indicated, 1-methyl-4- (cis-9-dioleyl) methyl-pyridinium chloride (SAINT-18) is added to the lipid mixture in a specific molar%, always due to the amount of POPC. If necessary, trace amounts of [3H] cholesteryl oleyl ether and cholesteryl [ ¹⁴ C] oleate are added to the composition as a non-degradable marker and a degradable marker, respectively. For fluorescence microscopy and confocal laser scanning microscopy, 0.5% mol of DiI is added to the lipid mixture as indicated. The lipids are then hydrated in Hepes buffer (10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 135 mM NaCl, pH 6.7) or in an aqueous solution containing 100 mM calcein. The obtained liposomes are adjusted to the required size by repeated extrusion (13 times) through polycarbonate filters (Costar, Cambridge MA, USA), pore size 50 nm using a high pressure extruder (Lipex, Vancouver, Canada).

Monoclonal mouse anti-human E-selectin antibody (H 18/7, kindly provided by other M. Gimbrone, jr., Boston, MA, USA) is thiolated with N-succinimidyl-S-acetylthioacetate and attached to the maleimide group at the distal end of the polyethylene glycol chain method sulfhydryl-maleimide compounds [2], exactly as described above for albumin [3]. Control immunoliposomes are prepared as previously described using irrelevant rat IgG (Sigma-Aldrich Chemie, Zwijndrecht, The Netherlands). Control antibody-free liposomes are obtained from a similar lipid mixture, but instead of incubating with antibodies, they are incubated with cysteine in a double molar amount of DSPE-PEG-Mal until the reactive maleimido groups are blocked. Immunoliposomes are characterized by a defining protein using mouse immunoglobulin G as standard 4 and phospholipids phosphorus content 5. Total liposome lipid concentrations are adjusted for the amount of cholesterol and SAINT present in the liposome compositions.

Particle size was analyzed by dynamic light scattering using a Nicomp Model 380 sub-micron particle analyzer in volumetric weighing mode (NICOMP particle size measurement systems, Santa Barbara, California, USA).

Cell targeting

HUVEC activate TNFα for a specific time. Liposomes, either labeled with isotope, or fluorescently labeled, are added to the cells over a period of time. To analyze the cellular binding of isotope-labeled liposomes, the cells are placed on ice at the end of incubation, the medium is removed and the cells are washed 5 times with PBS. Cells are then lysed and radioactivity determined by liquid scintillation activity measurement. For fluorescence microscopy and confocal laser scanning microscopy, incubation is performed as described above. Microscopy is performed within 1 hour after completion of the incubation. During the indicated time period, there are no changes in the morphology of the cell, and images of such incubations are reproducibly during this period.

RESULTS

Immunoliposomes targeting E-selectin or VCAM-1 on TNFα-activated endothelial cells readily undergo endocytosis in amounts comparable to macrophage endocytosis. However, unlike macrophages, endothelial cells do not process (do not destroy) liposomes (Fig. 1) and accumulate them inside the vesicles (Fig. 2). The lack of destruction of liposomes and / or destabilization leads to the retention of drugs inside the liposome and, therefore, to a decrease in pharmacological effectiveness.

Liposomes obtained using an increasing% mole of SAINT are dimensionally stable over a period of over 2 months when stored in argon at 4 ° C. Liposome compositions containing SAINT cause a slight increase in particle diameter from 90 nm for liposomes not containing SAINT to 130 nm for liposomes prepared using 20% mol of SAINT (FIG. 3). The results were compared with liposomes prepared using 20% moles of DOTAP, a non-pyridinium cationogenic lipid, often used in lipid-based nucleotide delivery vehicles.

Liposomes prepared using SAINT also show dimensional stability at 37 ° C for at least 24 hours (Fig. 4A). Upon incubation in the presence of 10% (v / v) sulfur, the size of liposomes increases after 7 hours of incubation, especially at room temperature (Fig. 4B). However, at a physiological temperature of 37 ° C, an increase in diameter after 24 hours of incubation was very limited for liposomes prepared using the artificial amphiphilic pyridinium derivative. For liposomes prepared using 20% mol of SAINT, the average diameter increased to 130 nm compared to time 0.

As follows from figure 5, liposomes obtained using up to 20% mol of amphiphilic pyridinium derivative, maintain size stability. An increase in the amount leads to a decrease in stability, an increase of up to 30% mol causes the formation of giant, coalesced particles having a diameter of approximately 500 nm.

The release of liposome contents is determined using calcein as a model for the water-soluble encapsulated compound. Calcein fluorescence is quenched at a concentration in which it is encapsulated inside liposomes (100 mM). When diluted, calcein fluoresces. This makes it possible to distinguish encapsulated calcein (without fluorescence) from the released calcein. The pathway of endocytosis by which (immuno) liposomes are involved in intracellular transport through endosomes occurs with a decrease in pH. Thus, the release of calcein from liposomes obtained using increasing amounts of the artificial amphiphilic pyridinium derivative is defined as a function of pH (see FIG. 6). Calcein retention in liposomes prepared using SAINT is stable at neutral pH. At a pH below 6, the release of calcein increases if the liposomes have 20% SAINT moles in their composition, demonstrating the highest release in the pH range 4.5-6. Liposomes prepared using 20% mol DOTAP show less variability in the release pattern (FIG. 6). In FIG. 7, release features for liposomes prepared using 20% mol of SAINT are compared at pH 7.4 and pH 5 (relevant pH relative to pH in endosomes). At pH 5, the release of calcein from liposomes is significantly increased. Additionally, release from SAINT liposomes at a given pH is better than from liposomes prepared using DOTAP.

The addition of 10% serum to liposomes obtained using SAINT leads to an increase with time of release of calcein for liposomes containing 10% mol of SAINT (Fig. 8).

Further, in vitro calcein release from liposomes was studied. Endothelial cells, HUVEC, activate TNFα and incubate with (a) anti-E-selectin immunoliposomes prepared using 20% SAINT mol, (b) anti-SAINT-free control E-selectin immunoliposomes, or (c) anti-liposomes -E-selectin) obtained using 20% mol of SAINT. Figure 9 clearly demonstrates that cells incubated with immunoliposomes prepared using 20% mol of SAINT exhibit the highest calcein fluorescence, revealing the preferred intracellular release of fluorophore from SAINT liposomes that are captured by cells. The absorption of immunoliposomes against E-selectin does not depend on the inclusion of SAINT in their composition (FACS is determined using DiI as a marker, see Fig. 10), therefore, the number of liposomes absorbed in Fig. 9A and Fig. 9B is the same. Liposomes obtained using SAINT, but without the use of cell targeting methods, are absorbed to a lesser extent (figs).

The result shown in FIG. 9 is confirmed in short-term incubations with liposomes that are double-labeled with DiI as a lipid label and calcein as an encapsulated label. HUVEC, which is activated for 4 hours, TNFα, incubated for 5, 15, 30 or 60 minutes with anti-E-selectin immunoliposomes not containing SAINT or obtained using 20% mol of SAINT. In cells incubated with SAINT-free liposomes, initially red fluorescence is observed at all time points. In contrast, in cells incubated with liposomes obtained using SAINT, in addition to red fluorescence, yellow fluorescence is also observed in the early stages. Yellow fluorescence reveals the release of calcein, the fluorescence of which is co-localized with liposomes. At 30 and 60 minutes, green fluorescence is also visible, revealing different intracellular accumulation of liposome contents compared to the liposomes themselves (data not shown).

The absorption of liposomes and the release of encapsulated calcein from liposomes is quantified using a (semi) quantitative method using FACS. The absorption of liposomes targeted by E-selectin, HUVEC activated by TNFα, is compared for liposomes that are prepared using 20% mol of SAINT, and for liposomes not containing SAINT, as determined by measuring the average fluorescence intensity (arbitrary units) of liposomal DiI (Fig. 10A ) The release of calcein from E-selectin-targeted liposomes prepared using 20% mol of SAINT, however, is 8–10 times higher compared to liposomes not containing SAINT (FIG. 10B).

E-selectin targeted liposomes prepared using 20% SAINT moles containing specific siRNA were very effective in downregulating target genes (FIGS. 11 and 12). In glomerular TNFα-activated glomerular endothelial cells, E-selectin-targeted liposomes prepared using 20% SAINT moles and containing siRNA anti-interleukin-8 (IL-8) inhibit IL-8 expression by almost 60%, while the liposome effect is absent obtained without SAINT or liposomes containing the control (encoded) siRNA (Fig. 11). The same applies to the expression of VE-cadherin, which can be effectively inhibited by more than 60% using liposomes targeting E-selectin obtained using 20% mol of SAINT in an endothelial mouse cell line.

LINKS

Claims (18)

1. A liposome containing at least one lipid bilayer surrounding the aqueous compartment, wherein said lipid bilayer contains at least one amphiphilic synthetic pyridinium derivative, in which the total amount of amphiphilic pyridinium derivative is from 2 to 25 mol% based on the total lipid content of the liposome. 2. The liposome according to claim 1, in which the total amount of amphiphilic pyridinium derivative is from 5 to 20 mol.%. 3. The liposome according to claim 2, in which the total amount of amphiphilic pyridinium derivative is 10-20 mol.%. 4. The liposome according to claim 1 or 2, having an outer diameter of from about 30 to about 300 nanometers (nm), most preferably from 50 to 150 nm. 5. The liposome according to claim 1, in which at least one of said artificial amphiphilic pyridinium derivative is a SAINT molecule, preferably a SAINT molecule selected from the SAINT group of compounds of general formula (I):

in which R ₁ represents (C ₁ -C ₅ ) alkyl, ap (alkyl) or an alkyl group with a cationic functional group, such as

or R ₁ represents (C ₁ -C ₅ alkylene) R ₅ in which R ₅ represents a structure with the general formula I, excluding R ₁ ;
X is a halide ion selected from Cl ^- , I ^- , Br ^- ;
R ₃ is hydrogen and R ₂ and R _{4 are} identical or different, and are selected from the group consisting of branched or linear (C ₁₀ -C ₂₀ ) alkyl, mono- or polyunsaturated (C ₁₀ -C ₂₀ ) alkenyl, O = C- O-alkyl

or ar (alkyl),
or R ₂ and R ₄ are hydrogen, and R ₃ is —CH (R ₅ ) ₂ with R ₅ containing (C ₁₀ -C ₂₀ ) alkyl, mono- or polyunsaturated (C ₁₀ -C ₂₀ ) alkenyl, O = C-O-alkyl

or aralkyl; or
from the SAINT group of compounds of the above formula (I), with the exception of compounds in which R ₁ represents CH ₃ , R ₂ and R ₄ represent hydrogen, R ₃ represents (C ₁₆ H ₃₃ ) ₂ CH, and X represents all designated counterions, and with the exception of compounds in which R ₁ represents CH ₃ , R ₂ and R ₄ represent C ₁₆ H ₃₃ —O — C (O), R ₃ represents hydrogen, and X represents all designated counterions. 6. The liposome according to claim 4, containing SAINT-18 (1-methyl-4- (cis-9-dioleyl) methylpyridinium chloride) and / or SAINT-12 (1-methyl-4- (pentacosan-13-yl) pyridinium chloride) . 7. The liposome according to claim 1, sterically stabilized, preferably by the inclusion of a monosialoganglioside (GM1) and / or poly (ethylene glycol) (PEG) derivative of a lipid in a lipid bilayer. 8. The liposome according to claim 1, containing on its surface at least one targeting agent capable of recognizing and binding to the surface molecule of an individual target cell or the intracellular compartment of the target cell. 9. The liposome of claim 8, wherein said targeting agents are specific for the surface of the target cell, which is not specialized for the removal and processing of particles, preferably for an endothelial cell. 10. The liposome of claim 1, wherein said targeting agents are an RGD peptide unit, an anti-E-selectin antibody, or an anti-VCAM-1 antibody. 11. The liposome of claim 1, wherein said at least one targeting agent is linked via a linker to a SAINT molecule incorporated into the lipid bilayer. 12. The liposome according to claim 1, containing in its internal compartment and / or associated with its lipid bilayer, at least one biologically active compound. 13. The liposome of claim 12, wherein said biologically active compound is selected from the group consisting of antibiotics, antibacterial, antiviral, antifungal, anti-inflammatory, antiproliferative and antitumor drugs; hormones; nucleic acids, including genes, recombinant nucleic acids, oligonucleotides, siRNA, viral genes and genes from microorganisms; and antigens. 14. A method of producing a liposome according to any one of claims 1 to 13, comprising mixing conventional lipids with a suitable amount of at least one artificial amphiphilic pyridinium derivative, and preparing the liposome in accordance with conventional methods. 15. A pharmaceutical composition comprising a liposome according to claim 12 or 13 and a pharmaceutically acceptable carrier. 16. The use of the liposome according to any one of claims 1 to 13 as a delivery vehicle for in vivo and in vitro introduction of the substance of interest into the target cell, which is not specialized for the removal and / or processing of lipid-based particles, in particular when the specified cell the target is selected from the group consisting of endothelial cells, epithelial cells, muscle cells, brain cells, nerve cells, skin cells, hair cells, undifferentiated progenitor cells and pericytes. 17. The use of liposomes according to any one of claims 1 to 13 as a means of drug delivery. 18. The use of the liposome according to item 12 or 13 in the treatment of cancer, chronic inflammation, atherosclerosis, tissue regeneration / healing, diabetes or metabolic disease associated with cell dysfunction.

Images