FR2836043A1 - LIPID VESICLES, PREPARATION AND USES - Google Patents

Abstract

The present invention relates to artificial lipid vesicles, their preparation and their uses. It relates in particular to artificial vesicles, the composition of which is based on the characteristics of natural vesicles, giving them particularly advantageous biological and / or physicochemical properties. It relates in particular to vesicles whose wall has a particular lipid composition, in particular a high content of disaturated phospholipids. The invention also describes the uses of these lipid vesicles, in particular as vectors for transporting molecules of interest, in the context of the preparation of vaccines, pharmaceutical compositions or cosmetic compositions.

Description

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LIPID VESICLES, PREPARATION AND USES
INTRODUCTION AND STATE OF ART
The present invention relates to artificial lipid vesicles, their preparation and their uses. It relates in particular to artificial vesicles whose composition is based on natural vesicle characteristics, giving them particularly advantageous biological and / or physicochemical properties. It relates in particular vesicles whose wall has a particular lipid composition, including a high content of disaturated phospholipids. The invention also describes the uses of these lipid vesicles, in particular as transport vectors of molecules of interest, in the context of the preparation of vaccines, pharmaceutical compositions or cosmetic compositions. The invention further relates to methods of preparing such lipid vesicles as well as compositions containing them. It also relates to tools and kits for the preparation of the vesicles and compositions mentioned above. The invention can be used for example in the technical fields of biology, pharmacology, diagnosis, cosmetology, medical imaging and food processing. Its applications concern in particular the fields of human and animal health.

 It has been demonstrated in different physiological contexts that cells can release membrane vesicles (called exosomes), involved in molecule transport, cell-to-cell communication, and so on. Thus, production and release of vesicles have been observed from different cell types such as: - B lymphocytes, which release exosomes carrying major class II histocompatibility complex molecules involved in antigen presentation (Raposo et al. al., J. Exp Med 183 (1996) 1161);

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 dendritic cells, which produce vesicles having particular structural and functional characteristics that play a role in mediating the immune response, particularly in the stimulation of cytotoxic T lymphocytes (Zitvogel et al., Nature Medicine 4 (1998) 594); tumor cells, which secrete particular vesicles carrying tumor antigens and capable of presenting these antigens or of transmitting them to the antigen-presenting cells (WO99 / 03499); the mast cell cells, which accumulate the molecules in the compartments; intracellular vesicles, which can be secreted under the effect of signals (WO00 / 28001).

 In general, it seems that the cells communicate with each other via membrane vesicles that they release, which may carry antigenic motifs, MHC molecules, or any other signal (cytokine, growth, etc.). These vesicles have specific and particularly interesting structural and functional characteristics, and therefore represent a particularly attractive product for diagnostic, vaccinal, therapeutic applications or for conveying molecules of interest.

Different approaches have been used to produce such vesicles and to purify them. These approaches use producer cells, possibly genetically modified, or biological fluids, from which the vesicles are purified (see, for example, WO99 / 03499, WO00 / 44389, WO 00/28001).

The present application now proposes artificial lipid vesicles having advantageous structural and functional properties, capable of replacing natural vesicles.

The present invention relates in particular to the analysis of the lipid composition of the membranes of lipid vesicles produced by different types

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 cell. The present application also reports the analysis of the structural properties of these vesicles and shows that the lipid phase of these vesicles is an essential element of their structure and their properties. The invention demonstrates for the first time the existence of a lipid structure specific for exosomes and structural parameters specific to these vesicles, which differ from the overall membrane composition of the cells from which they are derived. This information now makes it possible to reconstitute in vitro such vesicles, hereinafter referred to as exoliposomes, independently of any biological source and in particular of any cellular system, thus advantageously simplifying their production. Because of their composition, the vesicles of the invention have advantageous biological and pharmacodynamic properties compared to the liposomes of the prior art, such as greater stability, greater rigidity, increased plasma half-life, etc. In addition, these vesicles can be produced easily. These vesicles therefore represent particularly interesting products for conveying molecules of interest in vivo.

GENERAL DESCRIPTION OF THE INVENTION
The problem that the present invention proposes to solve consists in producing artificial lipid vesicles having improved properties in vitro or in vivo, in particular for the transfer of molecules of interest to cells or organisms. The present invention is based in particular on the analysis of the composition of natural lipid vesicles and on the detection of unexpected characteristics, allowing the preparation of artificial vesicles having improved behavior.

 A first particular aspect of the invention thus relates to an artificial lipid vesicle characterized in that it comprises a lipid wall (or membrane) comprising cholesterol and disaturated phospholipids. Preferably, the disaturated phospholipids represent at least 3% of the total constituents of the lipid wall.

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 The present application is based in part on the demonstration of the presence of particular phospholipids, especially disaturated phospholipids in the structure of the vesicle wall, to confer advantageous properties to these products. In a more preferred embodiment, the vesicles of the invention further comprise other lipids or phospholipids, such as sphingomyelin, phosphatidylserine and phosphoinositol.

 Another aspect of the invention relates to a method for producing a vesicle as defined above, characterized in that it comprises at least the following steps: a) bringing together, in a suitable medium, cholesterol and phospholipids, at least a portion of the phospholipids being disaturated, b) treating the mixture obtained in step a) to form vesicles comprising a lipid wall, and c) recovering the vesicles obtained at the end of the step b).

 Another aspect of the invention relates to a lipid vesicle (or a composition comprising lipid vesicles) obtained by assembling from a composition comprising cholesterol and phospholipids, at least a portion of the phospholipids being disaturated.

 The invention also relates to the use of the vesicles as described above as a carrier vector of one or more molecules of interest.

 Other aspects of the invention furthermore relate to a vaccine, a pharmaceutical composition and a cosmetic composition comprising a vesicle according to the invention, as well as kits comprising such vesicles or intended to carry out a method according to the invention. 'invention.

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DETAILED DESCRIPTION OF THE INVENTION
For the purposes of the invention, the term artificial lipid vesicle means a vesicle produced artificially or synthetically, that is to say for example by assembly or in vitro reconstitution of a vesicle from the constituents. An artificial vesicle is therefore typically produced independently of any cellular system. It is understood that certain lipids or other constituents used in the preparation of the vesicles can be of natural origin, and come from a cellular source. An artificial lipid vesicle according to the invention is more precisely a vesicle comprising a lipid wall surrounding or containing an aqueous solution (or phase).

 For the purposes of the invention, the term "lipid wall" designates any wall or membrane comprising an arrangement of lipids. The lipid wall or membrane of the vesicles of the invention may be in the form of a lipid monolayer (i.e., a single molecular layer of lipids) or a lipid bilayer (i.e., a double molecular layer of lipids). In the case of a double layer, the hydrophobic poles of the lipids are opposite one another. The wall (or membrane) of the artificial vesicles of the invention essentially comprises lipids (eg, phospholipids, sterols (eg, cholesterol), glycolipids, (di-) glycerides, etc.), even if other possible constituents, such as that proteins or carbohydrates, may be present.

 The vesicles according to the invention have a diameter generally of between 50 and 150 nm, preferably between 60 and 100 nm. It is understood that the dimension may be adapted by those skilled in the art depending on the intended applications, for example by modifying the preparation conditions. Of course, the vesicle compositions may comprise vesicles having varying sizes.

 As indicated, the present application relates to artificial lipid vesicles whose wall has a particular lipid composition. This composition is based on the analysis of natural vesicles, and gives the artificial vesicles of the invention biological and physical properties.

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 remarkable chemical properties, particularly in terms of stability, bio-compatibility, etc. More particularly, the inventors have proceeded to the analysis of the lipid composition of different biological membranes, and have demonstrated particular compositions allowing the reconstitution of artificial vesicles having advantageous properties.

 In particular, the results obtained show that physiological vesicles have a particular lipid structure, based mainly on: a balance of the molar proportions of the four main classes of phospholipids (PC, PE, PS / PI, MS), and / or presence of disaturated molecular species, especially PC and PE, and / or - a decrease in total diglycerides, conferring structural parameters of remarkable membrane fluidity and distinct cells.

 On the basis of these results, the present application now proposes artificial vesicles of particular composition having advantageous physiological properties.

 A first particularly advantageous feature of the vesicles of the invention lies in the fact that the lipid wall comprises di-saturated phospholipids. The term "di-saturated phospholipids" means, for the purposes of the invention, phospholipids bearing two saturated fatty acid chains.

The inventors have in fact demonstrated, by analyzing the composition of natural exosomes, the presence of such phospholipids and their enrichment relative to the global membranes of the cells.

 Phospholipids are constituents of biological membranes of living cells. Schematically, phospholipids are complex lipids characterized by a hydrophilic polar head (i.e., an end bearing a positive or negative electric charge interacting with water), and long hydrophobic apolary tails (which, n having no

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 electric charge, do not mix with water). These molecular characteristics mean that the phospholipids, immersed in an aqueous solution, are typically organized in the form of a vesicle. Depending on the conditions used, they can form a fluid bilayer in which the hydrophilic heads come into contact with water, while the long hydrophobic tails are arranged inward, isolating themselves from the aqueous medium. This lipid bilayer about 5 nanometers thick serves as almost impermeable barrier to the passage of substances soluble in water.

 Another characteristic of the vesicles of the invention lies in the presence of cholesterol. Cholesterol is the most abundant steroid in natural membranes. Its basic skeleton is composed of four carbon-based rings to which various lateral groupings are attached. It constitutes a stiffening element of the membrane.

 Particularly preferably, an artificial lipid vesicle according to the invention is a vesicle comprising a lipid wall (mono- or bilayer) surrounding an aqueous solution, the lipid wall comprising di-saturated phospholipids and cholesterol.

 More preferably, the disaturated phospholipids represent at least 3% of the total constituents of the lipid wall. As illustrated in the examples, the disaturated phospholipids advantageously represent more than 4% of the constituents, still more preferably more than 5%, preferably less than 15%. In a particularly preferred mode, the vesicles comprise from 5 to 7% of disaturated phospholipids.

 Advantageously, the vesicles of the invention comprise approximately 5 to 25% of cholesterol, more preferably 8 to 20%, even more preferably 12 to 18%, relative to the total constituents.

 Phospholipids are more precisely molecules of glycerides whose glycerol is esterified by two fatty acids and the last alcohol function

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lécithine),l'éthanolamine (phosphatidyléthanolamine (PE)), la sérine (phosphatidylsérine (PS)), l'inositol (phosphatidyl-inositol (Pl)), le glycérol (phosphatidyl-glycérol (PG)), etc. primary by a phosphoric acid. This structure, common to all phospholipids, is called phosphatidic acid. The phosphoric acid phospholipids are esterified a second time in most phospholipids by an alcohol function of another molecule: choline (phosphatidylcholine (PC) or

lecithin), ethanolamine (phosphatidylethanolamine (PE)), serine (phosphatidylserine (PS)), inositol (phosphatidylinositol (P1)), glycerol (phosphatidylglycerol (PG)), etc.

 In a particularly preferred manner, the vesicles of the invention comprise at least one di-saturated phospholipid chosen from phosphatidylcholine (PC) and phosphatidylethanolamine (PE), typically in the form of a mixture. In this respect, the respective proportions of PC and PE can be adjusted by those skilled in the art. They are generally between 1/10 and 10/1, more preferably between 3/10 and 10/3. More preferably, the PC-disaturated / PE-unsaturated ratio ranges from 0.5 to 2.

 Preferably, the fatty chains of the phospholipids used in the vesicles of the invention have a length, identical or different, of between 14 and 22 carbon atoms, preferably between 14 and 20, even more preferably between 16 and 20 carbon atoms. . It is thus possible to mention phospholipids having a fatty chain comprising 14,16, 18,20 or 22 carbon atoms, saturated or not.

 In a particular embodiment of the invention, the vesicles comprise disaturated phospholipids comprising two saturated fatty chains, identical or different, chosen from palmitic acid (C16: 0) and stearic acid (C18: 0).

 Palmitic acid is a long-chain fatty acid, the main product of lipid synthesis in our cells. It is often symbolized by the numbers 16: 0 to indicate that it contains 16 carbon atoms and no double bond: it is saturated fatty acid. It's also a white solid, which

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 at 64 C. Its name comes from palm oil, but it is abundant in all animal and vegetable fats and oils. Industrially, palmitic acid is used for the manufacture of margarines, hard soaps.

 Stearic acid is another long-chain fatty acid, which is symbolized by the numbers 18: 0 to indicate that it has 18 carbon and no double bond: it is saturated fatty acid. It is also a white solid, which melts at 70 OC. It is abundant in all animal fats (especially ruminants) or vegetable fats. Stearic acid is used industrially to make candles, soaps.

 A particular object of the invention relates to an artificial lipid vesicle whose lipid wall comprises phosphatidylcholine (PC) or phosphatidylethanolamine (PE) substituted with two saturated fatty chains, identical or different, chosen from C18: 0 and C16: 0 .

 It is understood that synthetic phospholipids may be used, in which the position of the fatty chains is modified (for example in position 2 and 3) and / or in which the length of the fatty chains is modified (for example 15.17). or 21 carbon atoms).

 Preferably, the wall of the vesicles further comprises other lipids or phospholipids.

 Thus, in a preferred embodiment, the artificial vesicles of the invention further comprise phosphatidylinositol (P1). The PI is formed of two fatty acids, linked by ester bonds to a glycerol whose third function is esterified with a phosphoric acid as in the other phospholipids. The other acid function esterifies a cyclic alcohol with six secondary alcohol functions, whose hydroxyls are oriented specifically on either side of the plane of the molecule. Phosphatidylinositol is located on the inner side of plasma membranes where it is the metabolic precursor of two secondary messengers: diacylglycerol (DAG) and inositol triphosphate. The results obtained by the inventors show that biological vesicles

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 Plas have natural properties, and this molecular species contributes to the properties of the vesicles. More preferably, the PI is present in the membranes of the vesicles of the invention in a proportion of 1 to 10% of the constituents, more preferably 3 to 10%, more preferably 4 to 8%.

 In another particular embodiment, the vesicles of the invention comprise phosphatidylserine (PS), preferably between 1 and 15% of the total of the constituents, still more preferably between 2 and 10%, typically between 3 and 8. %. The results obtained by the inventors show that the total amount of PS and PI is preferably between 10 and 20% of the total constituents.

l'invention à raison de 2 à 20 % des constituants, plus préférentiellement de 3 à 18 %, encore plus préférentiellement de 6 à 15 %. On the other hand, in a preferred embodiment, the artificial vesicles of the invention further comprise sphingomyelins. Sphingomyelins are membrane phospholipids found in different membranes but especially in the nervous system (myelin sheaths). They consist of a long chain (sphingosine), a fatty acid amide-amine function and a phosphate esterifying the primary alcohol function and itself esterified by an amino alcohol (choline). More preferably, the SMs are present in the membranes of the vesicles of

the invention in a proportion of 2 to 20% of the constituents, more preferably 3 to 18%, even more preferably 6 to 15%.

 The vesicles of the invention may further comprise diglycerides, although these are generally present at less than 10% of the total components. Diglycerides are fluidity agents and the results obtained show that they are less important in the vesicles than in the membranes of dendritic cell or mast cell type cells. More preferentially, the vesicles comprise less than 7% diglycerides, relative to the total of the constituents.

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In a particular embodiment, the artificial vesicles of the invention further comprise Iso-phosphatidylcholine (LPC) and / or lysophosphatidylethanolamine (LPE). These lipids may be present in variable amounts, preferably between 1 and 8% of the total constituents, preferably at least 3%
The vesicles of the invention may comprise, in addition to the disaturated phospholipids, polar, apolary, neutral, charged and / or unsaturated phospholipids. It can be synthetic or natural lipids. The lipids used in the context of the present invention can be obtained from commercial sources (Avanti Polar Lipids, Sigma, Inositol.com, Matreya, Alexis, etc.). By way of examples, mention may be made of phospholipids comprising two fatty chains, identical or different, having from 14 to 22 carbon atoms and having one or more double bonds, for example from 1 to 5. Examples of such phospholipids are given in Figures 4 and 5 in particular.

 A particular embodiment of the invention is an artificial lipid vesicle comprising a mono- or bi-layer lipid wall comprising, in% of the total constituents: at least 3% of PC and / or di-saturated PE, preferably 5 to 7%; . from 3 to 10% of Pi; . from 2 to 20% of SM, and. from 5 to 25% of cholesterol, more preferably from 12 to 18%.

 Even more preferably, it further comprises: from 1 to 15% PS, preferably from 3 to 8%, and / or from 1 to 8% of lyso-phosphatidylcholine and / or isosphosphatidylethanolamine.

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 The results obtained show that particularly advantageous lipid vesicles are obtained when the lipid wall comprises disaturated phospholipids and a similar proportion of the following constituents: cholesterol, sphingomyelin, PC and PE. Even more preferentially, the lipid wall comprises disaturated phospholipids and similar proportions of the following constituents: cholesterol, sphingomyelin, PC, PE and PS / PI.

 The term similar proportions indicates that said constituents are present in identical proportions or can vary with respect to each other by a gap generally less than 25%.

 As indicated above, the vesicle may further comprise other neutral, polar, saturated or unsaturated lipids or phospholipids, such as in particular unsaturated phosphatidylcholine and / or phosphatidylethanolamine and lysophosphatidylcholine (LPC), etc.

 On the other hand, the lipids or phospholipids may be glycosylated (glycolipids) or not, chemically or biologically modified, have a reactive group, etc. In this regard, the vesicle may comprise different types of carbohydrates present in the membrane in the form of oligosaccharides, short chains formed by the association of a few simple sugar molecules. These chains are linked to constituents (e.g., proteins or lipids) of the membrane, forming respectively glycoproteins and glycolipids. Both glycolipids and phospholipids are complex lipids with hydrophilic heads and hydrophobic tails. Another subject of the invention thus relates to vesicles as described above further comprising glycolipids.

 In a particular embodiment, the lipid wall is essentially free of proteins, in particular specific receptors.

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More specific compositions of vesicles are given in the table below (in% of the total constituents):

<Tb>
<tb> LYSO-PC <SEP> 3.75 <SEP> to <SEP> 6.21
<tb> SPHINGOMYELINE <SEP> 8.26 <SEP> to <SEP> 12.41
<tb> PC-DISATUREES <SEP> 3.00 <SEP> to <SEP> 4.81
<tb> PC-MIXTURE <SEP> 12.00 <SEP> to <SEP> 19.22
<tb> PS <SEP> 5.17 <SEP> to <SEP> 6.89
<tb> PI <SEP> 5.17 <SEP> to <SEP> 6.89
<tb> PE-DISATUREES <SEP> 2.61 <SEP> to <SEP> 2.85
<tb> PE-MIX <SEP> 13.42 <SEP> to <SEP> 14.65
<tb> CHOLESTEROL <SEP> 13.01 <SEP> to <SEP> 16.61
<tb> DIGLYCERIDE <SEP> 4.76 <SEP> to <SEP> 7.05
<tb> OTHER <SEP> LIPIDS <SEP> Qsp <SEP> 100%
<Tb>

The preferred vesicles of the invention have particularly advantageous biological and / or physicochemical properties. Thus, the lipid composition gives the vesicles a high membrane rigidity and creates, especially via phospholipids, a lateral surface pressure preventing the action of lipolytic enzymes or increasing the resistance to these enzymes. The membrane of the vesicles according to the invention thus has the particularity of being more rigid at 37 C than the membrane of the cells, thus increasing its stability and therefore its half-life in vitro but also and especially in vivo.

 Rigidity can be estimated by measuring the viscosity of the lipid walls. Preferably, the vesicles of the invention have a viscosity, measured by fluorescence anisotropy at 37 C, greater than about 0.15, preferably greater than 0.20.

 Another advantageous characteristic of the preferred vesicles of the invention, resulting from their high stability, is that these vesicles could

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 be frozen without adjuvant or stabilizer, without altering their properties. Thus, the natural exosomes can for example be stored at -80 C without any particular treatment.

 Without being bound by the theory, it is estimated that the particular properties of the vesicles of the invention derive in particular from the respective quantities of the major constituents of the lipid wall which are cholesterol, PCs, PE, MS and PS / PI. , enrichment of di-substituted phospholipids, and a low level of diglycerides.

 Based on an analysis, made by the inventors, of particular natural lipid membranes, the lipid vesicles of the invention should moreover have a better bio-compatibility and a much lower toxicity than synthetic products of the prior art. In addition, their composition could allow in vivo cell targeting particularly suitable for therapeutic or vaccine applications. The possible presence, in the lipid vesicles of the invention, of Iyso-PC also confers greater stability to these vesicles, reducing their fusogenic nature.

 The vesicles of the invention may further comprise different molecules of interest, such as proteins, polypeptides, peptides, chemical molecules, nucleic acids, etc. These molecules may be intended to improve the physicochemical properties of the vesicles, or to confer on them particular biological properties (targeting, biological activity, labeling, purification, etc.). These molecules may be inserted, in whole or part, into the lipid bilayer, anchored therein, exposed on the surface of the vesicle, or present in the aqueous phase. In this regard, another particular object of the invention relates to a vesicle as described above further comprising one or more molecules of interest.

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 In a particular embodiment, it is a question of proteins, of identical or different nature, which participate in the rigidity of the membrane, in particular by interaction with lipids.

 In another embodiment, they are molecules intended to be carried by the vesicles, in vitro or in vivo. Such molecules of interest carried by or contained in the vesicles of the invention may be any protein, polypeptide, peptide, nucleic acid or lipid, as well as any substance of interest (of a chemical, biological or synthetic nature). These molecules can be of recombinant nature, and be introduced directly into / on the vesicles. These molecules are for example bound to the vesicles by interaction with phosphatidylserine and / or phosphatidylethanolamine, optionally functionalized, present in the membrane. More particularly preferred types of molecules of interest are in particular MHC molecules, antigens (whole or in the form of peptides), receptor ligands, receptors (specific) of ligands, nucleic acids, pharmacological products, markers or peptides or proteins allowing purification of the vesicles.

 As antigen, there may be mentioned more particularly any protein, in particular cytoplasmic, of viral or tumor origin. As preferred examples of proteins of viral origin, mention may in particular be made of any cytoplasmic or membrane protein expressed by the EBV, CMV, HIV, measles, hepatitis, etc. viruses. It is more preferably cytoplasmic proteins, that is to say substantially invisible to the immune system in conventional infection processes, and therefore poorly immunogenic under natural conditions, or also proteins or fragments of membrane proteins. As preferred examples of proteins of tumor origin, mention may be made in particular of the p53 proteins (wild or any mutated form present in a tumor), MAGE (in particular MAGE 1, MAGE 2, MAGE 3, MAGE 4, MAGE 5 and MAGE 6), MART (in particular MART 1), Gp100, ras proteins (wild-type or mutated p21), etc. It is understood that any other protein of interest may be expressed in or on the surface of the vesicles of the invention, following the teaching of the present application. In addition, antigenic molecules

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 may be present either on the surface of the vesicles (exposed) or inside the vesicles.

 Among the ligand receptors, there may be mentioned, in general, any ligand receptor natural or derived from genetic manipulation. In particular, it can be any hormone receptor, growth factor, lymphokine, trophic factor, antigen, etc. There may be mentioned more particularly the interleukin receptors L1 to IL15, the growth hormone receptor, or the receptor for granulocyte and / or macrophage colony stimulating factors (G-CSF, GM-CSF, CSF, etc.). . A particular example of a ligand receptor is composed of an antibody or an antibody fragment or derivative, for example a single chain (ScFv), which allows interaction with a specific ligand. Another particularly advantageous example within the meaning of the invention is represented by the T cell antigen receptor (TcR). Vesicles of the invention expressing on their surface one or more defined TcRs constitute particularly advantageous analysis and diagnostic tools (cf. WO 00/28001).

 The invention also relates to a vesicle as defined above, characterized in that it comprises, typically in the aqueous phase, a pharmaceutical product. As a pharmaceutical product, there may be mentioned any active substance, of a chemical nature, such as, for example, pharmaceutical products prepared by conventional chemistry techniques. Any nucleic acid, any protein, polypeptide or peptide having a biological activity, such as, for example, a toxin, a hormone, a cytokine, a growth factor, an enzyme, a tumor suppressor, etc. may also be mentioned.

 The nucleic acid can be any DNA or RNA coding for a protein, polypeptide or pharmacological peptide as mentioned above, as well as any other nucleic acid having a particular property (antisense, antigen, promoter, repressor, site binding of a transcription factor, etc.). It can be an oligonucleotide, a coding phase, an artificial chromosome, etc.

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 The vesicles of the invention carrying a ligand receptor can be used for the detection of any receptor-ligand, particularly low affinity, interactions in any biological sample. On the other hand, the present invention also relates to the use of a vesicle as described above as a carrier vector of one or more molecules of interest. Such vesicles may in fact be used to convey to cells the substance or substances of interest, the latter being able to be active ingredients (protein, peptide, acid nucleic acid, chemical substance, etc.). The active ingredient may, in particular, be chosen from a molecule of a hormonal or neuroendocrine nature, a carbohydrate, a vitamin, an essential fatty acid, a protein and a nucleic acid. Thus, the vesicles of the invention can be used, in general, for the transport and transfer of any molecule in cells or organisms, in vitro, ex vivo or in vivo.

The invention therefore relates to any vesicle as described above comprising a heterologous molecule of interest, usable as a transfer vector of said molecule in a cell.

 In a more preferred embodiment, the vesicles of the invention are used for the oriented transfer of substances of interest to selected cell populations. Thus, it is possible to prepare vesicles of the invention comprising a substance of interest (a toxin, a hormone, a cytokine, a recombinant nucleic acid, etc.) and expressing on its surface a ligand receptor or a ligand of receptor, and to bring said vesicles into contact with cells expressing the corresponding ligand or receptor. This approach therefore allows a targeted and efficient transfer. In this regard, a particular object of the invention relates to a vesicle comprising at least one addressing molecule preferably chosen from an antibody, an antigen, a particular ligand, a particular receptor, a substrate and an enzyme.

Another particular object of the invention resides in a vesicle as defined above, characterized in that it expresses a ligand receptor and in that it comprises a heterologous molecule of interest. The heterologous term

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 indicates that the molecule of interest is not present, in this form, in the exosomes of the invention in the natural state.

 In a particular variant, the vesicles according to the invention also comprise at least one marker and / or one contrast medium. The marker may be of a different nature (enzymatic, fluorescent, radioactive, magnetic, paramagnetic, etc.) and present in the vesicle or on its surface. A preferred label is non-radioactive, such as fluorescent labeling. More preferably, the labeling used is a fluorochrome or a chromogenic substrate enzyme. The marking can be performed directly on the vesicles produced.

Production of artificial vesicles
The vesicles of the invention may be artificially produced by different methods, for example methods used for the production of liposomes. These include the methods described by Allen et al.

(FEBS Lett 223 (1987) 42), Gabizon et al. (PNAS 85 (1988) 6949) and Lentz et al (Biochemistry 26 (1987) 5389). A preferred method, which is also an object of the invention, comprises at least the following steps: a) bringing together the various constituents of the vesicles, in particular cholesterol and phospholipids, at least some of which are disaturated, b) the treatment of the mixture obtained at the end of step a) to form vesicles comprising a lipid wall, and c) recovery of the vesicles obtained at the end of step b).

Advantageously, step a) comprises contacting cholesterol, phospholipids, a portion of which are disaturated, phosphatidylserine and / or phophoinositol and / or sphingomyelin. The constituents are contacted in the desired proportions, as indicated above.

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Step a) of bringing together the constituents (e.g., cholesterol and phospholipids) can be carried out in any suitable medium, preferably any medium in which the constituents are soluble. Generally, it is carried out in an organic solvent. In this case, according to a preferred embodiment of the invention, there is carried out an additional step of removing the solvent, which is replaced by any aqueous medium suitable for biological or pharmaceutical use. This step can be performed before contacting the constituents or, preferably, after they are brought into contact.

In a particular variant embodiment, the method of the invention therefore comprises a step a ') of intermediate removal of the starting medium by evaporation (or drying), generally under vacuum, of the lipid mixture obtained at the end of the step a). This step makes it possible in particular to eliminate the solvent and / or to define a hydration medium. This hydration medium is important since it will be found, in part, within the vesicles of the invention (aqueous phase). It is therefore preferably biocompatible and may contain active agents or principles, additional structural constituents of the vesicles, etc. The medium or hydration buffer is for example made based on physiological saline or an isotonic buffered solution.

The mixture of the various constituents in the chosen medium (hydration buffer) is typically agitated to promote the suspension of the constituents and their reorganization. The phospholipids present in the hydration buffer are organized spontaneously into mono- or bi-lipid layer, reproducing for example the organization of a natural biological membrane. At this stage, the mixture obtained is composed of heterogeneous lipid structures.

Step b) of treatment of the mixture obtained at the end of step a) makes it possible to form vesicles of a more homogeneous size and / or shape, comprising a lipid wall surrounding an aqueous solution. According to a first particular embodiment of the invention, the treatment implemented during step b) is an ultrasonication process. According to a second mode, more preferred, he

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 this is an extrusion process. Extrusion is typically carried out through a membrane of defined porosity, making it possible to obtain vesicles according to the invention of homogeneous and pre-determined size. The extrusion can be carried out in any device known to those skilled in the art, such as in particular an Extruder (Lipex Biomembranes, Vancouver, Canada).

The methods according to the invention may further comprise a step d) of purifying the vesicles recovered at the end of step c). The purification may be carried out for example by gel-filtration, ultra-centrifugation, ultrafiltration, column filtration, chromatography, etc. It can still be an extraction carried out using a magnetic system.

The methods described above may also comprise a step of functionalization of the vesicles, for example by attachment or incorporation of a marker, an addressing molecule, a ligand, a modified lipid or a molecule. active, a drug, a nucleic acid, etc., more generally, any molecule of interest. This step can be carried out in different ways and at different stages of the process. Thus, the functionalization can be carried out on the final vesicles, for example by direct electroinsertion in the lipid bilayer or in the aqueous phase, or by interaction with a reactive or functionalized surface molecule. It can also be performed on the vesicles being formed, for example (i) using a detergent, when the lipid wall has already been formed, (ii) by adding one or more molecules of interest in the hydration solution, leading to their incorporation into the aqueous phase or into the vesicle membrane after the treatment carried out in step b), or (iii) by addition of lipids or functionalized constituents or reagents during step a).

According to a first advantageous embodiment of the invention, the vesicles are modified after their formation, by interaction between the molecule (s) of interest and a reactive or functionalized component of the lipid membrane. The reactive or functionalized component may be a protein, cholesterol or a

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 functionalised lipid, that is to say carrying a (chemical) reactive group for fixing a molecule of interest and not preventing the introduction of this component into the vesicle membrane. Thus, the negatively charged surfaces of the phospholipids, and in particular the amine function of phosphatidylethanolamine, may be adapted so as to comprise a reactive group capable of fixing a molecule of interest. According to another embodiment of the invention, the exoliposomes of the invention are functionalized by chemical conjugation with antibodies according to the method described by Holmberg et al. (<<Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions>>, Biochemical and Biophysical Research Communications, pp. 1272-1278, Vol 165, No. 3,1989).

According to another variant, a lipid having a particular affinity for a molecule of interest is introduced into the lipid composition. Thus, it is possible to introduce into the lipid membrane phosphatidylserine, which has a high affinity for lactadherin.

 In a particular embodiment, the method comprises, in step a), the introduction of a modified lipid carrying a reactive group having the ability to interact with a molecule of interest, the formation of vesicles according to b) then bringing the vesicles into contact with the molecule of interest, allowing its interaction with the vesicle.

 In another embodiment, the modified lipid is brought into contact with the molecule of interest prior to its introduction into the vesicle.

 The modified lipid is preferably a phosphatidylethanolamine in which the amine function has been chemically activated.

 In a particular embodiment, the method of the invention comprises, in step a), the introduction of phosphatidylserine, the formation of the vesicles according to b), and then bringing the vesicles into contact with a molecule of interest merged lactadherin, allowing its interaction with the gallbladder.

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 The methods described above may furthermore comprise the addition, during step a) or at the end of the latter, of an active ingredient of interest as defined above present in the solution of hydration.

 The vesicles thus prepared can be suspended and / or stored in different types of solutions or media. These include isotonic, buffered, sterile and / or biocompatible solutions.

 The invention also relates to any vesicle obtained by the implementation of a method as described above. In particular, it relates to any lipid preparation obtained by in vitro reconstitution from a mixture of constituents comprising cholesterol, disaturated phospholipids and, preferably, MS, PS and / or P1.

Compositions / Uses
Another subject of the invention concerns any composition comprising one or more vesicles as defined above. The compositions of the invention may further comprise a plurality of vesicles as defined above, carrying different molecules. In particular, a composition according to the invention may comprise vesicles as defined above, carrying different drugs, proteins, genes, peptides, antigens, markers, etc.

 The compositions of the invention generally comprise a pharmaceutically acceptable carrier, such as a buffer, saline, physiological solution, etc., to preserve the vesicle structure. They may further comprise any stabilizing agent, surfactant, etc., preferably compatible with a biological use (in vitro or in vivo). These compositions can be packaged in any suitable device, such as tube, flask, ampoule, flask, bag, etc., and stored at 4 ° C. As indicated above, a particularly advantageous property of preferred vesicles of

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 the invention lies in their stability, even in the absence of stabilizing agent.

Typical compositions according to the invention comprise from 5 to 500 μg of exoliposomes, for example from 5 to 200 μg.

 Another particular subject of the invention relates to the use of an artificial vesicle as described above as a carrier vector of one or more molecules of interest, and in particular of active principles, for example in the context of the preparation of a drug or a vaccine. Another subject of the invention relates to the use of vesicles for the preparation of a pharmaceutical composition. A vesicle according to the invention can thus be used in the context of the preparation of a composition intended for the prevention or treatment of a cancer, an allergy, a viral disease, a genetic disease, a dermatosis, a disease affecting the nervous system or the immune system. A vesicle according to the invention may also be used in the context of the preparation of a cosmetic composition.

 Another subject of the invention further relates to vaccines, pharmaceutical or cosmetic compositions comprising a vesicle according to the invention.

 The invention is particularly suitable for the transfer of molecules to cells of the immune system, in particular to antigen-presenting cells (dendritic cells, macrophages, etc.). A particular application of the invention therefore lies in the use of a vesicle as defined above for the transfer of antigens or other molecules to the presenting cells, in particular to dendritic cells. Another object of the invention is the use of a vesicle to stimulate or inhibit an immune response.

 The invention also relates to methods of transferring, administering, transporting or releasing molecules of interest in a subject, comprising administering to a subject a vesicle of

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 the invention comprising said molecule. The administration may be carried out systemically, in particular by intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal injection, etc. Injection or administration may be local (intra-tumoral, intracerebral, trans-dermal, etc.).

Application in medical imaging: Exoliposomes are advantageous tools for medical imaging because of their stability and their biodegradability. As previously indicated, they can be easily administered to patients, for example, intravenously. The markers used vary according to the imaging techniques. They can be coupled directly to lipids or indirectly via a protein of human albumin type.

<Tb>
<Tb>

Technical <SEP> Markers <SEP> Examples
<tb> Scintigraphy <SEP> radioactive tracer <SEP><SEP> Thallium <SEP> 201
<tb> Technetium <SEP> 99
<tb> Iodine <SEP> 131
<tb> Radiology <SEP> products <SEP> of <SEP> contrast <SEP> Derivatives <SEP> iodine
<tb> Imaging <SEP> with <SEP> resonance <SEP> paramagnetic <SEP> products <SEP><SEP> gadoteric <SEP> acid and
<tb> magnetic <SEP> derivatives
<Tb>
Exoliposomes allow advantageous stabilization of markers until administration by limiting their incompatibility with excipients or containers. They can also decrease the toxicity of certain markers and / or increase their bioavailability, and / or direct the markers to a defined target compartment if an addressing means is provided.

Applications in the pharmaceutical industry: Exoliposomes can be used to meet a number of pharmaceutical and pharmaceutical requirements and thus constitute a new drug delivery system. The active ingredient (AP) may be present on the surface or in the aqueous phase of

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 exoliposomes and be in free or bound form, for example lipids or carrier proteins. Specific addressing means may further be implemented as explained above. The exoliposomes can thus be used as simple "inactive" vectors and allow the solubilization of PAs that are poorly soluble in water, the protection of AP in the blood stream or in the tissues, or the decrease in the toxicity of AP.

They can also be used as "active" vectors and allow, for example, the controlled release over time of the PA, which is in fact progressively released during the degradation of the exoliposomes. They may also allow a controlled release in space, the addressing means used for cell or tissue targeting.

In this context, a preferred mode of administration is injection. The latter may be subcutaneous, intramuscular, intravenous or intraperitoneal.

les drogues anti-rétrovirales dans les réservoirs ganglionnaires du HIV et les produits de chimiothérapie dans les ganglions profonds Iymphomateux ou métastatiques. Dans ces cas, la voie d'administration préférée est également l'injection. Applications in Clinical Immunology: Due to their composition and properties, exoliposomes can have several major applications in clinical immunology. They can be used as adjuvant in vaccinology: the stability of exoliposomes makes these vesicles advantageous adjuvants that can potentially be used in human and animal vaccineology. They can also be used as vectors of active principles in lymphoid organs. By incorporating specific drugs and addressing the immune system, it becomes possible to target them to the lymphoid organs (spleen and ganglia). This property is useful for targeting especially

anti-retroviral drugs in HIV ganglion reservoirs and chemotherapy products in deep lymphoma or metastatic lymph nodes. In these cases, the preferred route of administration is also injection.

Applications in gene and cell therapy:

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 Exoliposomes can serve as a non-viral vector for transferring linear or circular fragments of nucleic acids (DNA, RNA). This transfer can be done in vitro, in vivo (direct injection to the patient of the preparation) or ex vivo (collection of cells, transfer of the sequence of interest and reinjection of the cells).

Applications in dermo-cosmetology: One of the major interests of exoliposomes resides in their stability and in their composition close to that of biological membranes. They are also easily administered by the dermal route. In this type of application, the active ingredient is preferentially coupled to the lipids or encapsulated in the exoliposomes.

Exoliposomes that are particularly stable can protect PA and increase its bioavailability. They also allow stabilization of the PA in the preparation until its application. They can also separate several incompatible PAs from each other. These properties are very useful for vitamins for example.

Exoliposomes are by themselves non-toxic. They can also reduce the irritation of some PA thus increasing the effectiveness and comfort of the preparations.

 Since the constituents of the exoliposomes may be the target of the skin enzymes, they may be modified to delay or, on the contrary, accelerate this degradation, thus allowing a programmed release of the AP.

Diagnostic applications:
Exoliposomes can serve as a stable support for ligands, receptors, substrates, enzymes or antibodies. They can be labeled with radioactive, fluorescent or magnetic molecules which allows to create original combinations of properties (ligands and fluorescence). These functionalized and labeled exoliposomes can be used to isolate a molecule, an enzyme or a cell population, to assay a substrate, to

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 measure an enzymatic activity, to mark in vitro or in vivo a cell population.

Applications in the agri-food industry: Exoliposomes can still serve as a stable support for various enzymes used in refining and maturation processes (proteases, lipases, decarboxylases, etc.) of agri-food preparations. This application allows better reproducibility and control of ripening processes.

Other aspects and advantages of the present invention will become apparent from the following examples, which should be considered as illustrative and not limiting.

LEGENDS OF FIGURES Figure 1: Quality control of exosome preparations by electron microscopy.

Photograph of a preparation of exosomes extracted from RBL cells. The exosome preparations contain vesicles 50 to 80 nm in diameter and much smaller vesicles (about 30 to 40 nm).

Bar = 200 nm Figure 2: Phospholipid composition of exosomes and RBL mast cells.

The figures on the left represent the exosome / RBL cell system. The results represent the average of 3 different experiences + 1- the standard deviation to the mean.

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The figures on the right represent the exosomes / dendritic cells system.

The results represent the average of 4 measurements +/- standard deviation from the same lot of exosomes and dendritic cells.

A. Proportion of phosphorus present in the major phospholipids (5PL) at the deposition and at the migration front. The separation of lipid extracts from exosomes (black bars) or from RBL cells at rest (gray bars) was performed by thin layer chromatography (TLC) in a Skipsky solvent (see Materials and Methods). The migration distances of the deposit and the front are plotted on the picture of the TLC plate below the histogram.

B. Proportion of phospholipid classes predominant in exosome membranes (black bars) or resting RBLs (gray bars). Separation of the major phospholipids is illustrated in the photo of the TLC plate after migration of the total lipid extracts in a Skipsky solvent. The migration of the lipid extracts in a basic solvent system (Chloroform / Methanol / Methylamine, 65/22/4, v / v / v) makes it possible to separate the phosphatidylserines from the phosphatidylinositols and shows an increase of the two phospholipids.

 Figure 3: Proportion of diacyl and alkylacyl subclasses, within phosphatidylcholines and phosphatidylethanolamines of exosomes and RBL cells.

 The results are expressed as a percentage of the total phospholipid class, PC or PE.

A. Determination, by normal phase HPLC, of the proportion of diacyls (DA) present in phosphatidylcholines (PC) or phosphatidylethanolamines (PE) of RBL cells (black bars) or exosomes (gray bars).

 B. Determination, by normal phase HPLC, of the proportion of alkylacyls (AA) present in PCs or PEs of RBL (black bars) or exosomes (gray bars).

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In both cases, the difference from 100% corresponds to the alkenylacyl subclass (not shown).

Figure 4: Analysis of the molecular species of phosphatidylcholines and phosphatidylethanolamines of exosomes and RBL.

A. Reverse Phase HPLC Analysis of the Diacylphosphatidylcholine Subclass (Top Panel; DA-PC) and Diacylphosphatidylethanolamine Subclasses (Bottom Panel; DA-PE) from Lipid Extracts RBL (black bars) or exosomes isolated from RBL (gray bars).

B. Reverse phase HPLC analysis of the alkylacylphosphatidylcholine subclass (top panel, AA-PC) and alkylacylphosphatidylethanolamine (bottom panel, AA-PE) from lipid extracts of the subclass RBL (black bars) or exosomes isolated from RBL (gray bars).

The results correspond to the average of two experiments and are expressed as a percentage of all the peaks obtained on the HPLC chromatograms. In bold are indicated the disaturated molecular species.

C. Comparison of proportions of disaturated molecular species involved in membrane stiffness (see Fig. 6), between RBL (black bars) and exosomes (gray bars). From the results obtained by HPLC (FIGS. 4A and 4B), the sum of the percentages corresponding to the disaturated molecular species (16: 0/16: 0 and 16: 0/18: 0) was carried out, taking into account the relative proportion of the two subclasses (AA and DA, see Figs 3A and 3B) within the PCs or PEs. The results are expressed as a percentage of the total molecular species in each class of phospholipids, PC or PE.

Figure 5: Gas chromatographic analysis of the neutral lipids of exosomes and RBL.

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Measurement of the proportion of cholesterol and total diglycerides (DG totals), - also involved in membrane stiffness - in the lipid extracts of RBL (black bars) and exosomes (gray bars). The results correspond to the molar ratio in the sample of cholesterol / total phosphorus (Ptot) or total DGs / Ptot. The histograms represent the average of 3 experiments +/- the average standard deviation for cholesterol, and 2 experiments for DGs.

Figure 6: Measurement of the viscosity (Ln h) of the membranes of exosomes (gray squares) and RBL at rest (black circles) as a function of the temperature (1 / T) by fluorescence polarization.

The results represent a typical experiment in obtaining Arrhenius diagrams.

Figure 7: Measurement of the membrane viscosity by fluorescence polarization.

The results show fluorescence anisotropy values at 25 C and 37 OC, for resting cells (n = 3), activated cells (n = 2) and exosomes (n = 3). From the intensity of the light polarized parallel (IX) and perpendicular (1) to the direction of polarization of the excitation beam, the fluorescence anisotropy r is calculated according to the following equation: r = ('// - ') / (' // + 2i) The anisotropy can be related to the microviscosity, the inverse term of the fluidity, by the relation of Perrin (1926). A high value of r is therefore associated with a high value of the microviscosity, and vice versa (Shinitzky et al., 1978). In motion asbence (rigid medium) the anisotropy has a maximum value of 0.4.

Figure 8: In vitro and in vivo labeling of exosomes with fluorescent phospholipids.

A. Exosomes isolated from RBL IAb-li cells were labeled in vitro with fluorescent phospholipids BODIPY-phosphatidylcholine (BPY-

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 PC), BODIPY-ceramide (BPY-Cer), NBD-phosphatidylcholine (NBD-PC) in ethanol, or incubated with ethanol alone (EtOH). The vesicles were then fixed on latex beads alone (beads alone) or immunoisolated with latex beads coated with anti-MHC antibody Class II lAb (Billes + Ac a MHC II), anti-CD63 antibody (beads + Ac a CD63) or anti-CD81 antibody (Billes + Ac a CD81). The fluorescence emitted by the exosomes fixed on the beads was then measured by flow cytometry. The histograms represent the results of a typical experiment, and correspond to the normalized fluorescence means with respect to labeling with irrelevant antibody.

B. Exosomes were isolated from labeled RIB lAb-1 cells, prior to stimulation, with BODIPY-PC fluorescent phospholipids, BODIPYCeramides, NBD-PC, or ethanol alone. The in vivo labeled exosomes thus obtained were then treated in the same way as in A.

EXAMPLES
Cell cultures
The cells used for the production of exosomes are murine mast cell cells called RBL 2H3 (Rat Basophil Leukemia). They are cultured at 37 ° C. under a 5% CO 2 atmosphere, either by adherence in culture flasks of 175 cm 2 or in suspension at a concentration of 2 to 5. 10 5 cells / ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 4mM final L-Glutamine and a penicillin / streptomycin mixture (penicillin: 140 U / ml, streptomycin: 140 ug / ml final).

Preparation of natural exosomes
Exosome preparations are made from 1. 1 09 cells in suspension. Exosomes are isolated by a set of differential centrifugations. First of all,

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 the cells are concentrated and washed by centrifugations of 300 × g for 10 'and twice 5', then the cells are stimulated by a calcium ionophore, the lonomycin at 1 μM final, at a rate of 108 cells in 5 ml of DMEM medium for 20 minutes. C. Activated cells and cell debris are then removed by centrifugation 3 times 300Xg for 5 ', then 2 000Xg for 20' and finally 10 000Xg for 30 '. The exosomes are then isolated from these supernatants by ultracentrifugation at 100 000 xg for 30 minutes. The vesicle pellet thus obtained is taken up in PBS and again ultracentrifuged at 100 000 xg for 1 h 1 O. This last pellet is taken up in PBS and subjected to various analyzes.

Exosome analysis by electron microscopy (Fig. 1) The pellet of exosomes is taken up and fixed in 2% PBS / Paraformaldehyde buffer and then deposited on electron microscopy grids. The electron density contrast is achieved with a mixture of uranyl acetate and methyl cellulose, then the grids are analyzed by a Philips TEM CM120 electronic microscope. The results obtained are shown in FIG.

Lipid extraction and phospholipid composition of exosomes (Fig. 2) Lipid extraction is performed from cells prior to stimulation or exosomes in PBS, by the method of Bligh and Dyer which consists of a mixture of chloroform / methanol / water or PBS (1/1/1, v / v / v). The organic phase containing the lipids is taken up, dried under a jet of nitrogen and stored in chloroform / methanol (1/1, v / v).

In order to know the proportion of each class of phospholipids in the membranes of exosomes or cells, the lipid extracts were analyzed by thin layer chromatography of silica. The mobile phase corresponds to a mixture of solvents chloroform / methanol / acetic acid / water (75/45/12/6, v / v / v / v), called Skipsky. The classes of phospholipids separated by this

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 The solvents are phosphatidylethanolamines (PE, Rf = 0.79), phosphatidylserines / phosphatidylinositois (PS / PI, Rf = 0.59), phosphatidylcholines (PC, Rf = 0.48) and sphingomyelins (SM, Rf = 0). 27), and finally Iyso-phosphatidylcholines (LPC, Rf = 0.14). Phospholipids are revealed by iodine vapors. A lipid extract of hepatocytes serves as a control to locate the phospholipids. Each silica spot is scraped off and its phospholipid content is measured. This quantification is carried out by phosphorus determination since each phospholipid molecule contains a single phosphorus atom. This assay consists first of all in a mineralization of the molecules by perchloric acid and then the released phosphorus is assayed by the method of Fisk and Subarov which gives rise to a colorimetric reaction readable by spectrophotometry at a wavelength k of 800nm .

et de phosphatidyléthanolamine d'exosomes (fig. 3 et 4) Les extraits lipidiques d'exosomes ou de RBL au repos ont été analysés par HPLC en trois étapes afin de séparer successivement les classes de phospholipides (PC, PE, PS,...), les sous-classes (alkylacyls, AA ; diacyls, DA), et enfin les espèces moléculaires de chacune des deux sous-classes. Analysis of subclasses and molecular species of phosphatidylcholine

and phosphatidylethanolamine exosomes (Figs 3 and 4) The lipid extracts of exosomes or resting RBL were analyzed by HPLC in three steps in order to successively separate the classes of phospholipids (PC, PE, PS, ... ), the subclasses (alkylacyls, AA, diacyls, DA), and finally the molecular species of each of the two subclasses.

 The separation of the classes of phospholipids is carried out in normal phase on a NUCLEOSIL type silica column (7 μm, 250 × 4 mm). The lipid extracts to be injected are taken up in hexane / isopropanol (3/2, v / v).

The mobile phase consists of the mixture of acetonitrile / hexane / methanol / phosphoric acid 85% (918/30/30/17, 5; v / v / v / v) passing over the column at a flow rate of 1.5 mL / min. . The phospholipids thus separated are detected by UV at a wavelength zu of 206 nm. The PC and PE classes are then subjected to an in vitro treatment consisting of phospholipase C hydrolysis. The diglycerides thus obtained are then derivatized with anthroyle chloride in order to obtain fluorescent compounds (anthocyanin diglycerides, DGA).

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 formed from the PCs and PE are taken up in the mixture cyclohexane / ether (98/2; v / v) which also corresponds to the mobile phase passing over the column at a flow of 0.5 mL / min. Subclasses (AA and DA) anthers thus separated are detected by fluorescence at a wavelength λ of 460nm.

 The separation of the molecular species is carried out in inverse phase on an apolare (octadecyl-silica) column of the EQUISORB ODS2 type (51 μm, 250 × 4, 6 mm). The anthrollated subclasses are taken up in the acetonitrile / ether mixture (1/1, v / v). The mobile phase consists of the acetonitrile / propanol-2 mixture (85/15, v / v) passing over the column at a flow rate of 1.5 ml / min. The different peaks are detected by fluorescence at a wavelength zu of 460nm.

For each separation, the identification of the peaks was performed by comparing the chromatograms of cells or exosomes with the chromatograms obtained after injection of a platelet standard whose subclass and molecular species composition is well established (Thevenon C. et al., J. of Chromatography B, 1998). Various commercial standards have also identified some peaks.

non naturels en quantité connue : le stigmastérol (3ug) dont la structure est proche de celle du cholestérol et le 1, 3 dimyristoyl glycérol (diglycéride 14 : 0- 14 : 0 ; 0, 5 ug). L'ensemble a été repris par de l'acétate d'éthyle puis les échantillons ont été analysés par chromatographie en phase gazeuse sur une colonne capillaire de silice de type Hewlett Packard Ultra 1 (5m X 0,31mm) recouverte par pontage covalent de diméthyl siloxane. La température du four a été programmée à partir de 205 C jusqu'à 345 C à raison de 6 C/min. Le gaz vecteur ici utilisé est l'hydrogène passant sur la colonne à une pression de 0,5 bar. Après intégration, les pics correspondant au cholestérol et aux Analysis of the neutral lipids of exosomes and RBL (Fig. 5) Total lipid extracts of exosomes or RBL, whose phosphorus content was known, were dried in the presence of 2 internal standards

non-natural in known quantity: the stigmasterol (3ug) whose structure is close to that of cholesterol and the 1,3 dimyristoyl glycerol (diglyceride 14: 0-14: 0, 0, 5 ug). The whole was taken up in ethyl acetate and the samples were then analyzed by gas chromatography on a Hewlett Packard Ultra 1 (5m × 0.31 mm) silica capillary column covered by covalent dimethyl bridging. siloxane. The oven temperature was programmed from 205 C to 345 C at a rate of 6 C / min. The carrier gas used here is hydrogen passing over the column at a pressure of 0.5 bar. After integration, the peaks corresponding to cholesterol and

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 Diglycerides were identified on the chromatograms and quantified using internal standards.

The typical vesicle composition is provided in Table 1.

Measurement of membrane lipid dynamics of exosomes and RBLs (fig.

6) and 7) The microviscosity (r1) of the membranes was measured by fluorescence polarization as a function of temperature, as previously described (Shinitzky M., Barenholz Y., Biochim Biophys.Acta, 1978) using of a fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) integrated into the membranes of exosomes or cells. The values of 11 obtained for different temperatures make it possible to draw a line Ln il = f (1 / T) (Arrhenius diagram). According to the equation previously established (Kauzmann W. et al., J. Am Chem Soc., 1940) Ti = Ae is Ln r) = LnA + AE / RT (A is a characteristic constant of the system, R is the constant of perfect gases, AE is the energy of activation of the flux), one can deduce the expression of the slope of the line is AE / R, which allows us to obtain AE, important structural parameter of the lipidic membranes . The fluorescence anisotropy measurements make it possible to compare the variations of microviscosity from one structure to another as a function of the temperature.

In this regard, Figure 7 reports fluorescence anisotropy values at 25 OC and 37 C, for resting (n = 3), activated (n = 2) and exosome (n = 3) cells. The fluorescence anisotropy corresponds to a ratio of fluorescence intensities; it is directly related to membrane microviscosity. There is thus a higher microviscosity of the exosomes at 25 ° C. as at 37 ° C., compared to the cells at rest or activated. Our results therefore demonstrate a higher stiffness of exosomes. When the medium is completely rigid, the anisotropy reaches a maximum value of 0.4.

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Use of fluorescent lipids to monitor the fate of exosomes (Fig. 8) - Fluorescent labeling of exosomes
RBL cells stably expressing the murine class II MHC molecule lAb as well as the invariant chain li were used to produce exosomes as previously described. Two types of fluorescent markings were made in parallel from the same batch of cells divided in two.

The first batch of cells was stimulated directly as previously described and labeling was carried out on these vesicles by incubating them with 3 types of fluorescent phospholipids: BODIPY-phosphatidylcholine, BODIPY-ceramide, or NBD-phosphatidylcholine for 30 minutes. 37 C in the dark. The 3 fluorescent phospholipids being stored in ethanol, an incubation with ethanol alone was carried out as a control. The exosomes thus labeled in vitro were then washed by two ultracentrifugations at 100 000 g in PBS.

The second batch of cells was first labeled with the three fluorescent phospholipids and ethanol separately for 30 to 37 C. The cells were washed and the fluorescence was then chased for 30 to 37 C in medium. DMEM alone. The cells were then stimulated to release the exosomes as previously described. The last pellet of exosomes thus labeled in vivo was washed once more and then taken up with PBS.

- Analysis of fluorescent exosomes by flow cytometry
The too small size of the exosomes (60-80nm) does not allow a direct analysis by flow cytometry. The exosomes were first fixed on aldehyde-sulphate-coated latex beads (dia = 1 μm) by a 15 'incubation at room temperature at the rate of 10 μg of exosomes per 0.5 μl of beads.

Then the mixture was diluted in PBS and again incubated for 1 h with stirring at room temperature. Glycine was then added to

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 100mM for 30 'at room temperature to saturate the aldehyde groups of the beads. In order to separate different populations of exosomes, 4 types of beads were used: beads alone to observe all the exosomes, beads coated with an anti-IAb antibody, beads coated with an anti-CD63 antibody and finally, beads coated with an anti-CD81 antibody. In order to normalize the fluorescence values, an unsuitable antibody coupled to a fluorochrome emitting in a different wavelength of fluorescent phospholipids, was incubated with the beads / exosomes complexes.

The beads coated with fluorescent exosomes labeled in vivo or in vitro were then analyzed by flow cytometry.

The results obtained reveal a particular lipid structure, based mainly on: - a balance of the molar proportions of the four main classes of phospholipids (PC, PE, PS / PI, MS) - a presence of PC and PE disaturated molecular species - a decrease in total diglycerides, - structural parameters of membrane fluidity distinct from the original cells.

The present invention thus demonstrates a lipid structure of the physiologically stable vesicular type, since isolated from biological vesicles capable of being carried by the body without being degraded.

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TABLE 1

<Tb>
<tb> AVERAGE <SEP> AND <SEP> VALUES <SEP> LIMITS <SEP> NORMALIZED <SEP> A <SEP> 100 <SEP>%
<tb> EXOSOMES <SEP> MASTOCYTES
<tb> EXOSOMES <SEP> DENDRITICS
<tb> RBL
<tb> Average <SEP> Average <SEP> Average <SEP> Average
<tb> - <SEP> average <SEP> - <SEP> - <SEP> average <SEP> -
<tb> -SD <SEP> + SD <SEP> -SD <SEP> + SD
<tb> PHOSPHOLIPIDS
<tb> 8.55 <SEP> 12.83 <SEP> 15.01 <SEP> 6.04 <SEP> 12.28 <SEP> 17.69
<tb> U, / <KK1
<tb> LYSO-PC <SEP> 3.75 <SEP> 5.49 <SEP> 5.08 <SEP> 4.59 <SEP> 4.53 <SEP> 6.21
<tb> SPHINGOMYELINE <SEP> 8.26 <SEP> 12.19 <SEP> 9.60 <SEP> 12.24 <SEP> 9.06 <SEP> 12.41
<tb> PC-DISATUREES <SEP> 4.81 <SEP> 3.17 <SEP> 3.84 <SEP> 3.52 <SEP> 4.27 <SEP> 3.00
<tb> PC-MIXTURE <SEP> 19.22 <SEP> 12.68 <SEP> 15.36 <SEP> 14.07 <SE> 16.45 <SE> 12.00
<tb> PS <SEP> 6.01 <SEP> 5.79 <SEP> 5.65 <SEP> 6.89 <SEP> 5.82 <SEP> 5.17
<tb> PI <SEP> 6.01 <SEP> 5.79 <SEP> 5.65 <SEP> 6.89 <SEP> 5.82 <SEP> 5.17
<tb> PE-DISATUREES <SEP> 2.81 <SEP> 2.58 <SEP> 2.85 <SEP> 2.62 <SEP> 2.85 <SEP> 2.61
<tb> PE-MIXTURE <SEP> 14.45 <SEP> 13.27 <SEP> 14.65 <SEP> 13.45 <SEP> 14.62 <SEP> 13.42
<tb> PHOSPHOLIPIDS
<tb> 3.80 <SEP> 6.42 <SEP> 4.29 <SEP> 6.04 <SEP> 4.09 <SEP> 5.17
<tb> 0 <Rf <0.14
<tb> CHOLESTEROL <SEP> 15.68 <SEP> 14, <SEP> 17 <SEP> 13.01 <SEP> 16.61 <SEP> 14, <SEP> 47 <SEP> 12.38
<tb> DIGLYCERIDES <SEP> 6.65 <SEP> 5.61 <SEP> 5.00 <SEP> 7.05 <SEP> 5.73 <SEP> 4.76
<tb> TOTAL <SEP> 100.00 <SEP> 99.99 <SEP> 99.99 <SEP> 100.01 <SEP> 99.99 <SEP> 99.99
<Tb>

Claims (39)

 1. An artificial lipid vesicle characterized in that it comprises a lipid wall comprising cholesterol and disaturated phospholipids, the disaturated phospholipids preferably representing at least 3% of the total constituents of the wall. 2. Vesicle according to claim 1, characterized in that the disaturated phospholipids comprise disaturated phosphatidylcholines (PC), disulfated phosphatidylethanolamines (PE) or a mixture thereof. 3. Vesicle according to one of claims 1 or 2, characterized in that the fatty acids phospholipids have a chain length, identical or different, between 14 and 22 carbon atoms, preferably between 14 and 20, even more preferably between 16 and 20. 4. The vesicle of claim 3, characterized in that the disaturated phospholipids comprise two fatty chains, identical or different, selected from C16: 0 and C18: 0. 5. The vesicle as claimed in any one of the preceding claims, characterized in that it comprises from 5 to 7% of disaturated phospholipids. 6. Vesicle according to any one of the preceding claims, characterized in that it comprises 12 to 18% of cholesterol. 7. Vesicle according to any one of the preceding claims, characterized in that it comprises phosphatidylserine (PS), preferably from 3% to 8%. <Desc / Clms Page number 40> 8. Vesicle according to any one of the preceding claims, characterized in that it comprises phosphatidylinositol (P1), preferably from 3 to 10%. 9. Vesicle according to any one of the preceding claims, characterized in that it comprises sphingomyelin (SM), preferably from 6 to 15%. 10. The vesicle as claimed in any one of the preceding claims, characterized in that it further comprises Iso-phosphatidylcholine (LPC) and / or Iso-phosphatidylethanolamine (LPE). 11. The vesicle according to any one of the preceding claims, characterized in that it further comprises glycolipids. 12. Vesicle according to any one of the preceding claims, characterized in that it further comprises polar phospholipids, apolary, neutral, charged and / or unsaturated. 13. The vesicle of claim 12, characterized in that it comprises unsaturated phosphatidylcholine and / or phosphatidylethanolamine. 14. Vesicle according to any one of the preceding claims, characterized in that it comprises less than 10% of diglycerides. 15. A vesicle according to any one of the preceding claims, characterized in that it comprises. at least 3% of PC and / or disaturated PE,

 . from 3 to 10% of P1. from 3 to 8% of PS,. from 6 to 15% of S) v), and <Desc / Clms Page number 41>  . from 12 to 18% of cholesterol. 16. Vesicle according to any one of the preceding claims, characterized in that it has a viscosity at 37 C, measured by fluorescence anisotropy, greater than 0.15, preferably 0.20. 17. The vesicle according to one of the preceding claims, characterized in that its diameter is between 50 and 150 nm, preferably between 60 and 100 nm. 18. The vesicle as claimed in any one of the preceding claims, characterized in that it comprises at least one molecule of interest, preferably chosen from a marker, an addressing molecule and an active molecule. 19. The vesicle according to claim 18, characterized in that the addressing molecule is chosen from an antibody, an antigen, a particular ligand, a particular receptor, a substrate and an enzyme. 20. The vesicle according to claim 18, characterized in that the active molecule is chosen from a protein, a peptide, a drug active principle, a nucleic acid and a lipid. 21. A method for producing a vesicle according to any one of the preceding claims, characterized in that it comprises at least the following steps: a) bringing into contact with the constituents of the vesicle, in particular cholesterol and phospholipids of which at least a portion is disaturated, b) the treatment of the mixture obtained at the end of step a) to form vesicles comprising a lipid wall, and c) the recovery of the vesicles obtained at the end of step b ). <Desc / Clms Page number 42> 22. Process according to claim 21, characterized in that the treatment carried out during step b) is an extrusion or ultrasonication process carried out on the mixture obtained at the end of step a). 23. The method of claim 22, characterized in that the method implemented in step b) is an extrusion process. 24. Method according to one of claims 21 to 23, characterized in that it further comprises a step d) purifying the vesicles recovered at the end of step c). 25. Production method according to one of claims 21 to 24, characterized in that the purification step d) is a step of gelfiltration, ultra-centrifugation, ultrafiltration, column filtration, chromatography or extraction by a system magnetic. 26. Method according to one of claims 21 to 25, characterized in that it further comprises an intermediate step a ') of suspending the lipid mixture in a hydration buffer. 27. Method according to one of claims 21 to 26, characterized in that it further comprises a step of introduction into the aqueous phase of the vesicles, in the lipid wall or on the surface thereof of a molecule interest. 28. The method of claim 27, characterized in that the molecule of interest is introduced by electroinsertion. 29. The method of claim 27, characterized in that the molecule of interest is introduced by adding it in the hydration buffer. <Desc / Clms Page number 43> 30. Vesicle obtained by the implementation of a method according to one of claims 21 to 29. 31. Use of a vesicle according to one of claims 1 to 20 and for the preparation of a composition for the transport of one or more molecules of interest. 32. Use according to claim 31, characterized in that the molecule of interest is an active ingredient. 33. Use according to claim 32, characterized in that the active ingredient is selected from a molecule of a hormonal or neuroendocrine nature, a carbohydrate, a vitamin, an essential fatty acid, a protein and a nucleic acid. 34. Use according to claim 32 for the preparation of a vaccine. 35. Use according to claim 32 for the preparation of a pharmaceutical composition. 36. Use according to claim 32 for the preparation of a cosmetic composition. 37. Use of a vesicle according to one of claims 1 to 20 and 30 in the context of one or more stages of ripening and / or maturation of agri-food preparations. 38. Vaccine comprising a vesicle according to one of claims 1 to 20 and 30. 39. Pharmaceutical composition comprising a vesicle according to one of claims 1 to 20 and 30.