WO1998029102A1 - Homogeneously charged particulate vector and pharmaceutical or cosmetic composition containing same - Google Patents

Abstract

The invention concerns a particulate vector comprising: a non-liquid hydrophilic nucleus; an amphiphilic lamella characterised in that the nucleus carries a global cationic, anionic or neutral charge and that the amphiphilic lamella carries a global charge of same polarity as that carried by the nucleus. The invention also concerns a pharmaceutical or cosmetic composition or a nutrient additive containing such a vector.

Description

HOMEOCHARGE PARTICULATE VECTOR AND PHARMACEUTICAL OR COSMETIC COMPOSITION CONTAINING THE SAME

The present invention relates to complex particles, useful for the transport and targeting of active principles, which are compatible with the biological functions of the organism and of the cells.

Particulate vectors have been described in EP 344 040; these vectors comprise a hydrophilic nucleus, surrounded by a first lipid layer, then by a second, more external layer, of amphiphilic compounds linked to the first lipid layer by hydrophobic interactions.

WO 94/20078 was filed for another generation of particulate vectors, or "BVSM-L", consisting of a hydrophilic nucleus and an outer layer composed at least in part of amphiphilic compounds, directly associated with the nucleus by ionic bonds or hydrophobic interactions. The nucleus of these vectors can be grafted with ionic, acidic or basic ligands, giving it a charge which can be used to change certain active ingredients. Unexpectedly, the Applicant has now found that particulate vectors, in which both the core and the outer layer carry an ionic charge can be obtained stably.

In addition, a generally neutral core can be associated with an unloaded external sheet.

In fact, contrary to the observations and knowledge generally accepted in the state of the art, charges of the same sign may be present on the various components of the vector.

This is why the present invention relates to a particulate vector comprising: a non-liquid hydrophilic core an amphiphilic sheet characterized in that the core carries an overall cationic, anionic or neutral charge and in that the amphiphilic sheet carries an overall charge of the same polarity as that carried by the core.

Such vectors, which associate an internal hydrophilic charged structure, with an external structure of similar charge and of amphiphilic character, will be called in the following "homeo-biovectors" or "homeocharged biovectors".

The layer of amphiphilic compounds is in an external situation with respect to the nucleus of the biomeector; it can be discontinuous or continuous, and / or present interdigitations with the constituents of the nucleus.

While one might expect that electrostatic repulsions between the amphiphilic sheet and the nucleus oppose this type of association, homeo-biovectors are characterized by a remarkable stability of the supra-molecular association in time.

They also have a specific organization of the amphiphilic membrane, with an increase in the molecular packing of lipids and a high degree of cooperativity. This organization is modulated by the ionic charge of the hydrophilic nucleus. It can in particular be objectified by measuring the phase transition temperatures.

Homeo-biovectors, cationic or anionic, can be used to transport molecules of interest. According to one of the preferred aspects of the invention, these vectors will contain at least one active principle, associated with the nucleus and / or the amphiphilic sheet.

The composition of the core and / or of the amphiphilic sheet will be adapted by a person skilled in the art according to the type of molecule to be associated with it and the application envisaged.

In one of the embodiments of the invention, the particulate vector will comprise at least: a non-liquid hydrophilic nucleus carrying a cationic or anionic charge an amphiphilic sheet carrying a cationic or anionic charge, of the same polarity as that carried by the nucleus. In another embodiment also included in the invention, the vector comprises: a non-liquid hydrophilic core, electrically neutral an overall neutral amphiphilic sheet, comprising zwitterionic phospholipids. In particular, the amphiphilic sheet may consist of one or more components, with a view to creating a physico-chemical environment adapted to the active principle and to the desired function.

Preferably, at least one constituent of the amphiphilic sheet is chosen from the group consisting of anionic, cationic or zwitterionic phospholipids, natural or synthetic, ceramides, fatty acids, steroids, lipids, glycolipids, lipoproteins and surfactants , carrying a charge of the same polarity as that of the core.

Among the constituents particularly suitable for the preparation of homeobiovectors, mention may be made of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cholesterol, and their derivatives. These constituents can be alone or as a mixture, possibly associated with other uncharged constituents. According to one of the aspects of the invention, at least one constituent of the amphiphilic sheet is chosen from dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, dioleyloxypropyl trimethylphosphyltrim dipalmitoyl phosphatidic acid, dipalmitoyl phosphatidylserine and stearylamine. The proportion of constituents of the amphiphilic sheet carrying a charge of the same polarity as that of the core is in all cases greater than 0%, in particular greater than 0.001%, and less than or equal to 100%. It can in particular be between approximately 5% and 30% of these constituents, depending on the charge carried by the hydrophilic core.

This core preferably consists essentially of compounds chosen from oligomers and hydrophilic polymers, naturally or chemically crosslinked. More particularly, the nucleus consists of saccharide derivatives, of different molecular weights, in particular of polysaccharides or oligosaccharides naturally or chemically crosslinked, carrying a cationic or anionic charge.

One can thus use starch, dextran or cellulose and their substituted derivatives, as well as their hydrolysis products such as in particular dextrins and maltodextrins. The salts and esters of these polymers or oligomers (which naturally contain hydroxy functions) will also be suitable for carrying out the invention. Crosslinking can be obtained by different techniques known to those skilled in the art. Methods for preparing nuclei which can enter into the composition of homeobiovectors are described in particular in WO 94/20078 and WO 92/21329. Advantageously, ionic, acidic or basic ligands are attached to the nucleus.

The binding of the ionic ligands to the nucleus will be carried out after this crosslinking step or at the same time, in certain embodiments. This second possibility is particularly applicable for "acid" nuclei, in particular by - use of a reagent simultaneously ensuring crosslinking and grafting ligands; in this regard, a reagent such as phosphorus oxychloride, used at low temperature (of the order of 0 ° to 10 ° C, preferably around 4 ° C), is particularly suitable.

In general, any compound capable of nucleophilic attack by the hydroxyls of the oligo or of the polysaccharide can be grafted onto the matrix; mention may thus be made of epoxides, acid chlorides, anhydrides, alkyl halides, isothiocyanates, chlorotriazines, etc.

Preferably, the ionic ligands carry at least one function chosen from the group comprising: phosphates, sulfates, carboxylic acids, quaternary ammonium, primary amines, secondary amines. The reagents can for example be chosen from succinic acid, phosphoric acid, citric acid, glycine, alanine, glutamic acid, aspartic acid or their derivatives, choline, 3-hydroxycholine, 2 - (dimethylamino) ethanol, 2- (dimethylamino) ethylamine and glycydyltrimethylammonium chloride.

The initial concentration of polysaccharides is preferably between 100 and 500 g / l, depending on the grafting and crosslinking yields desired, and depending on the type of gel sought (hardness, homogeneity, mesh size).

In any event, the functionalization step of the core will advantageously be carried out before grinding and homogenizing the gel to obtain particles of controlled size and hardness, in which the charges are distributed in a homogeneous and regular manner.

This homogenization can be carried out by high pressure (700 to 1,200 bars), and followed by one or more filtrations.

Preferably, the hydrophilic nucleus is directly associated with the amphiphilic sheet, by interactions which can in particular be of the hydrogen bond type. The homeo-biovectors are prepared by mixing the nuclei dispersed in an aqueous solvent with the constituent (s) of the sheet. The synthesis can in particular be carried out with stirring, at a temperature higher than the gel-liquid phase transition temperature of the amphiphilic constituents, or by pressure homogenization at a temperature higher than the gel-liquid phase transition temperature of the amphiphilic constituents, or any other suitable method.

After elimination of the residual organic solvents, the homeo-biovectors are sterilized in particular by filtration and stored under sterile conditions.

The average diameter of homeo-biovectors can vary from 10 nm to 100 μm.

In one of the embodiments of the invention, particularly suitable for pharmaceutical applications, this diameter is between 10 nm and 5 μm, in particular approximately 15 nm to 80 nm.

Different types of molecules could be associated with the Tiomeo-biovector, and in particular active principles carrying a global charge: anionic, cationic or zwitterionic. These molecules may in particular be chosen from peptides, proteins, lipoproteins, proteoglycans, lipopolysaccharides, fatty acids, triglycerides, phospholipids, glycolipids, steroids, oligosaccharides, nucleotide sequences, nucleoside sequences, nucleic acids, vitamins and porphyrins.

They can be useful in different fields, and homeo-biovectors can, in different embodiments of the invention, be associated with at least one active principle chosen from the following group: antibiotics, antiseptics, analgesics, anesthetics , anti-inflammatory drugs, antivirals, anticancer drugs, immunomodulators and cellular mediators, antihistamines, antimuscarinics, anti-anemic agents, hemostatic agents, anti-diabetics, cardiovascular agents, anti-depressants, anti-epileptics, anxiolytics, hypnotics, sedatives, antipsychotics, appetite suppressants, vaccines, natural or synthetic hormones and their derivatives, insecticides, fungicides, antimalarials, asthma medications, antibodies, antigens and their fragments, diagnostic agents, NMR diagnostic agents, and markers radioactive. Among cell mediators, mention may be made of cytokines, chosen from the group comprising: interleukins, interferons, TNF, G-CSF, M-CSF, GM-CSF and MIF.

The structure and the charge of the iomeo-biovector may be adapted according to the nature of the active principle, the effectiveness of which will be improved in particular by an accentuated membrane surface potential, due to the presence of the underlying nucleus of similar charge.

Such vectors are particularly advantageous since they make it possible to associate the targeting characteristics provided in particular by the hydrophilic nucleus and the specific cellular interactions of an amphiphilic membrane, in particular phospholipid. In addition, the lipid membrane structure is stabilized by the presence of the nucleus. The repulsive forces between homeo-biovectors limit interparticle interactions and therefore increase their lifespan.

Finally, depending on the physicochemical nature of the molecule transported, it could be associated with one and / or the other of the structures of the Tiomeo-biovector.

For example, in the case of association of molecules of interest such as proteins, model membrane systems described in the literature (phospholipid monolayers, liposomes) show that the interactions between the membrane and the proteins can be favored if the membrane is loaded with a large surface potential and has a strong molecular packing.

In particular, anionic homeo-biovectors can be associated with cationic drugs both at the nucleus and at the membrane (TNF, IL2). It has been widely described in the literature that an anionic membrane promotes the association, stability and cellular penetration of membrane proteins (cytochrome c, hemagglutinin). The anionic charge of the nuclei plays an important role in interactions with cell membranes.

The combination of these two factors (anion nucleus associated with the anionic membrane), within the framework of a therapeutic application, should confer a synergy of action for a favorable delivery of the drug. This effect would not be observable when using each system (anion nucleus or anionic liposomes) taken in isolation.

Schônhoff et al. (Progr. Colloid Polymer. Sri., 1992, 89, 243-248) has shown that the incorporation of membrane proteins into the lipid membrane is optimized with an anionic membrane which has a strong molecular packing.

Numerous publications referring to the interaction of Cytochromes (Cyt.) With model membranes have shown that: the binding of Cyt.c to a monolayer depends on the charge density of the lipid film. Penetration into the membrane is favored by the presence of surface charges. in the case of membranes made up of phospholipid mixtures, Cyt.c oxidase discriminates the various membrane constituents according to their polar head (with a preference for anionic charges) and according to the phase state of the membrane (preference for a membrane with high molecular packing) (de Cuyper, Eur. J. Biochem., 1980, 104, 397-405). the interactions between the membrane and Cyt.b5 are reinforced in the presence of anionic lipids (electrostatic interactions). The effect of these interactions is to promote membrane insertion (protection against denaturation) (Dufourcq-Faucon, FEBS Letters, 1975, 57 (1) 1 12-1 16). The presence of saturated lipids (membrane with high molecular packing) is recommended (Dufourcq-Bernon, Biochem. Biophys. Acta, 1976, 433, 252-263). In these reconstituted systems, it appears that the anionic polar head lipids are necessary to facilitate association and allow good orientation of the hemic groups (Chester, Biophys. J. 1992, 61, 1224-1243).

In the case of cyclic somatostatin, the presence of anionic charges in the membrane has the effect of promoting the association and the insertion of the peptide. This insertion is accompanied by a reorganization of the neighboring polar heads (Beschiaschvili Biochem. Biophys. Acta, 1991, 1061, 78-84).

The lipid mixture of phosphatidylcholine and phosphatidylglycerol (DPPC / DPPG) results in an anionic membrane which participates in the stabilization and orientation of surfactant proteins of the lung inserted into the membranes of the reconstituted systems (Shiffer, Biochemistry 1993, 32, 590-597 ).

A bacterial toxin like colicin A associates then inserts itself into an anionic membrane. In addition, a membrane potential allows a correct arrangement of the protein which then constitutes an active transmembrane channel (Massotte, Biochemistry (1993, 32, 13787-13794) (Stroud, Curr. Opinion Struct. Biol. 1995, 5, 51 1 -520), (Géli, Subcellular Biochem. 1994, 22, 21-69).

Taking the example of the ADP / ATP membrane transporter, it is shown that the surface potential generated by anionic lipids is a predominant parameter for modulating the affinity parameters of membrane proteins and for restoring their biological activity. in a reconstituted system (Krâmer, Methods in Enzymology, 1989,

171, 387-384).

The antibiotic Adriamycin enters the lipid bilayer thanks to the interactions between the polar heads of the phospholipids (phosphate group) and the aminoglycosyl group of the peptide.

This membrane penetration strongly depends on the molecular packing (Dupou (Eur. J. Biochem. 1989, 181, 695-702).

Anionic phospholipids have been shown to determine, in various cases, the interactions of proteins with membranes. These interactions result in protein insertions or translocations.

Reconstituted systems of proteoliposome type generally depend on the presence of specific phospholipids within the membrane (contribution of charges at the level of the polar heads, molecular cohesion and compaction).

In the case of cationic homeo-biovectors, it is possible to associate with the nucleus and / or the amphiphilic sheet drugs or anionic prodrugs such as nucleic arides (DNA, RNA, oligonucleotides), lipids, proteins. Indeed, cationic liposomes have been used for this type of molecule, in particular in gene therapy. In particular, they allow cytoplasmic delivery of DNA.

However, this work shows a certain number of limitations.

The gene expression is most often weak and transient. In addition, the DNA / liposome complex is too easily degraded and eliminated by the body. In some cases, the co-injection of promoters of the gene promoter is necessary in order to obtain the production of the protein. Finally, with regard to fusogenic proteoliposomes, the results are not very reproducible. In the context of the present invention, it has been shown that the type of association present in homeo-biovectors stiffens and stabilizes a phospholipid membrane. This type of stabilization could have an important role in vivo. In fact, cationic membranes interact strongly with their environment to promote (1) bioadhesion on the cell surface and (2) cytoplasmic delivery with the associated active ingredient.

Cationic homeo-biovectors are supramolecular structures which differ from liposomal structures, fusogenic proteoliposomal structures, viral vectors or soluble DNA complexes (Legendre-Skoza, Proc. Natl. Sri. USA, 1993, 90, 893-397), (Felgner-Rhodes, Nature, 1991, 349, 351-382): these homeobiovectors will make it possible to associate molecules (oligonucleotides, DNA, RNA) both on the surface (at the membrane level) and at depth (at the level of the polysaccharide nucleus cationic) where they will be stabilized and protected from enzymatic degradation. The presence of fusogenic proteins on the surface of cationic homeobiovectors can be envisaged: they will act in synergy with the charge effects to deliver the active molecules in the cytoplasm of cells.

Given the results of non-immunogenicity and biodistribution obtained in vivo with cationic biovectors, the intra-tissue residence time of homeo-biovectors is long enough to locally consider the delivery of exogenous genetic material as part of gene therapy. (example: compensate for hormonal deficits by injecting into the deficient tissue complexes "homeo-biovectors / DNA producing the hormonal protein").

The subject of the invention is also a pharmaceutical composition, characterized in that it contains a particulate vector as described above. According to another aspect, the subject of the invention is a cosmetic composition, characterized in that it contains a particulate vector as described above and cosmetologically acceptable excipients.

Homeo-biovectors also have applications as a food additive, or in the field of water treatment. The invention will be illustrated by the following examples.

In these examples, reference is made to the following figures. Figure 1: Separation of polysaccharide nuclei (NPS), liposomes and biovectors on sucrose density gradients (0% -35%). (A): separation of NPS labeled with fluorescein (O) and liposomes labeled with DPH (0). (B): separation of the biovectors whose NPS is labeled with fluorescein (•) and the membrane with DPH (^). Figure 2: Phase transition of lipids organized in the form of liposomes or iomeo-biovectors. Profiles obtained by measuring the fluorescence polarization p of DPH as a function of temperature for 1 ml of sample conditioned in water.

(A): DPPC liposomes and neutral homeo-biovectors. (B): DPPC / DPPG liposomes and anionic homeo-biovectors. (C): DPPC / stearylamine liposomes and cationic homeo-biovectors. Figure 3: Influence of cobalt ions on the fluorescence of the NBD-PC probe inserted in the liposomal membrane (0) and in the biovector membrane (•). The lipid concentration is 27.2 μM and the temperature is 20 ° C.

Example 1: Preparation of anionic homeo-biovectors The preparation of anionic homeo-biovectors takes place in two main stages: (1) the synthesis of anionic polysaccharide nuclei, (2) the association of lipids with anionic NPS.

1. Synthesis of anionic NPS 500 g of Glucidex maltodextrin (ROQUETTE, Lestrem, France) are introduced into a 10 liter reactor (TRIMIX) with 2 liters demineralized water. After solubilization at 4 ° C, 500 ml of 10M sodium hydroxide (NaOH) are added with mechanical stirring.

When the temperature of the solution is stabilized at 4 ° C, 1700 ml of 10M NaOH and 283.3 ml of phosphorus oxychloride (P0C1 ₃ ) are introduced under controlled flow rates. The crosslinking reaction takes place with mechanical stirring for 20 hours. At the end of the addition of the reactants, the reaction mixture is stirred for 15 minutes. A volume of 5 liters of demineralized water is then added. The pH is brought back to 7.0 by neutralization with glacial acetic acid. The iydrogel obtained is ground using a high pressure homogenizer (RANNIE Lab). The pressure exerted is 500 bars. At the end of this step, the dispersed matrices have an average diameter of 60 nm.

The purification is carried out by successive stages of (1) 0.45 μm microfiltration to eliminate particles of too large a size, (2) diai fi ltration at constant volume to eliminate small molecules (salts, polysaccharide fragments).

Finally, the anionic NPS are concentrated, recovered, in sterile bottles and stored at -20 ° C.

2. Synthesis of anionic homeo-biovectors

The thawed anionic polysaccharide nuclei are diluted with osmosis water in a glass receptacle in the proportion of lmg / lml (example: 250 mg NPS / 250 ml). The dispersion is subjected to magnetic stirring (5-10 minutes) then homogenized (RANNIE Minilab) at 400 bars for 3 minutes. The dispersion of NPS is placed in a water bath at 80 ° C.

The lipids (example: mixture of DPPC with DPPG), in powder form, are weighed so that the total mass represents, for example, 30% (w / w) of the mass of the NPS (75 mg of lipids per 250 mg of NPS). The constituent lipids of the membrane are mixed and solubilized with 2 ml of 95% ethanol (v / v). If the loaded lipids represent, for example, 5% (w / w) of the total lipids, weigh 3.75 mg of anionic lipid and 71.25 mg of DPPC.

The homogenizer is brought to a temperature of 60 ° C minimum by circulating water in a closed circuit. Concomitantly, the NPS dispersed at 80 ° C. are subjected to magnetic stirring and the ethanolic solution of lipids is injected into the dispersion of NPS.

The "NPS / lipids" dispersion at 80 ° C. is then introduced into the homogenizer and is homogenized at 450 bars for 25 minutes in a closed circuit at minimum 60 ° C. At the end of this step, the preparation is introduced into a glass flask and then subjected to low pressure at 60 ° C to remove the ethanol remaining in solution.

The anionic homeo-biovectors thus obtained are then stored under sterile conditions after filtration through 0.2 μm.

Example 2: Preparation of cationic homeo-biovectors

The preparation of cationic homeo-biovectors takes place in two main stages: (1) synthesis of cationic polysaccharide nuclei (NPS), (2) association of cationic lipids with cationic NPS.

1. Synthesis of cationic NPS

500 g of Glucidex maltodextrins (ROQUETTE) are dissolved with 0.880 liters of water in a reactor thermostatically controlled at 20 ° C with stirring. Then, about 7 g of NaBH 4 are introduced and the mixture is left stirring for 1 hour.

200 ml of 10 M NaOH are added and then 30.25 ml of epichlorydrine (Fluka). After 12 hours of reaction, 382.3 g of glycidyltrimethylammonium chloride (Fluka) are introduced and the mixture is stirred for 10 hours. The gel is diluted with 8 liters of demineralized water and neutralized with glacial acetic acid.

Lliydrogel obtained is ground using a high pressure homogenizer (RANNIE Lab). The pressure exerted is 400 bars. At the end of this step, the dispersed matrices have an average diameter of 60 nm.

The purification is carried out by successive stages of (1) 0.45 μm microfiltration to eliminate particles of too large a size, (2) diafiltration at constant volume to eliminate small molecules (salts, polysaccharide fragments).

Finally, the cationic NPS are concentrated, collected in sterile bottles and stored at -20 ° C.

2. Synthesis of cationic homeo-biovectors The thawed cationic polysaccharide nuclei are diluted with osmosis water in a glass receptacle in the proportion of 1 mg of NPS / 1 ml of water. The dispersion is first placed under magnetic stirring (5-10 minutes) and then homogenized (RANNIE Minilab) at 400 bars for 3 minutes. The dispersion of NPS is placed in a water bath at 80 ° C.

The lipids (example: mixture of DPPC with stearylamine), in the form of powder, are weighed so that the total mass represents, for example 30% (w / w) of the mass of the NPS (75 mg of lipids for 250 mg from NPS). The constituent lipids of the membrane are mixed and solubilized with 2 ml of 95% ethanol (v / v). If the loaded lipids represent, for example, 5% (w / w) of the total lipids, weigh 3.75 mg of cationic lipid and 71.25 mg of DPPC.

The homogenizer is brought to a temperature of 60 ° C minimum by circulating water in a closed circuit. Concomitantly, the NPS dispersed at 80 ° C. are subjected to magnetic stirring and the ethanolic lipid solution is injected into the NPS dispersion.

The heating water in the homogenizer circuit is removed and replaced by the "NPS / lipids" dispersion at 80 ° C. This new dispersion is homogenized at 450 bars for 25 minutes in a closed circuit at minimum 60 ° C. At the end of this step, the preparation is introduced into a glass flask and then subjected to low pressure at 60 ° C to remove the ethanol remaining in solution. The cationic homeo-biovectors thus obtained are then stored under sterile conditions after filtration through 0.2 μm.

Example 3: Preparation of neutral homeo-biovectors

The preparation of neutral homeo-biovectors takes place in two main stages: (1) the synthesis of neutral polysaccharide nuclei (NPS), (2) the association of zwitterionic lipids with neutral NPS.

1. Summary of neutral NPS

500 g of Glucidex maltodextrins (ROQUETTE) are dissolved with 0.880 liters of water in a reactor thermostatically controlled at 20 ° C with stirring. Introduce about 7 g of NaBH ₄ and leave for 1 hour with stirring. 220 ml of 10 M NaOH are added and then 30.25 ml of epichlorydrine (Fluka). After 12 hours of reaction, dilute the gel with 8 liters of demineralized water and neutralize by adding glacial acetic acid.

The hydrogel obtained is ground using a high pressure homogenizer (RANNIE Lab). The pressure exerted is 400 bars. At the end of this step, the dispersed matrices have an average diameter of 60 nm. The purification is carried out by successive stages of (1) 0.45 μm microfiltration to remove excessively large particles. important, (2) diafiltration at constant volume to remove small molecules (salts, polysaccharide fragments).

Finally, the NPS are concentrated, collected in sterile bottles and stored at -20 ° C.

2. Synthesis of neutral homeo-biovectors

The thawed neutral polysaccharide cores are diluted with osmosis water in a glass receptacle (250 mg NPS / 250 ml). The dispersion is first placed under magnetic stirring (5-10 minutes) and then homogenized (RANNIE Minilab) at 400 bars for 3 minutes. The dispersion of NPS is placed in a water bath at 80 ° C.

The lipids (example: DPPC), in powder form, are weighed so that the total mass represents, for example, 30% (w / w) of the mass of the NPS. The constituent lipids of the membrane are dissolved with 2 ml of 95% ethanol (w / w).

The homogenizer is brought to a temperature of at least 60 ° C by circulating water in a closed circuit.

The NPS dispersed at 80 ° C. are subjected to magnetic stirring and the ethanolic lipid solution is injected into the NPS dispersion.

The homogenizer circuit heating water is replaced by the "NPS / lipids" dispersion at 80 ° C. This new dispersion is homogenized at 450 bars for 25 minutes in a closed circuit at 60 ° C minimum. At the end of this step, the preparation is introduced into a glass flask and then subjected to low pressure at 60 ° C to remove the ethanol remaining in solution.

The neutral homeo-biovectors thus obtained are then stored under sterile conditions after filtration through 0.2 μm.

Example 4 Characterization of the Particulate Vectors

Homeo-biovectors have been characterized by: - the association of polysaccharide nuclei (NPS) with lipid molecules

- the organization of lipids (associated with NPS) between them

- the lipid layer surrounding the NPS. This supramolecular structure has remarkable properties of stability over time.

1. Materials and Methods

The anionic and cationic particulate vectors are prepared according to the technique described in Examples 1 and 2.

The liposomes were prepared by injection of an ethanolic solution of lipids in water as described in Batzri et al (Biochem. Biophys. Acta, 1973, 298, 1015-1019) and Kremer et al (Biochemistry, 1977, 16 , 3932-3935). All the samples prepared are stored in sterile tubes after sterilizing filtration through 0.2 μm.

1.1. Determination of the size of nanoparticles

The average diameter of the polysaccharide nuclei, the liposomes and the particle vectors is determined by a Coulter N4MD nanoparticle analyzer (Coultronics, Margency, France). The measurements were carried out in triplicate, at a temperature of 25 ° C., after dilution of the sample in 50 mM PBS.

1.2. Fluorescent labeling of biovectors

The labeling of the polysaccharide nucleus with fluorescein was carried out by addition of 10 mg of an aqueous solution of DTAF (5 ([4,6- dichlorotriazine-2λL] amino) fluorescence 2 mg / ml) to 100 mg of polysaccharide particles to pH 10, with magnetic stirring. After 2 hours at room temperature, the reaction mixture is washed by ultrafiltration, first with 1 M NaCl and then demineralized water until no fluorescein can be detected in the ultrafiltrate. The fluorescein-labeled polysaccharide nuclei (1 mg / ml) are then stored in sterile tubes after sterilizing filtration over 0.2 μm. The labeling of the phospholipid layers with DPH (1, 6-diphenyl-1,3,5-hexatriene) is carried out by adding the probe (10 ³ M in tetrahydrofuran) to aqueous suspensions of homeo-vectors or liposomes. The temperature is maintained at 60 ° C for 30 minutes. The final ratio of DPH to phospholipids is _ 0.5%.

For the double labeling of homeo-biovectors with fluorescein and DPH, homeo-biovectors were first prepared with polysaccharide nuclei labeled with fluorescein and then the external lipid layer was labeled with DPH as described above. All fluorescent preparations were stored in the dark.

1.3. Fluorescence spectroscopy

The fluorescence excitation and the emission spectrum were recorded with an SLM-Aminco spectrofluorimeter, Model 500. In these experiments, as in the fluorescence polarization experiment described below, the absorbance of the suspensions homeo-biovector and liposome was never greater than 0.1. For the fluorescence emission spectrum, the wavelength was 360 nm for DPH and 490 nm for fluorescein. For the excitation spectrum, the emission wavelength was 426 nm for DPH and 522 nm for fluorescein.

1.4. Fluorescence polarization The experiments were carried out on a formatted automatic device connected to a microcomputer. 1.5. Fluorescence quenching experiments

The homeo-biovectors or the liposomes were prepared with DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) supplemented with NBD-PC (l-acyl-2- [12 - [(7-nitro-2 - 1,3-benzoxadiazole-4-yl) amino] dodecanoyl] -sn-glycero-3-phosphocholine, (2 mol%), and dispersed in 1.5 M NaCl. Aliquots of thiomeo-biovector or liposome suspension have was put in a quartz tank (phospholipid concentration: 27.2 μM) and the fluorescence was measured to give the value Fo. Then a given volume of quenching solution C0CI2 (0.3 M in water) is added in order to obtain the desired concentration. The reagents are mixed for 15 seconds and the fluorescence is measured immediately to give the value Fi. The quenching efficiency is calculated as (l-Fι / Fo) xlOO. excitation and emission were 474 nm and 535 nm respectively, and the temperature 20 ° C.

1.6. Isopycnic separation on sucrose gradients

The sucrose gradients were prepared by melting 9 ml of 20% (w / w) sucrose solution at room temperature. 1 ml of the sample is placed at the top of the tube and centrifugation is carried out for 4 hours at 250,000 g, 8 ° C. After centrifugation, 0.5 ml fractions are collected from the top to the bottom of the tube with a pipette and analyzed in fluorescence spectroscopy.

2. Results

2.1. Association of polysaccharide nuclei (NPS) with phospholipids As shown in Figure 1A, NPS without lipid

(Img / ml) sediment over a large area of the density gradient (from 2 to 20%, sucrose, with a peak at 7%) indicating a heterogeneity of density of these NPS. The liposomes (0.2 mg / ml) are denser and more homogeneous than the NPS (narrow isopycnic position between 18% and 20% o sucrose). As for the biovectors (FIG. 1B), marked both with fluorescein at the level of the NPS and with DPH at the level of the membrane, they are positioned between 14 and 19% sucrose (peak at 17%). The two fluorophores being found in the same fractions, the association of NPS with lipids is thus demonstrated. It leads to an increase in the density and homogeneity of the constituent NPS.

Controls show that the synthesis of Biovectors results in a pure product. Indeed, the presence of free NPS and free liposomes is not detected in a preparation of biovectors. In addition, the procedure applied to synthesize the biovectors makes it possible to vary the lipid / NPS weight ratio from 20 to 100% without causing the formation of liposomes.

2.2. Determination of the size of the homeo-biovectors The measurements carried out with the control NPSs, the control liposomes and the homeo-biovectors are reported in Table 1.

It appears that the size of the neutral, cationic and anionic homeo-biovectors depends on the size of their constitutive NPS (example: for a cationic NPS of approximately 80 nm, the resulting cationic iomeo-biovector measures approximately 80 nm in diameter). On the other hand, the size of the control liposomes is smaller. The phospholipids of the liposomes organize themselves so that the structure is thermodynamically stable. The NPS core of homeo-biovectors promotes better stability of the lipid system. Table 1: Measurement of the size of nanoparticles (polysaccharide nuclei (NPS), liposomes and homeo-biovectors) by LASER light scattering.

[nucleus] = 1 mg / ml; [lipids] = 0.2 mg / ml; the weight ratio of the lipid mixtures DPPC / DPPG or DPPC / SA is 95/5. Measurements made in 50 mM PBS.

Sample Average diameter + standard deviation (nm)

NPS neutral 42.3 ± 26

Zwitterionic liposomes (DPPC) 39.4 + 13

Neutral homeo-biovectors 32.9 + 09

Cationic NPS 74.7 + 29

Cationic liposomes (DPPC / SA) 22.3 ± 1 1

Cationic homeo-biovectors 78.5 + 42

Anionic NPS 34.6 + 13

Anionic liposomes (DPPC / DPPG) 18.5 + 09

Anionic homeo-biovectors 45.4 + 26

(DPPC = dipamitoyl-phosphatidylcholine; DPPG = dipamitoyl- phosphatidylglycerol; SA = stearylamine)

These size measurements reveal a single homogeneous population, in correlation with the studies on density gradient showing the high degree of purity of the homeo-biovector preparations. The lipids alone give relatively monodispersed liposomal suspensions with an average size depending on the 40 nm and 20 nm composition for the DPPC and DPPC / DPPG mixture respectively.

2.3. Influence of the polysaccharide nucleus on lipid organization

Having demonstrated that lipids are associated with NPS, the aim is to show that these amphiphilic molecules are organized together to form the constitutive membrane of homeo-biovectors. The phase behavior of the lipids makes it possible to study this organization. The phase behavior of the lipid crown organized around the NPS is studied by visualizing the gel-liquid phase transition of lipids as a function of temperature by measuring the fluorescence depolarization of the DPH probe inserted within the hydrophobic membrane zone. . The profiles obtained (FIG. 2) for the homeo-biovectors and the control liposomes of the same lipid composition make it possible to raise the transition temperature of the gel-liquid phase (Tm) (Table 2). The results obtained make it possible to know the influence of the NPS and of the saline composition of the aqueous phase on the lipid organization. The gel-liquid phase transitions of lipids organized in the form of homeo-biovectors are very well defined. This observation is explained by a high degree of intermolecular cooperativity and therefore by a degree of organization of the membrane of homeo-biovectors quite comparable to liposomal membranes. The neutral homeo-biovectors and the control liposomes are analyzed in FIG. 2A, which shows a characteristic profile of the phase transition of the DPPC organized in the form of a liposome making it possible to note a Tm value of 40 ° C. The neutral homeo-biovectors have a membrane whose phase transition is as clear as that of the liposomes but exhibiting a shift towards the highest temperatures. The anionic homeo-biovectors (FIG. 2B) have a very marked and shifted phase transition towards higher temperatures compared to the control liposomes.

The cationic homeo-biovectors (FIG. 2C) offer similar characteristics: the effect of cationic NPS on the associated cationic membrane is such that the phase transition of the lipids is shifted towards higher temperatures compared to the control liposomes.

Homeo-biovectors therefore show a higher phase transition than that of control liposomes. By way of comparison, the light biovectors constituted by a cationic NPS and an anionic membrane have a lower phase transition than that of the control liposomes (temperature difference of -1 ° C). In this case, the NPS nucleus has an effect on its membrane which is the opposite of that of the homeo-biovector.

Table 2: Temperatures of gel-liquid phase transition (Tm) of lipid membranes organized in the form of liposomes or homeo-biovectors.

[nucleus] = 1 mg / ml; [lipids] = 0.2 mg / ml; the weight ratio of the lipid mixtures DPPC / DPPG or DPPC / SA is 95/5. Measurements carried out with 1 ml of nanoparticles dispersed in water or in PBS.

Tm (° C)

PBS Water Samples

DPPC zwitterionic liposomes 40.0 ± 0.2 39.2 + 0.3 Neutral homeo-biovectors 40.8 + 0.2 39.8 + 0.1

Cationic liposomes DPPC / SA 43.4 + 0.1 42.8 + 0.1

Cationic homeo-biovectors 44.9 + 0.2 43.4 + 0.1

Anionic liposomes DPPC / DPPG 41, 1 + 0, 1 38.3 + 0.2 anionic homeo-biovectors 44.8 + 0.19 39, 1 + 0.2

(DPPC = dipamitoyl-phosphatidylcholine; DPPG = dipamitoyl- phosphatidylglycerol; SA = stearylamine)

The temperature reading Tm (table 2) of each system makes it possible to characterize the phase transition offset.

These temperatures Tm, for samples dispersed in water or in the presence of an ionic force (PBS 50 mM), show that the membranes associated with NPS of similar ionic charge have a lipid organization such that the molecular packing is more significant (Tm of homeo-biovectors higher than the temperature of control liposomes), whatever the type of omeo-biovector.

The addition of salts in the dispersions of homeo-biovectors has the effect of reducing the effect of NPS on the organization of the lipids between them. This observation makes it possible to conclude that the NPS has an influence on the molecular packing of lipids by means of electrostatic and hydrogen type interactions with the polar heads of phospholipids.

Thus whatever the driomeo-biovector type envisaged, this supramolecular structure promotes better membrane organization than that of the liposomes resulting in an increase in the molecular packing of the lipids between them.

2.4. Number of lipid bilayers surrounding the polysaccharide core

The lipids associated with the NPS are organized together to form a membrane. It is a question of characterizing the structure of this membrane. Fluorescence quenching experiments were carried out using an NBD-PC probe, for which it has been shown that the NBD group is localized in the glycerophosphate region of the lipid bilayer. Cobalt ions have been used as fluorescence extinguishers. The biovectors were prepared in the presence of 1.5 M NaCl, in order to avoid excessive changes in ion concentration after addition of the extinguisher in the aqueous phase. The fluorescence curves obtained respectively for the DPPC liposomes and the DPPC anionic biovectors are shown in FIG. 3. The modifications of the curve are detected around 50% -55% extinction. The fact that the molecular probes located in the external lipid layer are the first affected, suggests that the homeo-biovectors as well as the DPPC liposomes are composed of a single lipid bilayer. Consequently, for a calculation on the base of a thickness of the bilayer of 5.5 nm and a molecular surface of 0.47 nm ² in the gel phase, indicates that for unilamellar vesicles of 40 nm and 80 nm in diameter, approximately 65% and 55 % of the lipid molecules respectively are present in the outer layer.

2.5. Stability over time of homeo-biovectors

Is the particular structure of homeo-biovectors (nanoparticles made up of a lipid membrane organized in a bilayer and supported by an underlying NPS of similar charge) stable over time?

The stability of the nanoparticulate size is followed at regular time intervals for preparations stored sterile at 4 ° C. The measurements are reported in Table 3. No significant change in size of the anionic homeo-biovectors and of the cationic homeo-biovectors n 'is detected for more than 9 months.

These structures have remarkable stability over time.

Table 3: Stability over time of the size of nanoparticles (liposomes and homeo-biovectors) at 4 ° C.

[nucleus] = 1 mg / ml; [lipids] = 0.2 mg / ml; the weight ratio of the lipid mixtures DPPC / DPPG or DPPC / SA is 95/5.

Measurement made in 50 mM PBS.

Average diameter + standard deviation (nm)

Sample 24 h 1 month 3 months 6 months 9 months 12 months

Cationic liposomes (DPPC / SA) 22.3 + 1 1 28.3 + 08 25.0 + 09 Aggr. Aggr. Aggr. Cationic homeo-biovectors 78.5 ± 42 76.2 + 1 1 82, 1 + 15 77.0 + 25 77.2 + 67 Aggr.

Anionic liposomes (DPPC / DPPG) 18.5 + 09 22.3 + 0.9 Aggr. Aggr. Aggr. Aggr. Anionic homeo-biovectors 45.4 + 26 52.3 + 1 1 48.2 + 0.8 55.0 + 22 42, 1 + 15 49.2 + 12

(DPPC = dipamitoyl-phosphatidylcholine; DPPG = dipamitoyl-phosphatidylglycerol; SA = stearylamine) (Aggr. = Aggregation).

EXAMPLE 5 Association of Cationic Homeobiovector with DNA

1. Preparation of cationic homeobiovectors: Cationic homeobiovectors prepared as in Example 2 are used, but here the constituent lipids of the membrane are a mixture of DPPC (dipalmitoyl phosphatidylcholine), DOTAP (dioleyloxypropyl trimethylammonium) and cholesterol according to a weight ratio of 35/35/30, the positively charged lipid being DOTAP. The lipids have a total mass representing 30% by weight of the mass of the NPS. The cationic nucleus has an overall charge of 0.8 mEq / g dry NPS and an average diameter of 1 10 nm.

The complete cationic homeobiovector obtained has an average diameter of 130 nm.

2. DNA preparation

The DNA used is 6.7 kb plasmid DNA (plasmid 1633). It is packaged in PBS and stored at 4 ° C.

3. Preparation of the biovector / DNA association

In a test tube, a volume of DNA solution is mixed, the concentration of which is calculated as a function of the DNA / NPS ratio which it is desired to obtain (1.1 to 5.5% for example) and a volume of dispersion. of homeobiovector at 1.8 mg / ml in PBS. For example, to obtain a DNA / NPS ratio equal to 5.5%, a volume of cationic homeobiovector at 1.8 mg / ml will be mixed with a volume of DNA at 100 μg / ml in PBS. The mixture is maintained for 1 hour with magnetic stirring and stored at 4 ° C.

4. Characterization of homeocharged / DNA biovector formulations

The parameters measured to characterize the biovector / DNA preparations are summarized in Table 4 below. TABLE 4

(dppc = dipamitoyl-phosphatidylcholine; dotap = dioleyloxypropyl trimethylammonium)

Physicochemical characteristics of homeobiovectors and homeobiovector / DNA preparations

5. Results

- the size of the resulting nanoparticles makes it possible to verify that there is no aggregation (the technique used is light scattering). The formulation mode allows a homogeneous nanoparticulate dispersion.

- the rate of association of DNA on homeocharged biovectors (determined by isopycnic separation on a sucrose density gradient and electrophoresis on 1% agarose gel) is greater than 95%. - homeobiovectors favor a spontaneous association with DNA by electrostatic interactions without any external energy supply.

It is remarkable to note that the “home-charged biovector / DNA” formulations subjected to an electric field during electrophoresis on agarose gel retain their associated DNA. Indeed, no free DNA is detectable at the end of the electrophoresis. The interactions between DNA and cationic homeobiovectors are very stable. Furthermore, we tested the conservation of the DNA structure after formulation: the DNA associated with the biovectors is released by ionic competition. Analyzed by electrophoresis on 1% agarose gel, it is found that the DNA has well retained its plasmid structure. The formulation process used therefore allows the structural integrity of DNA to be maintained. LEGEND OF FIGURES

Figure 2

LIPOSOMES 7WITTERÉRI0NI0LΕS O NEUTRAL HOMEO-BIOVECTORS α

ANIONIC LIPOSOMES * ANTONIOUS HOMEO-BIOVECTORSTE

CATIONIC LIPOSOMES o CATIONIC HOMOEBIOVECTORS a

Claims

1. Particulate vector comprising: - a non-liquid hydrophilic core an amphiphilic sheet characterized in that the core carries an overall cationic, anionic or neutral charge and in that the amphiphilic sheet carries an overall charge of the same polarity as that carried by the core . 2. Particulate vector according to claim 1, characterized in that it also contains at least one active principle, associated with the core and / or the amphiphilic sheet. 3. Particulate vector according to one of claims 1 or 2, characterized in that it comprises at least: - a non-liquid hydrophilic core carrying a cationic or anionic charge, of the same polarity as that carried by the core. 4. Particulate vector according to one of claims 1 or 2, characterized in that it comprises: a non-liquid hydrophilic core, electrically neutral - a generally neutral amphiphilic sheet, comprising zwitterionic phospholipids. 5. Particulate vector according to one of claims 1 to 4, characterized in that at least one constituent of the amphiphilic sheet is chosen from the group consisting of anionic, cationic or zwitterionic phospholipids, natural or synthetic, ceramides, acids fatty, steroids, lipids, glycolipids, lipoproteins and surfactants, carrying a charge of the same polarity as that of the nucleus. 6. Particulate vector according to one of claims 1 to 5, characterized in that the proportion of constituents of the sheet amphiphilic carrying a charge of the same polarity as that of the core is greater than 0% and less than or equal to 100%). 7. Particulate vector according to one of claims 1 to 6, characterized in that the nucleus consists of compounds chosen from oligomers and hydrophilic polymers, naturally or chemically crosslinked. 8. Particulate vector according to one of claims 1 to 7, characterized in that the nucleus consists of polysaccharides or oligosaccharides naturally or chemically crosslinked, carrying a cationic or anionic charge. 9. Particulate vector according to one of claims 1 to 8, characterized in that the core comprises constituents chosen from: dextran, starch, cellulose, their substituted derivatives, their hydrolysis products, salts and esters of these compounds. 10. Particulate vector according to one of claims 7 to 9, characterized in that ionic, acidic or basic ligands are fixed on the nucleus. 1 1. Particulate vector according to claim 10, characterized in that the ionic ligands carry at least one function chosen from the group comprising: phosphates, sulfates, carboxylic acids, quaternary ammonium, primary amines, secondary amines. 12. Particulate vector according to one of claims 1 to 1 1, characterized in that at least one constituent of the amphiphilic sheet is chosen from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, cholesterol, and their derivatives. 13. Vector according to one of claims 1 to 12, characterized in that at least one constituent of the amphiphilic sheet is chosen from dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol, diole trimethylammonium, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dipalmitoyl phosphatidylserine and stearylamine. 14. Particulate vector according to one of claims 1 to 13, characterized in that the amphiphilic sheet comprises at least two different lipid compounds. 15. Particulate vector according to one of claims 1 to 14, characterized in that the amphiphilic sheet is directly associated with the core. 16. Particulate vector according to one of claims 1 to 15, characterized in that its average diameter is between approximately 10 nm and 100 μm. 17. Particulate vector according to one of claims 2 to 16, characterized in that the active principle also carries an overall charge, or is lipophilic 18. Particulate vector according to one of claims 2 to 17, characterized in that at least one active principle is chosen from peptides, proteins, lipoproteins, proteoglycans, lipopolysaccharides, fatty acids, triglycerides, phospholipids, glycolipids, steroids, oligosaccharides, nucleotide sequences, nucleoside sequences, nucleic acids, vitamins and porphyrins. 19. Particulate vector according to one of claims 2 to 18, characterized in that at least one active principle is chosen from the following group: antibiotics, antiseptics, analgesics, anesthetics, anti-inflammatories, antivirals, anticancer drugs, immunomodulators and cellular mediators, antihistamines, antimuscarinics, anti-anemia agents, hemostatic agents, anti-diabetics, cardiovascular agents, anti-depressants, anti-epileptics, anxiolytics, hypnotics, sedatives, antipsychotics, appetite suppressants, vaccines, natural hormones or synthetics and their derivatives, insecticides, fungicides, antimalarials, asthma medications, antibodies, antigens and their fragments, diagnostic agents, NMR diagnostic agents, and radioactive markers. 20. Pharmaceutical composition, characterized in that it contains a particulate vector according to one of claims 1 to 19. 21. Cosmetic composition, characterized in that it contains a particulate vector according to one of claims 1 to 19 and cosmetologically acceptable excipients. 22. Food additive characterized in that it comprises a particulate vector according to one of claims 1 to 19.