CA2157384C - Synthetic particulate vectors and process for their preparation - Google Patents

Abstract

The subject of the present invention is a synthetic particulate vector, characterized in that it comprises: a non-liquid hydrophilic nucleus, an external layer consisting at least in part of amphiphilic compounds, associated with the nucleus by hydrophobic interactions and / or ionic bonds . It also relates to a process for the preparation of a particulate vector by encapsulation of an ionizable active principle, as well as the vectors which can be obtained by this process. Finally, it relates to pharmaceutical, cosmetological or food-use compositions containing such vectors.

Description

WO 94/20078 ~} 15 ~~ 3 QTl PCT / FR94 / 00228 ~ + ü

SYNTHETIC PARTICULATE VECTORS AND PREPARATION METHOD.
The present invention relates to new types of particles, used alone or as vectors of different compounds. She also relates to a method of preparing vectors particulates allowing improved control of loading in principle active.
SUPRAMOLECULAR BIOVECTORS or BVSM are particles, biomimetics of the endogenous vectors of the body, able to encapsulate and vectorize a large number of active ingredients for a particular use pharmaceutical, cosmetic or agro-food.
A first type of BVSM has been described in application EP 34-1 0-10. Their structure is very well suited to the role of vector, especially makes the possibility to change their size and composition according to the or the molecules transported and their use.
The BVSM are synthesized in three successive stages:
- synthesis of a central core consisting for example of cross-linked natural polysaccharide, which can be derived by ionic groups, and brought back, in particular by ultra-grinding, to the desired size (between 10 nanometers and a few microns next the desired use) - establishment of a crown of fatty acids grafted covalently only at the periphery of the central nucleus, in order to give it a hydrophobic peripheral character while retaining its internal hydrophilic character stabilization of one or more external lipid layers, consisting in particular of phospholipids or ceramides, sometimes addition of other constituents, for example constituents of biological membranes.
Active ingredients, according to their physical characteristics can be transported either in the lipidic leaflets ~ external (case of lipophilic or amphiphilic compounds) either inside the hydrophilic nucleus (case of polar compounds).
The encapsulation of active ingredients of a polar nature can be to proceed, according to the structure of the latter, before the establishment of the crown of fatty acids, either between this step and the stabilization of the leaflet external.

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Despite their adaptability to many uses, the synthesis of the BVSM can sometimes cause problems and in particular:
it requires a delicate step to control in order to graft the crown of acids fat;

- this grafting, only carried out on the periphery of the nucleus, must be performed in a homogeneous manner, which requires in particular a step prior drying under very specific conditions;
if the active ingredient is encapsulated before grafting the crown fatty acids, some of these molecules, localized, after their encapsulation, at the periphery of the core, may be derived by fatty acid, leading to the modification of the properties of this active ingredient;
if the active ingredient is encapsulated after grafting of the crown of fatty acids, this can be a gene for penetration of the active ingredient into the hydrophilic core.
The Applicant has shown that, surprisingly, for some applications it was possible to reduce the schema by not grafting the ring of fatty acids to the periphery hydrophilic mesh core.
The Applicant has shown that the particles polysaccharides thus obtained could be used as such.
They are then named, by analogy with the Biovectors Supramolecular, BVSM type-PS.
The Applicant has indeed shown that the particles polysaccharides, even small ones, could be used through the adoption of adapted loading protocols.
They can also be used in combination with naturally occurring amphiphilic compounds, including phospholipids and Applicant has shown that the outer lipid sheets have the possibility to organize around this core in the absence of a crown graft lipid as in the case of BVSM. These particles have a supramolecular character and are called BVSM type-L.

WO 94/20078 6 ~ 15738I ~ PCT / FR94 / 00228 ~ ~ =

This is why the subject of the present invention is a vector synthetic particle, characterized in that it comprises a hydrophilic non-liquid core, an outer layer consisting at least in part of compounds amphiphilic, associated with the nucleus by hydrophobic interactions and / or ionic bonds, either by the outer ring of the nucleus hydrophilic, using a special process avoiding encapsulation of the active ingredient in this crown and concentrating the principle active in the inner part of the kernel.
The notion of vector must here be understood in a broad sense, that is to say, it comprises particles having a supporting role, for example example when they are incorporated into a composition, or such whether for the transport, presentation and / or stabilization of active compounds.
The non-liquid hydrophilic nucleus (or matrix) may be a hydrophilic polymer. The hydrophilic matrix may in particular be consisting of polysaccharides or oligosaccharides, naturally or chemically crosslinked. Preferably, the polysaccharide is chosen from dextran, starch, cellulose and their derivatives.
Two types of interactions can explain the stabilization phospholipids for example on a polysaccharide core crosslinked and derived in its mass by ionic groups: these are either hydrophobic type interactions, ie ionic type bonds, two modes that can cooperate in the stabilization of these sheets.
Indeed, the phospholipids are constituted on the basis of a glycerol unit, a polar head comprising a phosphate group, strong anionic charge, possibly derived by groups different polar and two fatty acids constituting the hydrophobic tail.
The polar head has the ability to bind by interaction ionic with the ionic groups grafted into the matrix polysaccharide, either via phosphate or by intermediate ionic groups grafted onto the phosphate of the phospholipid (quaternary ammonium phosphatidylcholines example).

215738d ~

On the other hand, it is known that polysaccharides, if they have a globally hydrophilic, have a hydrophobic groove, established in that the polar hydroxyl groups are orientated in a given direction, leaving accessible the basic structure of sugars, consisting of carbon-carbon bonds, of hydrophobic nature. In the case of polysaccharide particles constituting the core type-L, the stabilization of compounds such as phospholipids may be due to a phenomenon of cooperativity between two connection modes.
The combination phospholipid-polysaccharide nuclei could be demonstrated by fluorescence energy transfer techniques.
The theory of energy transfer is based on the interactions that exist between two fluorophores when the emission band of the first fluorophore (F1), covers the excitation band of the second fluorophore (F2). If the two elements are close to the energy of a photon absorbed by the fluorophore F1 can be transferred to fluorophore F2 which will then fluoresce as well as if he had been excited directly. Fluorescence F1 can then decrease to total extinction. Transfer efficiency of energy between the two fluorophores is therefore dependent on their spatial separation. After performing a marking with the help of Rhodamine isothiocyanate on polysaccharide nuclei and using of Nitrobenzodiazole (NBD) on phospholipids a transfer of energy to could be highlighted between the two fluorophores whether in the case 1Mm, 200nm or 20nm L-type LMVs. This transfer remains stable after incubations at -4 C, 37 C and even at 100 C.
The hydrophilic core can be obtained by different methods and in particular if it is a natural kernel polysaccharide, using a biodegradable, branched polysaccharide or linear. This polysaccharide may be for example starch or one of its derivatives. Crosslinking processes are known to those skilled in the art and can be performed using bi or trifunctional agents, such as epichlorohydrin or phosphorus oxychloride.

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The properties of the polysaccharide can be modified in substituting sugars with ionic, acidic or basic functions, which are important in the stabilization of the external lipidic leaflet and for encapsulation of ionic active ingredients.
The encapsulation of the hydrophilic active ingredients can be performed at this stage of the synthesis. The gel obtained during the synthesis step is then partially washed and dehydrated by means of, for example, centrifugation techniques, then put in the presence of the active ingredient and rehydrated slowly. Since the gel has the capacity to inflate with water, the active ingredient is trained within the polysaccharide network where it can bind through ionic bonds with the grafted groups within the gel.
The gel obtained, whether or not it contains an encapsulated compound, must be mechanically milled to obtain large particles desired. Ultra-milling methods are known in the state of the technical and may include high extrusion pressure by means of a homogenizer.
Encapsulation of hydrophilic compounds within BVSM type-L is usually performed at this stage. For that, the particle suspension is previously dried using drying methods, such as lyophilization or atomization.
The dried particles are, for example, mixed with the active ingredient also in dry form. Progressive rehydration allows, as in the case of the gel, to solubilize the active ingredient and then to drag it inside the particle where it binds by ionic bonds.
The external lipidic leaflet of the L-type BVSM can be made with different types of natural or synthetic lipids, among which phospholipids and ceramides, but also surfactants ionic or non-ionic agents capable of organizing into micelles, to which can also be added other compounds either lipid or amphiphiles such as cholesterol, fatty acids or vitamins soluble.

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This leaflet is preferably obtained using methodes which are used for the preparation of liposomes, that is to say the preparation in reverse phase, detergent dialysis or high pressure extrusion. The active ingredients to be vectorized or presented on the surface of BVSN1 of type-L can be introduced during this step, mixed with assets.
The present invention also relates to a method of preparation of a particulate vector, characterized in that:
a) the encapsulation of at least one ionizable active principle is carried out basic, in a cross-linked hydrophilic matrix grafted by acid ligands, at a pH lower than the pKa of the active principle, b) the pH of the medium is increased to a value greater than pKa active ingredient, c) recovering the hydrophilic matrix comprising the active ingredient located mainly in the center of said matrix.
Indeed, the adoption of a protocol for loading cores hydrophilic adapted, allows the topological control of the loading.
The hydrophilic matrix is preferably composed of polysaccharides or oligosaccharides, naturally or chemically crosslinked.
This process that can be used with BVSM is more particularly important with particles with lipidic layers have been reduced (BVSM type-L) or suppressed (BVSM type-PS) compared to the previously described model. The Applicant has observed that it is difficult to use such reduced lipid-reduced BVSM
as vectors for the encapsulation of ionic active ingredients, with conventional loading methods.
Indeed, if molecules of the active ingredient bind with the polysaccharide particle of the nucleus while staying at the periphery thereof, it may result in instability of the suspension of particles, which can be aggregated together by means of links interparticulates due to the active ingredient. This phenomenon is relatively minor for low loading rates of active ingredients, whereas becomes very important with high active ingredient loading rate.

Similarly, particle size is extremely important. With some large particles (eg greater than 100 nanometers), the ratio between the surface and the internal volume of the particle is very small; from this in comparison with the total amount of encapsulated active ingredient, the amount of active ingredient bound to the periphery of the particles is very small, thus limiting the possibilities of interparticle links. Conversely, when the particles have a very small size, this phenomenon aggregation is very sensitive. It should also be noted that this phenomenon is not very marked with BVSM having a layer of acids fat grafted on the nucleus, which then acts as an insulator for interactions interparticle.
To remedy this, the Claimant has shown that it is possible to control topologically the penetration of the active ingredient to inside the particles by mastering the ionic conditions of encapsulation.
Polysaccharide nuclei can be considered as polyelectrolyte matrices and, as such, they exhibit a Differential pH between the inside and the outside of the particle. This phenomenon is due to the more or less important dissociation of ions and the immobilization of ionic functions on the network polysaccharide. This property allows you to control the location of the active ingredient to be incorporated, forcing encapsulation only internal or only at the surface or in the periphery of the nucleus.
When the active ingredient to be incorporated is of a basic nature, its solubilization in a pH environment lower than its pKa leads it to present in ionized form; he can then become anchored with the groupings anionic grafted on the polysaccharide nucleus. When the pH goes up above the pKa, the active ingredient is in a deionized form, which does not does not interact with the matrix. So we use, to control the location of the encapsulated active ingredient, the existing pH differential between the inside and the outside of the particle: if the outside environment pH is too high, the active ingredient can not interact with the _2. ~~~~ 8.1 ionic groups placed on the periphery of the nuclei. The internal pH of nuclei derived by acidic ligands being lower than the external pH, the active ingredient, entered into the particle in deionized form, becomes ionic and therefore binds with the anionic groups of BVSM type-L.
In this case, the active ingredient will be located only in the heart of the particle, excluding the peripheral zone. This guy encapsulation is therefore very favorable to an optimal dispersion of particles.
In the case of acidic active ingredients, the process with nuclei derived from basic ligands, so symmetrical, according to the following steps:
a) the encapsulation of at least one ionizable active principle is carried out acid, in a cross-linked hydrophilic matrix grafted by basic ionic ligands, at a pH above the pKa of the principle active, b) the pH of the medium is lowered to a value lower than the pKa of the active ingredient, c) recovering the hydrophilic matrix comprising the active ingredient located mainly in the center of said matrix.
This type of loading with topological control of the localization of the active ingredient in the polysaccharide nucleus is particularly interesting for vectorization applications with MSBs with external lipid layers reduced (BVSM
type-L) or deleted (BVSM type-PS), but it is also adaptable and Desirable for BVSMs already described in the foregoing patents to increase the loading rate or minimize disturbances made to the structure of the phospholipid outer leaflet in the case of external anchoring of macromolecules, for example recognition and in particular of an apoprotein.
The subject of the invention is also a particulate vector consisting of a crosslinked hydrophilic nucleus, grafted by groups ionic, called here BVSM type-PS. Ionic groups can be anionic groups, such as phosphates succinates, carboxymethylates, or cationic groups, by example of quaternary ammoniums or amines. Preferably, the size of the BVSM type-PS is between 20 nm and 200 nm.

The crosslinked hydrophilic core may consist of natural or synthetic polymers, naturally or chemically crosslinked. In particular, polysaccharides or oligosaccharides are used, such as aniidon, dextran, cellulose and their derivatives.
Advantageously, an active ingredient is encapsulated in the PS-type PSM mainly in the center of the matrix; the external part of the core is practically devoid of active principle, which allows to avoid the phenomena of aggregation generally occurring for small particles.
One of the objects of the invention is therefore a vector particulate consisting of a crosslinked polysaccharide matrix containing an active ingredient, said active ingredient being preferably located mainly in the center of the matrix.
According to yet another aspect, the subject of the invention is a loading process, which allows the encapsulation of the active ingredient in a complete BVSM, a PS-type BVSM or an L-type BVSM, which can be in the form of suspension.
Loading is carried out on the particulate vector on which, where appropriate, the different lipid layers and / or amphiphilic. For this purpose, the hydrophilic core must have ionic groups. The process therefore requires the following steps:
a) a crosslinked hydrophilic core is prepared in which are fixed ionic groups, b) optionally, lipid compounds are example of the fatty acids, on the cross-linked nucleus and / or, one establishes a amphiphilic slip (for example, phospholipid) on the nucleus, optionally acylated, c) the active ingredient is loaded inside the vector at a suitable pH
to the active ingredient and by providing energy, d) recovering the vector having incorporated the active ingredient.
In the case of PS-type BVSMs, it is difficult to use the conventional loading methods to incorporate active ingredients Ionic. It is true that methods with topological control allow to overcome this problem. However, topological control requires a game end of pH which must be compatible with the active principle and the vector.

4b In the case of normal or L-type BVSN.t, methods developed so far consist in incorporating the active ingredient in the nuclei and then establish the phospholipidic sheet. These certainly have very interesting incorporation results but require the manipulation of the active ingredient at all stages of the process of preparation. In the case of very toxic or very expensive active ingredients, these methods should be discouraged for reasons of safety or cost.
The applicant has therefore developed an alternative method to overcome these problems. Ionic ligands grafted into the network polysaccharide vectors induce significant affinity for ionic active ingredients of opposite charge. However, this affinity, when of incorporation, must be controlled to avoid the aggregation of vectors and allow the localization of the active principle mainly to inside the particles. For preloading, this control requires a end of pH play or a low incorporation rate. This aggregation is due in large part to the surface location of the active ingredient which it is due to the presence of ligands on the surface of the particles.
To develop this new type of loading, three factors come into play:
a) a strong affinity of the active ingredient for the vector for ensure incorporation of the active ingredient: this affinity is created ionic ligands, acidic or basic, which are grafted into the crosslinked polymer; the density and strength of the ligands can be adjusted according to the active ingredient, (b) a significant dispersion of the vectors during incorporation for avoid interactions between particles that promote aggregation:
this dispersion can be ensured by the dilution of the vectors in the reaction medium at a concentration sufficient to reduce interparticle interactions but also at a concentration compatible with pharmaceutical applications, (c) the implementation of any means to promote the entry of the principle active within the vector: the energy input, in form by example of agitation or heat, will accelerate the kinetics active ingredient but also to promote the dispersion of ~

vectors; the proper form of the active ingredient that must be Ionic enough to allow anchoring of the active ingredient but also the least loaded to avoid interactions superficial.
For the BVSM, the type L or type PS, it is possible to implement incorporation protocols that meet the these requirements. The presence of grafted ionic ligands in the polymer crosslinked to ensure the anchoring of the active ingredients for the three species. The vector dispersion can be achieved by suspending in the water of the BVSM type PS. MSBs or Type B MSVs are prepared for from nuclei, acylated or polysaccharide, and phospholipids, previously dispersed in an aqueous medium and are thus found in suspended in water. The supply of energy, for example in form agitation or heat, is not degrading for the BVSM. It is possible to play with pH and set pH ranges compatible with this type loading.
In the case of BVSMs and BVMS type L, the slips lipids and / or phospholipids represent a barrier that must be crossed by the active principles. But, in the loading process described, two additional factors intervene to promote this crossing:
a) the supply of energy that can fluidify the lipid layers and phospholipid and increase the kinetics of entry of the principle active inside the vectors, b) the form of the active ingredient that can be very weakly ionic fact of the existence of the gradient existing between the interior and the outside of the vector.
In addition, the strong affinity between the active ingredient and the Crosslinked polyelectrolyte polymer ensures its anchoring and high dispersion avoids the aggregation of vectors between them.
This new process makes it possible to prepare the BVSM, of any type, loaded in active principle while keeping the size of the basic vectors and has many advantages. This method consists in preparing the vectors, without active ingredient, before incorporation, which allows to process the white vectors according to conditions adapted to the vectors and not depending on the active ingredient to encapsulate and then charge.

~~~~~ 811 These conditions can therefore be more or less drastic. She permits also to be able to characterize the white vector as a basic entity.
The incorporation step is the final step of the process, which leads to manipulating the active ingredient, which can be toxic and expensive, during only one step during the process. This process therefore reduces manipulations and possible losses of the active ingredient. It therefore allows to be safer at the security level but also more profitable.
In addition, for certain active ingredients, the conditions of incorporation may be relatively simple, allowing consider loading the vectors with the active ingredient so extemporaneous. This mode of extemporaneous preparation can eliminate the storage problems in the liquid state.
This new loading mode is based on the important affinity between the vectors and the ionic active principles but also on the simple control of the incorporation by the dispersion of the vectors and the ionic form of the active ingredient. It has very advantages interesting: preparation of the white vector independent of the principle active ingredient, manipulation of the active ingredient in a single step, possibility of extemporaneous preparation.
The subject of the invention is also a particulate vector which has from inside to outside a matrix crosslinked polysaccharide containing an ionizable active ingredient, a first lipid layer attached to the matrix by covalent bonds, and a second layer of amphiphilic compounds on which are optionally grafted protein or peptide molecules.
The particulate vectors according to the invention have preferably a diameter between 10 nm and 5 m, still preferred between 20 and 70 nm.
These particulate vectors are intended to convey or present on their surface one or more molecules with biological activity.
Among these molecules, it should be mentioned, without this list being limiting:
- antibiotics and anti-virals, proteins, proteoglycans, peptides, polysaccharides, lipopolysaccharides, the antibodies, antigens, - insecticides and fungicides, ~

the compounds acting on the cardiovascular system, - anti-cancer, - anti-malarics, - anti-asthmatics, the compounds having an action on the skin, - the constituents of the milk fat globules.
In the examples below, we describe the loading of different products according to their characteristics, and in particular a hydrophilic product of small size intended for administration systemic, an active ingredient with anti-cancer activity, - two enzymes with antibacterial activity, lactoperoxidase and glucose oxidase a plant extract composed of procyanidolic oligomers, possessing an antioxidant activity.
- constituents of the fat globule of the milk.
As well as BVSM possessing a crown of fatty acids transplant recipients, L-type BMSMs can be sterilized either by filtration or by by autoclaving. It can also be frozen or freeze-dried, by example in the presence of an additive or atomized.
The benefits of type-L or PS-type BVSMs are especially :
- a modular construction allowing adaptation to the product to vectorize or present - a biomimicry vis-à-vis natural structures such as lipoproteins or dairy fat globules - greater ease of synthesis and industrialization by report to the BVSM with a fatty acid crown - high chemical and thermal stability due to the structure polymeric core - a defined size and the possibility of obtaining homogeneously very small sizes (20 nm).
- their ability to stabilize encapsulated compounds within the nucleus and especially enzymes or antioxidant compounds.

1 ~
The subject of the invention is therefore a composition pharmaceutical composition, characterized in that it contains a vector particle according to the invention, and a pharmaceutically acceptable carrier acceptable for his administration. The vectors according to the invention are especially useful for therapeutic applications and in immunology.
The invention also relates to a composition cosmetological, characterized in that it contains a vector particulate matter as described above, and excipients cosmetologically acceptable.
Finally, food compositions containing vectors Particles according to the invention are part of the invention.
The following examples are intended to illustrate the invention without limiting its scope.
In these examples, reference is made to the following figures Figure 1 energy transfer on L-type BVSMs labeled with two types of fluorescent ligands. The polysaccharide core is labeled using Rhodamine and the phospholipid layer is labeled with NBD.
Figure 2 energy transfer between BVSM type-PS
labeled with Rhodamine and liposomes marked with NBD.
Figure 3 Energy transfer on BVSM marked by two types of fluorescent ligands. The acylated heart is marked using Rhodamine and the phospholipid layer is marked using the NBD.

EXAMPLE 1 Preparation of Polysaccharide Particles [QUES, OF AVERAGE DIAMETER OF 20 NANOMETERS, PER DOUBLE
CROSS-LINKING DEXTRIN BY PHOSPHORUS OXYCHLORIDE
In a reactor with a double jacket of 3 liters is introduced 100g of dextrin (Rocket) that is dissolved in 350m1 of water demineralized and 100m1 of soda lON.

After homogenization, 35.3 ml of POCI3 and 225m1 of ION soda.
After the addition of the reagents has been completed, the reaction mixture is stirred for another 15 minutes then neutralized by addition of acid Hydrochloric acid.
The gel is diluted in 2 liters of demineralized water and homogenized at 900 bar using a high-pressure homogenizer (Westfalia). This step makes it possible to obtain matrices of average diameter of 20nm.
The matrices are then washed by ethanol precipitation.
to remove the salts and then dried by lyophilization at a concentration 30g / l in matrices and 20g / l in ammonium bicarbonate.
75 g of freeze-dried matrices are recovered (yield of 75% reaction) EXAMPLE 2 Preparation of Polysaccharide Matrixes GRAFTED BY AMMONIUMS CATIONIC GROUPS
Quaternary In a 5-liter reactor, 200 grams are dispersed of amvlopectin (Roquette, Lille, Fr.) in 500 milliliters of 2N sodium hydroxide.
When the solution is homogeneous, 93.6 grams of glycidyltrimethylammonium chloride (Fluka, CH), corresponding to 0.5 equivalents / glucose residue, solubilized in 1 50 milliliters of water, and 11.4 grams (or 9.7 milliliters) of epichlorohydrin (Fluka, CH) corresponding to 0.1 equivalents / glucose residue. The mixture is homogenized for 1 to 2 hours then allowed to stand for 8 hours. The polymerized starch preparation is then brought to pH 6 by addition of acetic acid. The gel obtained is then washed several times with water distilled until the elimination of all salts and by-products of the reaction.
After lyophilization, 244 grams of cross-linked gel are obtained, a reaction yield of 80%.

21 ~~~ 811 .4b EXAMPLE 3 Preparation of Polysaccharide Matrixes GRAFT BY TYPE ANIONIC GROUPS
CARBOXYMETHYL ("CM TYPE") In a 5 liter reactor, 200 grams of dextrin (Rocket) in 300 milliliters of 7N sodium hydroxide. When the solution is homogeneous, we simultaneously introduce 9.6 milliliters of epichlorohydrin (Fluka, CH) corresponding to 0.1 equivalents / residue glucose and 117.2 grams of chloroacetic acid solubilized in 80 milliliters of water.
After stirring for 1 hour, 9.6 milliliters are added of epichlorohydrin and 150 milliliters of 20 N sodium hydroxide while stirring vigorously. After the end of the addition, the preparation is homogenized 6 hours then left to rest one night. The gel thus obtained is suspended in 1 liter of water and acidified to pH 3-4 by adding acid The gel is then filtered and washed with distilled water.
After lyophilization, 276 grams of Carboxymethyl-type gel are obtained.
a yield of more than 80%.

AVERAGE DIAMETER OF 11 & M, BY RECYCLING OF STARCH BY
PHOSPHORUS OXYCHLORIDE

In a reactor with a double jacket of 3 liters is introduced 100g of wheat starch (Roquette) which is dissolved in 375m1 of distilled water and 100m1 of 10N sodium hydroxide.
The mixture is stirred for 15 minutes at room temperature room.
Once the mixture homogenized we add simultaneously 11m1 of POCl3 and 50ml of 10N sodium hydroxide. After the end of the reagent addition, the reaction mixture is stirred again for 15 minutes and then neutralized at pH7 by addition of acetic acid.
The gel is washed in a wringer (Rousselet) during 30 minutes with distilled water to remove excess salts and by-products of the reaction.
The gel thus obtained is then homogenized at high pressure (500bars, Westfalia minilab homogenizer). This step allows obtaining matrices with an average size of 1 m. The titration of 1y, gel crosslinked using an automatic titrimeter (N [ethrorri1682]
reveals a degree of crosslinking of 0.3meq of phosphodiester functions by gram of crosslinked gel.
This gives PS-PSMS of 1 m diameter.
EXAMPLE 5: OBTAINING POLYSACCHARIDE PARTICLES

15 grams of gel obtained according to Example 2, or gel type CN1 obtained according to Example 3, are dispersed in 500 milliliters of distilled water TM
and homogenized using a Rannie MiniLab 12-5I homogenizer (APV Rannie, Copenhagen, Dk). Homogenization pressure applied is 600 bar for 12 minutes.
A fluid suspension of particles of crosslinked, basic or acidic polysaccharides, the size of which is measured by a Coulter N4® Nanosizer, is centered around 200 nanometers. The nanoparticles are then dried by lyophilization in the presence of 20 grams / liter of ammonium bicarbonate.
PS-PSMS of 200 nm diameter are thus obtained.
usable as is or transformed into L-type BVSM

Preparation of white-type BVSM-L

lOmg of polysaccharide nuclei 20nm are dispersed in 2m1 of Octyl Glucopyranoside OGP (Fluka) SOmM. We mix them under ultrasound to a solution of 10mg of a mixture of lecithins of yellow purified eggs (Sigma) and cholesterol (Sigma) 80/20 dispersed in 2ml of OGP 5OmM. This preparation is then diluted ultrasound to a final 5mM OGP molarity and then dialyzed extensively for 48 hours. The size analysis is performed at using a Coulter N4 nanosizer indicates that 97% of the particles have a diameter of 23nm. =

A - Preparation of Fluorescence-labeled L-type LMVBS
a - Preparation of polysaccharide nuclei labeled with Rhodamine (x ex540nm - x em580nm) 50mg of polysaccharide nuclei 20nm prepared according to Example 6 are dispersed in 1 ml of sodium bicarbonate (Sigma) 100mM pH10. 0.5 mg of Rhodamine B isothiocyanate (Sigma) is added solubilized in dimethyl formamide (SDS), ie 1% of Rhodamine per relative to the weight of polysaccharide rings. After 18 hours stirring at room temperature several ethanol washes are performed to remove unreacted Rhodamine. The nuclei are then dried by lyophilization.

b - Establishment of the phospholipidic leaflet labeled with Nitrobenzodiazole (NBD) (X ex470nm -) ~ em530nm) * From fluorescent polysaccharide nuclei lOmg of nuclei labeled with isothiocyanate Rhodamine of 20nm are dispersed in the presence of 2m1 of Octyl OPG Glucopyranoside (Fluka) 50mM. We then bring this suspension lOmg of a mixture of purified egg yolk lecithins (Sigma), Cholesterol (Sigma) and phosphatidylethanolamine labeled with Nitrobenzodiazole NBD (Sigma) 79/20/1 dispersed in 2m1 OGP 50mM, ie 100% phospholipidic mixture compared to nuclei polysaccharide. This solution is then diluted a final 5mM OGP molarity under ultrasound then dialyzed extensively for 48 hours.
* From non-fluorescent polysaccharide nuclei BVSM type-L are prepared identically but with non-fluorescent polysaccharide cores. This preparation serves as a control, it represents the zero level of energy transfer.

WO 94/20078 '~ 15738 ~ PCT / FR94 / 00228 ~, =

B - Preparation of fluorescent polysaccharide nuclei witnesses: BVSM type-PS

10mg of 20nm fluorescent nuclei are dispersed in presence of 2m1 50mM OGP. this solution is then diluted brutally up to a final 5mM OGP molarity under ultrasound then dialyzed extensively for 48 hours.

C - Preparation of control fluorescent liposomes 10 mg of the previous phospholipid mixture are dispersed in 2ml OGP 5OmM. The solution is diluted abruptly to a final molarity in OGP of 5mM under ultrasound then dialyzed extensively for 48 hours.
D - Preparation of control BVSM

BVSM labeled with two types of fluorescent ligands (acylated heart labeled with Rhodamine and phospholipids marked with using NBD) as well as BVSMs with non acylated hearts fluorescents and the phospholipid layer labeled with NBD were prepared according to the methods described in the previous patents (EP 344 040).
The energy transfer is quantified by the decrease in NBD fluorescence (emission wavelength at 530nm) .Transfer fluorescence energy of 20% was highlighted on the BVSNt's type-L labeled by two types of fluorescent ligands with respect to BVSM type-L having only the phospholipidic layer labeled in fluorescence (Fig. 1). This transfer remains stable after incubation of 4 hours at 4 C and 37 C and after incubation for 2 hours at 100 C.
This tends to demonstrate that phospholipids and nuclei polysaccharides are spatially close. The close association Phospholipid-polysaccharide nuclei is confirmed. The simple mixture of control fluorescent polysaccharide nuclei (BVSM
type-PS) with control fluorescent liposomes does not lead to energy transfer between the two fluorophores, even under the conditions previous incubation.

~~~~~~~~ 'ib The energy transfer observed on the L-type BVSMs is comparable with the energy transfers observed on the BVSM in the same experimental conditions (Fig. 3).

EXAMPLE 8: PREPARATION OF TYPE BVSM FROM

10 grams of PS-PSM prepared according to Example 5 are dispersed in 500 milliliters of distilled water and placed in a Homogenizer Rannie Mini Lab 12-51 (APV Rannie, Copenhagen), under a pressure of 300 bar at a rate of 10 liters / hour. We inject 0.5 grams of hydrogenated soy phosphatidylcholine (Nattermann, Fr) directly into the recirculating suspension in the homogenizer.
This preparation is homogenized for 12 minutes.
The suspension obtained contains type L-LMVS of about 200 nm scattered.

EXAMPLE 9 PREPARATION OF A SUCCESSION OF FATTY MATTER:
ANCHORING OF PROTEIN AND PHOSPHOLIPID CONSTITUENTS
MILK AT THE SURFACE OF PREPARED HYDROPHILIC MATRICES

100 g of hydrophilic matrices prepared according to Example 4 are put in a mixer of 3 liters. 10g of protein constituents and phospholipids are added.
1 liter is gradually introduced with stirring of water.
The whole is homogenized at a speed of 801 / h and 200bars (Westfalia homogenizer).
We obtain a creamy preparation containing almost no free protein and phospholipid components: two controls are carried out with protein and phospholipid constituents alone.
A control (# 1) and the preparation are centrifuged. The rate of proteins, by the Bradford method, in the non-centrifuged control, in the supernatant of the centrifuged control and in the supernatant of the preparation. The analyzes are carried out on 100m1. We measure 0.5g of proteins in the supernatant of the centrifuged control and 0.05g in the WO 94/20078 215rJ3Op i ~ PCT / FR94 / 00228 i supernatant of the preparation. From these results, the quantity of protein present in the centrifugate of control No. 1, ie 0.05 g, and the quantity of proteins anchored on the hydrophilic matrices is 0 -lg.
The percentage of anchored proteins is 80% compared to the amount of protein introduced initially.
On the other hand, the centrifugate of the preparation is dried and washed, by filtration with chloroform, so as to extract the phospholipids. The chloroform is then evaporated and the phospholipids remaining. The measurement gives 0.4g or 80% compared to the amount of phospholipid components initially introduced.
EXAMPLE 10: INCORPORATION OF AN ENZYME, GLUCOSE
OXIDASE (GO) IN THE BVSM TYPE-L
Glucose Oxidase (GO) is an acidic protein with a point isoelectric of 3.5 and an average molecular weight of 180,000 daltons.
0.3 grams of QpE-type nuclei are mixed in the dry state, obtained according to Example 2 and 5, and 0.6 grams of GO. The mixture is then gradually hydrated by adding 1.5 milliliters of buffer to pH 7, above the pH of the GO, with stirring at room temperature, then left agitated all night in a cold room (4 C). The pH is then adjusted to 3, below the pI of the GO and incubated for 30min additional. The preparation is then dispersed in 48.5 milliliters distilled water, the pH adjusted to 7, and lyophilized in the presence of bicarbonate ammonium. The nuclei loaded with GO thus obtained are mixed with 0.15 grams of hydrogenated soy phosphatidylcholine (Nattermann, Fr.) powdered. The mixture is hydrated with 150 milliliters distilled water and homogenized in a Rannie Mini Lab homogenizer 12-51 (APV Rannie, Copenhagen, DK) under a pressure of 300 bar. We thus obtains type-L-type LMVSM loaded with GO, with a yield encapsulation rate of 92% and an incorporation rate of 184% compared to weight of the nuclei, determined by UV at 278 nm after ultrafiltration of suspension.

T) LACTOPEROXYDASE (LP) IN PS-TYPE BVSM

Lactoperoxidase (LP) is an antibacterial enzyme. It is a basic protein having an isoelectric point of 9.6 and a weight average molecular weight of about 80,000 daltons.
0.5 grams of BVSM type-PS are suspended Anionic compounds (CM), obtained according to Example 3 and then 5, in 100 milliliters of a buffer adjusted to pH 7, below the pH of the LP, in a 250 ml flask.
0.5 g of LP (BioSerae) solubilized in 1 milliliter are then introduced.
of water, with stirring.
The mixture is stirred overnight in a room cold (4 C). The pH is then adjusted to 9.8, above the pH of the LP, and incubated for 30min. The pH is then reduced to 7 and the suspension is lyophilized in the presence of ammonium bicarbonate (20 grams / liter).
PS-type BVSMs loaded with LP are obtained with a yield of 99% loading and 99% incorporation rate by weight nuclei according to the UV assay at 412 nm.

EXAMPLE 12: ANTI-BACTERIAL ACTIVITY OF THE LP MIX
ENCAPSULATED IN PS-TYPE PSMS AND GO ENCAPSULATED
IN L-TYPE BVSM

* Assay of in vitro activity:
1 ml of a 1M glucose solution, 0.2 ml of a ortho-phenylenediamine solution (1 mg / ml in pH citrate buffer 5.6 concentration 10 mM) and 1.6 ml of a citrate buffer solution (10 mM, pH
5.6).
In addition, 0.2 mg of BVSM type-PS containing lactoperoxidase prepared according to Example 11 and 1.8 mg of BVSM type L containing Glucose Oxidase prepared according to Example 10 in 1 ml of water.
0.2 ml of this suspension is added to the 2.8 ml prepared more high and stirred for 2 minutes at room temperature.
The color reaction that develops is stopped by 1 ml 2N sulfuric acid and the color is read at 490 nm against a white containing no BVSM.

~ 2157384.

The presence of an intense coloration shows that both Enzymes encapsulated in the L-type BVSN1 retain their activity. The Assaying the activity of encapsulated enzymes gives a comparable result to that obtained with the enzymes in aqueous solution to the same concentration.

* Anti-bacterial activity against a strain of Escherichia coli:
A glucose LB culture medium mixed with a Agar agar, poured into antibiogram boxes. The strain of E. coli is seeded on the surface of the agar at the rate of 200 l / box. Some discs sterile paper are impregnated with enzyme suspensions, encapsulated or no and deposited on the agar plates seeded. Let incubate 2-4h at 37 ° C. and the inhibition diameters around the disks are measured.
The discs have been impregnated with concentrations of enzymes ranging from 0.05 to 0.6 mg / ml LP and from 0.8 to 10 mg / ml of GO. The Inhibition diameters range from 12 to 20 mm and are comparable enzymes were encapsulated or not. EXAMPLE 13: STABILIZATION OF THE ACTIVITY
ANTIBACTERIAL

ENZYME ENCAPSULATED IN BVSM

Three lots of glucose oxidase were prepared. Lot 1 is consisting of an aqueous solution of GO at 0.2 mg / ml, the batch 2 by a aqueous suspension of GO encapsulated in BVSM prepared according to previous patents, with an encapsulation rate of 200% compared to particle weight and whose GO concentration is 0.2 mg / ml and the batch 3 by an aqueous suspension of GO encapsulated in BVSM type-L
according to Example 10, and whose concentration in GO is also 0.2 mg / ml.
These three suspensions are left at 4 C and dosed every weeks then every month by the method described in Example 12.
The activity of the GO solution decreases over time and becomes zero at after 250 days. Conversely, the activity of the two suspensions of GO
encapsulated in either the BVSM or the L-type BVSM remains constant and remains equal to 100% of the initial activity after 320 days.
Furthermore, an aqueous solution is also prepared of lactoperoxidase at 0.1 mg / ml and a suspension of LP encapsulated in PS-PSV prepared according to Example 11 and whose concentration in LP is also 0.1 mg / ml.

~ - 21 ~~~ 8 he These two suspensions are left at 4 C and dosed every weeks then every month by the method described in Example 12.
The activity of the LP solution decreases over time and, after 90 days, the residual activity is only 35% of the activity initial. Conversely, the activity of the LP suspension encapsulated in the BVSM PS-type remains constant and remains equal to 100% of activity initial after 90 days.

(OPC) IN L-TYPE OR COMPLETE BVSM

A - Incorporation of OPC into an ammonium gel quaternary Procyanidolic oligomers (OPC) are agents antioxidants from the family of hydrolysable tannins (flavonoids). They have anti-free radical properties. Their molecular weight average is about 2,000 daitons. They are soluble in water at a pH
less than 7.
5 grams of OPC are solubilized in a minimum quantity distilled water (approximately 5 milliliters) and 10 grams of gel quaternary ammonium type, prepared according to Example 2. The mixture is hydrated with 180 milliliters of buffer at pH 6, above the average pKa of OPC, with stirring to obtain a homogeneous paste. The mixture is left restless one night. The pH is then reduced to 3.5, below the average pKa of OPC, left incubated 30 min. stirred and then washed with distilled water centrifugation to eliminate free OPC. The amount of free OPC in the supernatant is determined by UV at 280 nm. We get a return encapsulation rate of 82% and a loading rate of 41%
weight of the gel.
This loaded gel is then dispersed in 500 milliliters of water distilled and homogenized in a Rannie Mini Lab 12-51 homogenizer (APV Rannie, Copenhagen, DK) under a pressure of 600 bar for 15 minutes at a rate of 10 liters / hour. We obtain a suspension of BVSNi PS type loaded with OPCs at a size centered around 200 nm.

2 15 "138 4 B - Obtaining 200 nm L-type BVSM loaded in OPC

The PS-PS suspension obtained previously is placed in a Rannie Mini Lab 12-51 homogenizer, under pressure 300 bars.
0.5 grams of soy phosphatidylcholine is then injected hydrogenated (Nattermann, Fr.) directly into the suspension in recirculation in the homogenizer, under a pressure of 300 bar and homogenizes for 12 minutes with a flow rate of 10 liters / hour. The obtained supension contains particles of about 200 nm dispersed.
C - Obtaining full 200 nm BVSM 'loaded in OPC:
"Complete" BVSM are prepared as previously described by means of PS PSMs loaded with OPC by drying (lyophilization), regioselective acylation in an organic solvent and establishment of an outer leaflet of phospholipids.

D - Stabilization of procyanidolic oligomers (OPC) in the BVSM type L

An oil-in-water emulsion is prepared and divided in three lots. OPCs are incorporated in the first batch at the concentration final 0.05%. In a second batch are embedded encapsulated OPCs in BVSM type L and in the third batch are incorporated OPC
encapsulated in "complete" BVSMs. The final concentration of these two last lots is also 0,05% in OPC; These three emulsions have a uniform creamy white color.
A sample of each of the three lots is placed under one UV lamp (254 nm) for 48 hours and then protected from light.
After 10 days, the emulsion containing the free OPCs has a very intense brown color, the emulsion containing encapsulated OPCs in BVSM type L has a slightly creamy white color slightly more sustained while the emulsion containing OPCs encapsulated in BVSM "complete" has not evolved.
In the same way, the stability of the OPCs in these emulsions is tested for 6 months at 40 C. The cream containing the OPCs encapsulated in "complete" BVSM did not significantly change staining;
containing OPCs encapsulated in BVSM type L has a color slightly more intense whereas that containing free OPC is become brownish.

ANTICANCER, DOXORUBICIN, IN L-TYPE BVSM
USING THE CONTROLLED LOADING MODE
TOPOLOGICAL
Doxorubicin is an anticancer antibiotic belonging to the family of anthracyclines. It is an amphiphilic product characterized by a polyaromatic aglycone conferring on the molecule characteristic fluorescence properties and by an amino sugar, the daunosamine. The molecular weight of the hydrochloride is 580 and its pKa is 8.5.
Polysaccharide nuclei prepared as previously.

1- Incorporation of doxorubicin without topological control Doxorubicin (0.1g) in aqueous solution is added gradually to the polysaccharide nuclei (0.5 g) with stirring magnetic. The suspension obtained is then left stirring for 17 hours at room temperature and protected from light.
The polysaccharide nuclei thus loaded into doxorubicin completely precipitated. Even in the presence of detergent and phospholipids, they can not be dispersed properly with a size of 20nm.
Incorporation of doxorubicin without topological control leads to the total aggregation of polysaccharide nuclei and can only be used to form 20nm L-type LMVMS.

2- Incorporation of doxorubicin with topological control Doxorubicin (0.1g) in aqueous solution is added gradually to the polysaccharide nuclei (0.5 g) with stirring ~~~~~ 811 28 magnetic. The pH is adjusted to 7, below the pKa of doxorubicin, during the addition. The suspension obtained is stirred for 17 hours at ambient temperature and protected from light. The pH is then adjusted to 9, above pKa of doxorubicin, and incubated for 30min. The polysaccharide ring suspension is then divided into two: one part is analyzed as polysaccharide nuclei, the other part polysaccharide cores are prepared in L-type BVSM

a- BVSM type PS
After the incubation step at pH 9, the suspension obtained is diluted in 11 of water and brought back to pH 7. The nuclei thus loaded are analyzed: the 0.2 m filtration of the suspension shows a greater than 95% doxorubicin, indicating that the size of the nuclei polysaccharide is 20 nm, and the centrifugal ultrafiltration of an aliquot suspension shows the absence of free doxorubicin. The results indicate the presence of 4mg of doxorubicin in the ultrafiltrate and 46mg of doxorubicin in the polysaccharide nuclei, which corresponds to a yield of 92% and an encapsulation rate of 18% in doxorubicin. The 0.2 m filtration of the suspension obtained shows a yield greater than 95%, indicating that the size of the BVSM is 20nm.

b- BVSM type L
The polysaccharide nuclei are then dispersed with phospholipids (0.25 g) (EYPC / cholesterol 80/20 by weight) predispersed in hMameg SOmM at a concentration of 10 g / l. Suspension obtained is diluted sharply in 11 of water. The detergent is then removed by ultrafiltration. Doxorubicin is then assayed in ultrafiltrate and in the BVSM obtained. The results indicate the presence 2.5 mg of doxorubicin in the ultrafiltrate and 47.5 mg of doxorubicin in the BVSM, which corresponds to a doxorubicin yield of 95%
and a rate of encapsulation in doxorubicin of 19% compared to polysaccharide nuclei. 0.2 m filtration of the suspension obtained has a yield greater than 95%, which indicates that the size of the BVSM is 20nm.

the POLYSACCHARIDE CORES

Doxorubicin is an anticancer antibiotic belonging to the family of anthracyclines. It is an amphiphilic product characterized by a polyaromatic aglycone, conferring on the molecule characteristic fluorescence properties, and by an amino sugar, the daunosamine. The molecular weight of the hydrochloride is 580 and its pKa is 8.2-8.5.
* Incorporation of doxorubicin in nuclei polysaccharides -The polysaccharide cores used were crosslinked and functionalized by POCI3 and have a size of 20 nm. Their density ionic is 1.59mequivP04 / g ..
The polysaccharide nuclei (10 mg) are dispersed in water (10ml) under ultrasound. The pH of the suspension of nuclei is adjusted to 7 with 0.1N NaOH. Doxorubicin (4.6mg) in solution in water at 5mg / ml is slowly added under sonication by 1. The pH is adjusted to 7 if necessary with 0.1N NaOH.
The polysaccharide nuclei thus loaded are characterized by:
- their ability to filter over 0.2 m: the filtration efficiency is determined by the ratio of concentrations before and after filtration.
After incorporation, 100 1 of the suspension of nuclei charged polysaccharides are taken to determine the doxorubicin concentration. The rest of the suspension is filtered on 0.2 m porosity membrane. An aliquot of 1001il is again taken for the determination of doxorubicin. Doxorubicin is dosed by HPLC after release of the polysaccharide nuclei.
doxorubicin concentration after filtration Filtration efficiency over 0.2 m = ------------------------------------ x 100 (%) concentration of doxorubicin before filtration the unincorporated fraction that is determined by ultrafiltration centrifugal. After filtration, 1 ml of the suspension of nuclei polysaccharide diluted 1/2 is deposited on the system CM
of centrifugal ultrafiltration (Nlicrosep) then centrifuged at 7500g For 30 minutes. The ultrafiltrate obtained is determined by HPLC in doxorubicin.

doxorubicin concentration in 1'ultrafiltrat 10 Unincorporated fraction = -------------------------------------------- - x (96) concentration of doxorubicin after filtration The filtration yield is 97% and the fraction incorporated is less than 5%, leading to a 99% incorporation.

* Comparison of behavior under physiological conditions of doxorubicin incorporated into the nuclei polysaccharide and in BVSM
2p Particles post-loaded with doxorubicin, nuclei polysaccharide or BVSM are incubated in PBS at 37 ° C
concentration of doxorubicin of 50 g / ml at time 0h and 4h, 1 ml of particle suspension is removed and ultrafiltered by centrifugation (7500g, 30min) on Microsep to determine the doxorubicin fraction 25 released. The ultrafiltrate obtained is then dosed into doxorubicin by HPLC.
The results are presented in the following table:

% of doxorubicin Type of particles Type of particles remaining incorporated NPS BVSM
30 hours 0h 67 +/- 1 62 +/- l time 4h 64 +/- 3 55 +/- S
Behavior in physiological conditions of nuclei polysaccharide and BVSM, incorporated into doxorubicin by post-,_loading ~ 31 The results obtained indicate a difference in behavior of doxorubicin incorporated in these two types of particles: doxorubicin remains incorporated more strongly in the polysaccharide nuclei only in BVSM.

BVSM

* Incorporation of doxorubicin into BVSM (type L or no) BVSM are prepared from acylated nuclei on which the phospholipidic layer (100% by weight) is established by the detergent method.
Briefly, phospholipids (10 mg) of EYPC composition 80/20 cholesterol (by weight) are dispersed in Hecameg SOnM (2m1).
The acylated nuclei (10 mg) are added to the phospholipids and then subjected to Ultrasonic bath for 5min. The suspension obtained is diluted in 8m1 distilled water under the CMC of Hecameg which is 20nM then dialyzed extensively. The BVSM obtained are filtered on 0,2 m and preserved sterile.
Type LMSVs are prepared according to the same protocol but using polysaccharide nuclei instead of nuclei acylated.
The pH of the suspension of BVSM (20mg) is adjusted to 7 with 0.1N NaOH Doxorubicin (4.6 mg) in solution in water at Smg / ml is slowly added under sonication by 20 1. The pH is adjusted to 7 if necessary with 0.1N NaOH.
The BVSM thus loaded are characterized by:
- their ability to filter over 0.2 m: the filtration efficiency is determined by the ratio of concentrations before and after filtration.

After incorporation, 100 1 of the loaded BVSM suspension are taken to determine the concentration of doxorubicin. The The remainder of the suspension is filtered through a 0.2 m porosity membrane. A
aliquot of 100 l is again taken for the determination of the doxorubicin. Doxorubicin is assayed by HPLC after release BVSM;
doxorubicin concentration after filtration Filtration efficiency over 0.2 m = ----------------------------------- x 100 (%) concentration of doxorubicin before filtration the unincorporated fraction which is determined by ultrafiltration centrifugal. After filtration, 1 ml of the suspension of BVSM diluted 1/2 is deposited on the centrifugal ultrafiltration system (Microsep) then centrifuged at 7500g for 30min. The ultrafiltrate obtained is measured by HPLC in doxorubicin.
doxorubicin concentration in the ultrafiltrate Unincorporated fraction g --------------------------------------------- - x 100 (%) concentration of doxorubicin after filtration The filtration yield is 95% and the unincorporated fraction is less than 5% leading to an incorporation efficiency of 99%.

* Behavior in physiological conditions of the Doxorubicin incorporated into type L-LMHBs Post-loaded particles of doxorubicin, L-type BVMS
or not, are incubated in PBS at 37 C at a concentration of doxorubicin of 50 g / ml at time 0h and 4h, lml of the suspension of particles is removed and ultrafiltered by centrifugation (7500g, 30 min) on Microsep to determine the fraction of doxorubicin released. The ultrafiltrate obtained is then dosed into doxorubicin by HPLC.

~.

The results are shown in the following table ~ Yo doxorubicin BVSM BVSM
remaining incorporated type L
time 0h 62 +/- 1 time 4h 54 +/- 3 55 +/- 5 Behavior in physiological conditions of BVSM, type L or not, incorporated into doxorubicin by post-loading The results obtained indicate that L-type LMVs, loaded with doxorubicin, exhibit behavioral conditions physiological similar to that of the BVSM.

Legend of FIG. 1 = 37 C
~ 100 C
Legend of FIG. 2 = 37 C
~ 100 C
0 37 C Sonicated Legend of FIG. 3 = 37 C

Claims (18)

1. Synthetic particulate vector, characterized in that it comprises:
a hydrophilic non-liquid core, an outer layer consisting at least in part of amphiphilic compounds, associated with the nucleus by hydrophobic interactions and / or bonds Ionic. 2. Particulate vector according to claim 1, characterized in that the core hydrophilic is made up of a matrix of polysaccharides or oligosaccharides naturally or chemically crosslinked. 3. Particulate vector according to one of claims 1 and 2, characterized in what ionic ligands are grafted onto the hydrophilic core. Particulate vector according to one of Claims 1 to 3, characterized in what the amphiphilic compounds of the outer layer are chosen from the group including phospholipids and ceramides and ionic surfactants and nonionic surfactants. Particulate vector according to one of Claims 1 to 4, characterized in that what the outer layer further comprises at least one selected lipidic compound in the group consisting of cholesterol and natural fatty acids and fat soluble vitamins. 6. Particulate vector according to one of claims 1 to 5, characterized in what he further comprises an active ingredient, located in the core or in the layer external. 7. Particulate vector according to claim 2, characterized in that includes in addition, an active principle located in the heart of the matrix. Particulate vector according to one of claims 1 to 7, characterized what he has a diameter of between 10 nm and 5 μm. Particulate vector according to one of claims 1 to 7, characterized what he has a diameter of between 20 and 200 nm. 10. Particulate vector according to one of claims 1 to 9, characterized what he contains an active ingredient selected from the group consisting of:
- antibiotics and anti-virals, proteins, proteoglycans, peptides, polysaccharides, lipopolysaccharides, the antibodies, antigens, - insecticides and fungicides, the compounds acting on the cardiovascular system, - anti-cancer, - anti-malarics, - anti-asthmatics, the compounds having an action on the skin, - the constituents of the milk fat globules. 11. A process for preparing a particulate vector according to one of claims 1 to 10 characterized in that:
a) the encapsulation of an acidic or basic ionisable active ingredient is carried out, in a cross-linked hydrophilic matrix grafted with ligands of a species ionic complement of that of the active ingredient, at a pH at which the active ingredient is in ionized form, b) the pH of the medium is varied, with respect to the pK a of the active principle, until a value at which the active ingredient is not in ionized form, c) recovering the hydrophilic matrix comprising the localized active ingredient at center of said matrix, d) a layer of amphiphilic compounds is fixed on the matrix obtained at the end of of step c). 12. Process for the preparation of a particulate vector, according to the claim characterized in that a) the encapsulation of at least one basic ionizable active ingredient is carried out, in a crosslinked hydrophilic matrix grafted with ionic ligands acids, at a pH below the active ingredient pK a, b) the pH of the medium is increased to a value greater than the pK a of active ingredient, c) recovering the hydrophilic matrix comprising the localized active ingredient at center of said matrix, d) a layer of amphiphilic compounds is fixed on the matrix obtained at the end of of step c). 13. Process for the preparation of a particulate vector, according to the claim characterized in that a) the encapsulation of at least one acid ionizable active ingredient, in a crosslinked hydrophilic matrix grafted with basic ionic ligands, a pH greater than the pK a of the active principle, b) the pH of the medium is lowered to a value lower than the pK a of principle active, c) recovering the hydrophilic matrix comprising the localized active ingredient at center of said matrix, d) a layer of amphiphilic compounds is fixed on the matrix obtained at the end of of step c). 14. Method according to one of claims 11 to 13, characterized in that the matrix hydrophilic is composed of polysaccharides or oligosaccharides, naturally or chemically crosslinked. 15. Pharmaceutical composition, characterized in that it contains a vector particle according to one of claims 1 to 10 or obtained by a method according to one of claims 11 to 14, and a pharmaceutically acceptable carrier acceptable for his administration. Cosmetic composition, characterized in that it contains a vector particle according to one of claims 1 to 10 or obtained by a method according to one of claims 11 to 14, and cosmetologically excipients acceptable. 17. A composition for a food use, characterized in that it contains a vector according to one of claims 1 to 10 or obtained by a method according to one of claims 11 to 14. 18. Use for the production of a drug of a vector according to one of the Claims 1 to 10 or obtained by a method according to one of the claims 11 to 14.