MXPA97007392A - Particles based on polyaminoacido (s) for use as vectors of active principle (s) and methods for preparation - Google Patents

Abstract

The present invention relates to vectors useful for the administration of active principles (PA), preferably medicinal or nutritional, particularly orally or parenterally. The technical problem solved by the invention is to provide vectors consisting of (nano) or (micro) particles based on polyamino acids, and which are inert for PA (proteins), of a controllable granulometry, resistant and economical. According to the invention, the particles have an average size of less than 200 microns, and consist of a polyamino acid of the Leu / Glu type, where Leu / Glu + Leu is greater than or equal to 3 percent, and Mw is greater than 400

Description

PARTICLES BASED ON MINOACIDO POLY (S) TO BE USED AS VECTORS OF ACTIVE PRINCIPLE (S). AND METHODS FOR ITS PREPARATION The field of the present invention is that of vectors useful for the administration of active principles (PA) through cell membranes. These vectors allow the transport under protection of the PA, inside an organism, to its site of action. The PA is preferably a medicament or a nutrient for administration to an animal or human organism orally, nasally, vaginally, occularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, intracerebrally, parenterally, etc., but can also be a herbicide, a pesticide, an insecticide, a fungicide, etc., for the treatment of agricultural crops as a phytosanitary application. For all these applications, PA vectors aim to improve the bioavailability of PAs. These vectors can be, for example, prolonged release systems of PA. The APs to which the invention makes special reference, although this is not limiting, are for example, proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, oligonucleotides and polynucleotides. The present invention relates more specifically to particles - mostly sub-icronic and / or micronic - based on polyamino acids and intended to be used as PA vectors, in particular medicaments. Therefore, they are Vectorization Particles (PV), among which, throughout the present exhibition, on the one hand, Vectorization NanoParticles (NPV) and, on the other hand, Vectorization Microparticles (MPV), according to their own nomenclature, will be distinguished. to the invention and that will be defined later. The present invention aims both at the naked particles as such, as at the PA vector systems, constituted by the particles charged by the considered PA (s). The invention also relates to a process for preparing said particles. The advances in genetic engineering and biotechnologies, as well as the discoveries of genetic tools, proteins and biologically active peptides, which result from these, allowed the rise of new active medicinal principles (PA) that offer an intrinsic activity and a high selectivity. Said PAs are, on the other hand, easily degraded in the organism before reaching its therapeutic action site, being therefore its bioavailability very weak. In the case of oral administration, the gastrointestinal tract constitutes a fearsome chemical and physical barrier for PA that must, on the one hand, resist degradation by the digestive system and, on the other, pass through the epithelial membrane gastrointestinal. In this regard, you can consult, for example, the magazine of M.J. HUMPHREY (Delivery System for Peptides Drugs, edited by S. DAVIS and L. ILLUM, Plenum Press, N.Y., 1986), which takes into account the low bioavailability of peptides and peptides administered orally. Naturally, these avatars of transport and permanence in the organism are not limited to proteins, but also affect PAs formed by genetic tools (oligonucleotides, polynucleotides, plasmids) susceptible to be applied in gene therapy techniques. To alleviate this, it was proposed to encapsulate PA in PA vectorization particles, also called PV. The interest of these encapsulation techniques is to protect and / or transport the PA to its site of therapeutic action, safeguarding it against the aggressions of the organism in order to increase its bioavailability. Among all the materials considered for the encapsulation of PA, polymers are increasingly used, because of their intrinsic properties. In the case of the requirements that the PV must meet, they are demanding and include, above all, the following specifications. 1. It should be possible to have PV of average diameter comprised between a fraction of micron and a few microns, with a narrow granulometric distribution, so as to be able to adapt the granulometry of the PVs to the chosen administration mode and / or the desired therapeutic site. For example, if an oral mucous immunization is sought, the size of the PVs should be between 0.5 μm and 10 μm, in order that the PV can penetrate the Pleyer plaques and reach the lymphatic tissues. In the case of a subcutaneous administration, it has the advantage of having PV of size greater than 10 microns so that the particles do not enter the general circulation, where they are rapidly internalized by the reticuloendothelial system, but are progressively disseminated from their site. of injection. This specification implies a dimensional control of the PVs, at the same time on the distribution of the granulometry of the PVs and on their average diameter, which constitutes a very delicate operation in the technological plane. 2. It is desirable that PVs ensure protection of the PA to the site of release. For example, in an oral administration of a PA formed by a vaccine, the latter would gain by being protected, along the gastrointestinal tract. 3. It is preferable that the polymer, which constitutes PV, be biocompatible and biodegradable and, furthermore, that it be metabolized into non-toxic products for the organism. 4. It is also advantageous that the polymer, constitutive of the PVs, does not induce an immune (immunogenic) response. 5. Finally, it is also preferable that the PV can be obtained by a non-denaturing process for the PA. In this way, the use of organic solvents and / or elevated temperatures is prohibited. Numerous previous technical proposals tried in vain to satisfy the set of these specifications, so the answers provided until then are partial and incomplete. Among these unsuccessful proposals, mention may be made of the one presented in U.S. Patent No. 5,286,495, which relates to a process for encapsulating proteins in aqueous phase, with materials constituted by alginate and polylysine. This procedure is presented as non-denaturing for protein PAs, since there is no organic solvent, aggressive chemical reagent or elevated temperature. However, the PV manufacturing technique, by applied vaporization, does not allow the production of particles smaller than 35 microns, which does not allow their penetration by the body's cells. On the other hand, emulsion techniques are commonly used to prepare mycoparticles of a few microns. For example, Patent Applications WO 91/06286 and WO 91/06287 disclose methods of forming emulsion particles in which a hydrophobic protein chosen from collagen, casein, keratin and, preferably, prolase, or a biocompatible polymer is used as the polymer; biodegradable, such as poly (lactic) or poly (orthoester). The PA can be hydrophobic or hydrophilic, but in the latter case, the double emulsion technique is recommended. The size of the microparticles is approximately 100 microns and, preferably, between 50 nanometers and 100 microns. Patent Application WO 89/08449 also refers to emulsion encapsulation, to incorporate PA into poly (lactic) microparticles less than 10 microns in size. And in this document it is specified that said size is a maximum limit for the absorption through the lymphatic tissues of the mucous membranes (oral, nasal, rectal, ophthalmological administration). The emulsion techniques are very attractive a priori, since they allow the application of most PA in microparticles, whose granulometry can be controlled up to sizes of the order of 1 microns. But in these techniques, organic solvents are used to solubilize the constituent polymers of the particles. These solvents are, for example, ketones, alcohols, amides or their mixtures. And regrettably, it turns out that said solvents can be denaturants, especially for peptide or polypeptide PAs. Biocompatible PVs, formed in aqueous solution without excessive elevation of temperature and called proteinoids, are also known. They were described from 1970 by W. FOX and K. DOSE in "Molecular Evolution and the Origin of Life", Ed. Marcel DEKKER Inc. (1977). On the basis of these works, Patent Application WO 88/01 213 ('1213) proposes a system for the release of PA based on proteinoids. The polymer used is a mixture of artificial polypeptides obtained by thermal condensation of synthetic or natural amino acids and / or small peptide chains. The mode of condensation chosen leads to branched oligomers and, therefore, very little soluble. Then we proceed to a selection by filtering those branched oligomers, in order to recover the water-soluble fractions. This fraction is necessarily composed of branched crosslinks of very small mass. The microparticles according to this invention are obtained by changing the pH which causes the precipitation of the branched oligomers in proteinoids.

When the solution in which the precipitation is carried out contains PA in solution, a part of these is taken to the proteinoid at the time of its formation. The drawbacks of this system are: - a low percentage of encapsulation, a delicate purification procedure, a non-regular (non-alpha peptidic) chaining of the amino acids, due to the synthesis mode that does not allow affirming that the enzymatic degradation reactions will be identical to those of an alpha-polyamino acid, finally, the use of a large number of different amino acid monomers, which can induce an immune response. Patent Application WO 93/25 589 refers to the improvement of the process of synthesis of proteinoids by thermal condensation of amino acids. The proteinoids are formed, here too, by branched oligomers of small molar masses, constituted by irregular chains of amino acids. The water-soluble character of said branched oligomers obtains: on the one hand, by the use of very small masses (between 250 and 2,400), which corresponds to very short chains of 2 to 20 amino acids, on the other hand, by the choice of amino acids of departure.

As above, the proteinoids are formed by precipitation triggered by lowering of the pH of the water-soluble branched oligomers. When this precipitation occurs in the presence of water-soluble PAs, a part of these is carried in the proteinoid at the time of its formation. The encapsulation percentages remain modest: from 20 percent to 40 percent. On the other hand, the decrease in pH can be harmful for some PA. Furthermore, the fact that the encapsulation has to be carried out at a particular pH constitutes an annoying methodological limitation, and limits the use of these microparticles with pH of precipitation of the proteinoids that does not necessarily correspond to the biological pH. For example, the pH can vary from 2 to 7. in the gastrointestinal tract. It should also be remembered that United States Patent Number 4,351,337, coming from a field different from that of the vectorization of PA own to the invention. This patent discloses fixed and localized implants in very precise sites of the organism. Therefore, such implants have nothing to do with administrable forms, for example, oral or by injection. Said implants may be, inter alia, spherical spherical or gangue spherical capsules, whose dimensions are of the order of 400 to 800 microns (Figures 8 and 9) and, therefore, well above the dimensions of the order of 0.5 microns and 10 microns required for the microparticles to be internalized by the cells of the organism. These implants are made from polymer materials of the polyamino acid type (Leu / Glu, specifically). The conformation of these implants is effected, for example, by means of solutions of polyamino acids in dioxane, which are evaporated in fine form. In this state of knowledge, one of the essential objectives of the present invention is to provide PV, in particular submicronic or micronic, based on polyamino acids and capable of serving as vectors of an active principle (PA), in particular medicament and / or nutritional , for the administration of said PA to a human or animal organism, satisfying fully such PV the requirements listed above and which are repeated below: 1. It should be possible to have PV of average diameter, comprised between a fraction of micron and a few microns , with a narrow granulometric distribution, in order to adapt the granulometry of the PVs to the chosen administration mode and / or the desired therapeutic site. For example, if an oral mucosal immunization is sought, the size of the PVs should be between 0.5 microns and 10 microns, in order that the PVs can penetrate the Pleyer plaques and reach the lymphatic tissues. In the case of subcutaneous administration, it has the advantage of having PV larger than 10 microns so that the particles do not enter the general circulation, where they are rapidly internalized by the reticuloendothelial system, but progressively spread from their injection site. This specification implies a dimensional control of the PVs, at the same time on the distribution of the granulometry of the PVs and on their average diameter, which constitutes a very delicate operation from the technological point of view. 2. It is desirable that PVs ensure protection of the PA to the site of release. For example, in an oral administration of a PA formed by a vaccine, the latter would gain by being protected, along the gastrointestinal tract. 3. It is preferable that the polymer, which constitutes PV, be biocompatible and biodegradable and, furthermore, that it be metabolized into non-toxic products for the organism. 4. It is also advantageous that the polymer, which is a constituent of PV, does not induce an immune response (immunogenic). 5. Finally, it is also preferable that the PV can be obtained by a non-denaturing process for the PA. In this way, the use of organic solvents and / or elevated temperatures is prohibited.

Another essential objective of the invention is to provide PV based on polyamino acids having a controllable and adjustable average granulometry within orders of size ranging from 200 microns (MPV) to a few nanometers (NPV). Another essential objective of the invention is to provide PV that are simple to prepare (non-aggressive pH), stable at all pH comprised between 4 and 13 and non-immunogenic. Another essential objective of the invention is to provide PV based on polyamino acids that are industrially feasible and economical and that are suitable for loading into PA with high load percentages. Another essential objective of the invention is to provide a process for preparing MPV and / or NPV based on polyamino acids and susceptible to being used as PA vectors, said economic process having to be simple to apply, non-denaturing for PAs, and also allow a fine control of the average particle size of the particles obtained (maximum 200 microns). Another essential objective of the invention is the use of the mentioned particles for the preparation of medicaments (for example, vaccines) and / or nutrients, in particular for oral, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal administration , intracerebral or parenteral, active ingredients, such as proteins, glycoproteins, peptides, polysaccharides, lipopolysaccharides, oligonucleotides and polynucleotides. Another essential objective of the invention is to provide a medicament of the type prolonged release system of PA, which is biocompatible and which presents a high bioavailability of PA. Another essential objective of the invention is to provide a vaccine vectorization system that is non-immunogenic intrinsically and in combination with one or more antigens.

BRIEF DESCRIPTION OF THE INVENTION The objectives relating to the products, among others, are achieved by the present invention, which refers to vectorization nanoparticles (NPV) of active principle (s), of the type based on polyamino acid (s) characterized: because, in an average size, they comprise between 0.01 and 0.5 microns, preferably between 0.03 and 0.4 microns, because these NPV are obtained by contacting the polyamino acids (PAA) with an aqueous solution, because their polyamino acids are linear in α-peptide chains, and comprising at least two recurring amino acid types AAN and AAI: * the AAN type corresponding to a hydrophobic neutral amino acid, * and the AAI type corresponding to an ionizable side chain amino acid, at least one being part of the recurring amino acids of type AAI in ionized form, being the recurring amino acids of each type, AAN and AAI, identical or different from each other, and because the mass The weight of the polyamino acids is greater than or equal to 4,000 D, preferably 5,000 D, because these PAAs are not water soluble at an acid pH and at a pH between 3 and 12. The merit of the Applicant is to have proceeded to a selection among the polyamino acids to retain only those which are characterized by being water-insoluble, by forming stable colloidal suspensions in a wide pH range compatible with the pH of the physiological media for the applications considered, and by containing a first type of AAN monomers formed by a neutral hydrophobic amino acid and at least a second type of monomer formed by an amino acid AAI, characterized by a side chain of carboxyl functionality (Glu, Asp), ionizable with non-denaturing physiological pH for the proteins.

DETAILED DESCRIPTION OF THE INVENTION According to a feature of the invention, these polyamino acids (PAA) are linear and, more preferably, exhibit α-peptide linkages. The PAA, selected as constitutive elements of the PVs of the invention, present the advantage of being able to be PAA "blocks" and / or "statistical" PAA. The PAA "blocks" are those that have an ordered, sequential and alternating structure, in which the amino acids are distributed in blocks along the polymer chains. The "statistical" PAAs are those that have an ordered, sequential and random structure, in which the amino acids are distributed irregularly along the polymer chains. Regarding the molar ratio AAN / AAI + AAN, it is a function of the "block" or "statistical" structure of the PAA. In this way, said molar relation is: >; 6 percent, preferably > 15 percent for PAA "blocks", > 20 percent, preferably > 25 percent for "statistical" PAAs. According to another characteristic of the invention, the mass of the selected polyamino acids is high. In this regard, it should be noted that the preferred weight molar mass (Mw), for the polyamino acids applied in the context of the invention, is defined differently depending on the type of polyamino acid considered.

This is how M ^ > 5,500 D, preferably it is comprised between 6,500 D and 200,000 D and, more preferably still, between 8,000 and 20,000 D, for the polyamino acid "blocks", as already defined, while, for the "statistical" polyamino acids, and also defined , M ^, > 10,000 D, preferably comprised between 20,000 D and 500,000 D and, more preferably, between 20,000 D and 150,000 D. Said polyamino acids form amphiphilic polymers which can interact both with hydrophobic substances and hydrophilic substances, which gives them remarkable properties as surfactants or as dispersants. But, in addition to their amphiphilic properties, these polyamino acids are distinguished by a new and completely unexpected property, since the polyamino acid chains in aqueous solution spontaneously associate and form particles that can be associated with proteins. In practice, such particles preferably form matrices, within which the PA (or) is dispersed. The preferred linear structure by α-peptide linkages and high molar mass are also important characteristics of these polyamino acids. Said water-insoluble PAAs are distinguished by a totally unexpected new property. When placed in contact with an aqueous solution, they spontaneously form a colloidal suspension of nanoparticles (NPV) that can be added to microparticles (MPV). In addition, proteins in solution can spontaneously associate with said particles to form charged PA particles. This discovery is all the more surprising because the teaching of Application WO 93/25 583 was more likely to incite the specialist to direct his research on an ideal material for the "encapsulation" of proteins towards products other than polyamino acids. Indeed, the numerous tests, carried out and presented in the Patent application WO 93/25 583, suggest that, among all the polyamino acids tested, only those that have been selected and claimed are suitable. And it was on the basis of an inventive procedure that the Applicant was able to prove that this was not the case, by proposing another selection of polyamino acids with a different behavior from those of the Application WO 93/25 583, these PAA being, specifically: linear PAA and with high molar masses (greater than 4,000 D), rather than small branched oligomers, insoluble PAA, rather than soluble, it is surprising that such insoluble PAAs spontaneously form a colloidal suspension of NPV and that proteins spontaneously associate with these. Preferred polyamino acids are linear synthetic polymers, with the advantage of being composed of a-amino acids linked by peptide bonds. There are numerous synthetic techniques for forming block or statistical polymers, multi-chain polymers and polymers containing a certain amino acid sequence (see Encyclopedia of Polymer Science and Engineering, Volume 12, page 786, John Wiley &Sons). Many amino acid derivatives and peptides have been used as monomers for the preparation of the polyamino acids. However, the monomers that most commonly serve are the anhydrides of the N-carboxy-a-amino acids, the preparation of which is found, for example, in Biopolymers, JL5, 1869 (1976). The polymerization techniques of these monomers are known to the specialist and are detailed in the work of H.R. KRICHELDORF "a-Aminoacid-N-Carboxy Anhydrides and Related Heterocycles" Springer Verlag (1987). Synthesis techniques generally involve protecting the reactive functions of the amino acids of ionizable side chains, so as not to interfere with the polymerization step. This results in the need for a deprotection step to restore the functionality of the ionizable side chains of the polymer. Mention may be made, for example, of the deprotection processes by saponification of the methyl esters (STAHMAN et al., J. Biol. Chem., 197, 771 (1952), KYOWA HAKKO, FR 2 152 582) or of debenzylation [BLOUT and collaborators; J. Amer. Chem. Soc., 80, 4631 (1858)].

The PVs have the advantage of having an average concentration in polyamino acids ranging from 0.01 percent to 25 percent by dry weight, preferably from 0.05 to 10 percent by dry weight According to a preferred embodiment of the particles according to the invention , the AAN (or the AAN) is (are) chosen (s) within the following list: Leu - lie - Val - Ala - Pro - Phe - and their mixtures and the AAI (or the AAI) is (are) (s) by Glu and / or Asp. More preferably, the particles of the invention are characterized in that their constitutive polyamino acids comprise a single type of AAI monomers corresponding, preferably, to Glu and a single type of monomers corresponding, preferably, to Leu. The fact of limiting the number of comonomers to only two, one of type AAN and one of type AAI; it allows to minimize the immunogenicity of the particles. This is a remarkable advantage of this preferred embodiment of the invention. The size of the selected polyamino acid particles forms part of the fundamental elements of the present invention. These particles have the advantage of having an average size between 0.01 and 200 microns, with a narrow granulometric distribution. One of the advantages of the invention is to have achieved a good control of the average granulometry of the particles and their granulometric distribution. This control goes through the achievement of extremely small particle sizes, of the order of a few nanometers and of very low polydispersity, knowing that it is possible to increase the size of these nanoparticles by aggregation. Without this limiting, two populations of particles can be distinguished according to their sizes. The first of these populations groups particles of type NPV nanoparticles, of medium size, comprised between 0.1 microns and 0.5 microns, preferably between 0.03 microns and 0.4 microns. The second population comprises the MPV type particles, with an average size greater than 0.5 microns, preferably less than or equal to 20 microns. In the sense of the invention, mean size or particle size is understood as the arithmetic mean of the diameters in volume (D4.3) established by laser diffraction, in the case of MPV, and the diameter of rotation measured by elastic diffusion of light in the case of NPV. The MPV microparticles have the advantage of being obtained from the NPV nanoparticles, for example, by aggregation. According to a variant, the microparticles comprise at least one aggregation agent.

According to a preferred feature of the invention, the particles comprise at least one active ingredient. The control of the size of the MPV and NPV is operated equally by means of the composition of the polyamino acids, but also, for the same composition, of the ordered structure (sequential alternating, that is, blocks: Sx) or disordered (sequential random, that is, statistics: S2). The nomenclature that will be used in the present exhibition to name the polyamino acids is the following: poly AAN1 / ANN2 ... AAI1 / AAI2 /...- A / B / C / D ..., being A, B, C , D ... the molar percentages of the amino acids. In addition, the structure ordered in blocks of the disordered or statistical structure is distinguished by adding the term "blocks". For example, the statistical copolymer composed of 30 percent leucine and 70 percent glutamic acid is poly Leu / Glu-30/70 and the copolymer with the same composition and block structure (Leu) n- (Glu) m is poly Leu / Glu blocks-30/70. According to a preferred embodiment of the invention, the particles are characterized in that AAI = Glu and AAN = Leu. In addition to the particles described above as a new product per se, the present invention also relates to a process for the preparation of particles based on polyamino acid (s) and capable of being used as vectors of active principle (s). , characterized: because polyamino acids (PAA): * are applied which comprise at least two recurring amino acid types AAN and AAI: >; the AAN type which corresponds to a neutral hydrophobic amino acid, > and the AAI type corresponding to an ionizable side chain amino acid, the recurring amino acids of each type being AAN and AAI identical or different from each other, * the molar ratio being AAN / AAI + AAN > 3 percent, preferably > 5 percent, * the molar mass being by weight of the polyamino acid (s) greater than or equal to 4,000 D, preferably 5,000 D, because a dispersion of said polyamino acids is carried out in a liquid, preferably in a solution aqueous salt, whose pH was adjusted to a chosen value, so that at least a part of the amino acids of type AAI is in ionized form, and because a colloidal solution of particles is thus obtained. The description of the characteristics of the PAA carried out previously, in the framework of the presentation of the particles, can be transposed entirely in the present statement relating to the procedure. This procedure is one of those that allows to obtain the NPV particles presented previously. Therefore, these particles can be those in which the AAN (or ANN) is (are) chosen in the following list: Leu - lie - Val-Ala-Pro-Phe - and its mixtures and those in which the AAI (or AAI) is (are) formed by the Glu and / or the Asp Therefore, the formation of NPV occurs in a simple manner, in an aqueous saline solution (for example) and with a pH chosen in such a way that at least a part of the AAI monomers (of identical or different nature between Yes) is in ionized form. This spontaneous generation of nanoparticles, by dispersion of copolyamino acids in saline medium, stands out for its simplicity, economy and therefore industrial feasibility. Furthermore, it is possible by this method to avoid the organic solvents, generally used to prepare this type of particles and which are known to cause protein denaturation. The conditions for obtaining these NPV are easily controlled by the specialist. The formation of NPV depends, on the one hand, on the nature of the aqueous dispersion solution and, on the other hand, on the characteristics of the polyamino acid. Aqueous dispersion solutions of polyamino acids must comply with certain pH and ionic strength conditions. Indeed, it is easily conceived that the stability of polymer nanoparticles containing ionized groups depends on the ionic strength. As for the pH, it is certainly a function of the nature of the ionizable groups, whose fraction f is of fixed ionization. In this way, for carboxyl groups f it grows with the pH. However, one of the certain advantages of the process according to the invention is to allow the spontaneous formation of the NPV independently of the pH, in an extended field of pH comprised between 3 and 13, which widely covers the field of the biological pHs, thus opening a large field of applications. NPV polyamino acids form colloidal solutions. For these polyamino acids, the discriminant characteristics of NPV formation are: i) the molar mass, 2i) the nature of the amino acids, 3i) the proportions in amino acids, 4i) the presence of linear chains, preferably α-peptidic, 5i) the distribution of the amino acids along the polymer chains, on a regular or random basis, according to the "block" or "statistical" structures, respectively. These characteristics are discussed later. Regarding the influence of the molar mass, it can be indicated that the formation of the NPV results from the association of the amino acids between the chains of the polyamino acids and that this operates differently according to the structure and the molar mass of the polymer. For structure polyamino acids "statistics", polymers of molar masses greater than or equal to 10,000 D, preferably comprised between 20,000 D and 500,000 D, and more preferably comprised between 20,000 D and 150,000 D, are easily dispersed in aqueous solution and form stable NPV colloidal suspensions. Under the same conditions, polymers of lower molar mass do not form stable colloidal suspensions, a part of the particles precipitate and NPV; maintained in dispersion, they are little diffusers. Therefore, the association of the polymer chains in NPV is all the more favorable the higher the molar mass of the polyamino acids. In the case of polyamino acids of "block" structure, the interchain association between blocks of identical amino acids is favored, which allows the use of polymer with lower molar mass than the polyamino acids of "statistical" structure. Polymers of molar masses greater than or equal to 5,000 D, preferably comprised between 6,500 D and 200,000 D and, more preferably, comprised between 8,000 D and 20,000 D, are easily dispersed in aqueous solution and form stable NPV colloidal suspensions. Regarding the influence of the nature of the amino acids and their proportion, it can be specified that, in the case of PAAs of leucine and glutamic acid, the leucine fraction must be sufficiently high to prevent the polymer from being totally soluble and ensure sufficient hydrophobic interactions for the polymer chains to associate in NPV. These interchain interactions are favored with the polyamino acids "blocks" and the minimum leucine fraction necessary to form NPV is lower with the "block" polymers than with the "statistical" polymers. It has been demonstrated, for example, that the critical concentration below which the polymer is soluble is between 20 percent and 30 percent for the "statistical" polymers of leucine and glutamic acid. For the application of the NPV preparation according to the invention, the molarity of the saline solution is set between 10 ~ 4 and 1 M, preferably 10"2 M to 0.5 M approximately In accordance with another practical embodiment of the invention, the polymer concentrations in the solution are chosen, expressed in percent weight / volume, greater than or equal to 10"2, preferably comprised between 0.05 and 30, and, more preferably, between 0.05 and 5. To the extent that one of the most notable applications of the particles and its method of obtaining according to the invention is the transport under protection of active principles in the human or animal organism, it is convenient to provide, for this purpose, that at least one active principle is dissolved in the liquid medium for the formation of particles. This dissolution of the active principle, in particular protein and polypeptide, is preferably carried out before the introduction of the polyamino acids into the medium, in order to obtain, after this introduction, a colloidal solution of charged particles in active principle. Without wanting to be subject to the theory, it can be assumed that the interaction between PA and polyamino acids comes from hydrophobic and electrostatic associations. In summary, the encapsulation according to the invention consists in: placing the PA to be encapsulated in aqueous solution, and dispersing the inoacid polyacid in an aqueous solution, then in mixing the colloidal suspension of nanoparticles thus formed with the PA solution, or, and preferably, in directly dispersing polyamino acid in the PA solution, so as to spontaneously obtain nanoparticles loaded in PA. One of the essential and main features of the invention is that the phenomenon of association of the (or) PA with the particles is independent of the pH. It has been previously observed that the dispersion of the copolyamino acid in the liquid medium, preferably saline, constitutes a key step of the process for the preparation of possibly charged particles in PA according to the invention. The process according to the invention is also characterized in that it comprises at least one additional stage of aggregation of the nanoparticles (NPV) into microparticles (MPV), preferably by means of a salt and / or a polymer (especially a polyelectrolyte) . Thanks to this characteristic of the process of the invention, it is possible to add NPV of size comprised between 0.01 and 0.05 microns, in MPV of size comprised between 0.05 and 200 microns, preferably between 0.05 and 20 microns and, more ideally still, between 0.05 and 10 microns. mieras This aggregation must be carried out under non-denaturing conditions for the PA, and the Applicant has found that the addition, especially of salts or acids or cationic polymers, causes the aggregation of the NPV in MPV.

The addition of salts allows to increase the ionic strength of the medium and causes the aggregation of the NPV, separating the electrostatic repulsions between the particles. In addition, the salt can also act as crosslinking agent of the carboxylic functions of the polyamino acids present on the surface of the particles and thus cause their aggregation by addition of various carboxylic acids on the cation of the salt. In this case, it will be preferred to choose polycationic salts, among which they form complexes with the carboxylic acids, such as the Fe2 +, Fe3 +, Zn2 +, Ca2 +, Al2 +, Al3 + and Cu2 + salts. The addition of acids decreases the fraction f of ionization by neutralizing the carboxylic functions of the polyamino acids and thus causes the aggregation of the NPV in MPV. The ionization fraction in which the aggregation is operated depends on the composition of the polyamino acid AAN / (AAN + AAI). This is lower the higher the proportion in AAI. The acid that is added has the advantage of being a strong acid of pKa lower than that of the carboxylic functions in the polyamino acids. The cationic polymers act as aggregation agents associating NPVs: they form complexes between the carboxylic functions on the surface of the particles that are thus connected to each other by the molecules of cationic polymers.

The conditions of aggregation of the NPV in MPV are developed in the examples. At the end of the procedure, with or without PA encapsulation, the (nano) and (micro) particles are recovered by any means known per se and appropriate. In practice, it is possible, for example, to make use of centrifugation and lyophilization. The active principle, which can be included or incorporated (preferably according to a matrix-like configuration) in the particles according to the invention, obtained or not by the process described above, is medicament and / or nutritional. It is preferably chosen from: proteins and / or peptides among which are preferred: hemoglobins, cytochromes, albumins, interferons, antigens, antibodies, calatonina, erythropoietin, insulin, growth hormones, factor IX, interleukin or mixtures thereof, polysaccharides, especially heparin, nucleic acids and, preferably, RNA and / or DNA oligonucleotides, and mixtures thereof. PAs that can be classified in the category of drugs and that are suitable to be vectorized by the particles according to the invention are vaccines.

As an example of a nutritional product, vitamins, amino acids and trace elements may be mentioned. According to another of its aspects, the invention also aims at the use of those NPV and MPV loaded in PA, for the manufacture of medicines of the controlled release systems type of PA. Finally, the invention relates to medicaments or pharmaceutical or nutritional specialties comprising the PVs loaded in PA and defined above. In the case of drugs, it may be, for example, those that are administrable preferably orally, nasal, vaginal, ocular, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intracerebral or parenteral administration. The applications of the invention are not limited to vectorization, to the transport of PA of a medicinal or nutritional nature. Indeed, it is entirely conceivable that the PA, capable of being included or incorporated in the PV, is at least a cosmetic or phytosanitary product. Cosmetic applications that can be considered are, for example, compositions applicable by transdermal routes. Plant protection products in question can be, for example, herbicides, pesticides, insecticides, fungicides, etc .. The present invention also relates to phytosanitary and cosmetic compositions comprising PV PA loaded in kind considered above. The examples detailed below will allow a better understanding of the invention in its different product / procedure / application aspects. Said examples illustrate the preparation of polyamino acid particles loaded or not with active ingredients, as well as having the structural characteristics and the properties of such particles.

EXAMPLES I. PREPARATION OF THE PROVEN POLYAMINO ACIDS The polymers applied in the examples are synthetic linear copolymers, block structures or statistics, based on leucine and glutamic acid. Its weight-average molar mass M ", determined by elastic diffusion of the light in the trifluoroacetic solvent, is between 50,000 D and 150,000 D. The polyamino acids have molar masses by weight M ^ determined by elastic diffusion of the light in the acidic solvent trifluoroacetic, comprised between 50,000 D and 150,000 D. These polymers are obtained from the copolymer of leucine and methyl glutamate, whose functionality of the ionizable side chains of sodium glutamate is restored using the known deprotection procedures of methyl esters described, for example, by STAHMAN et al., J. Biol. Chem., 197, 771 (1952) or in the KYOWA HAKKO Patent, FR 2 152 582. The copolymer of leucine and methyl glutamate is obtained from the anhydrides of the N-carboxy-a-amino acids (NCA) of leucine and of methyl glutamate, the preparation of which is presented, for example, in Biopolymers, 15, 1869 (1976). The techniques used for the polymerization of the NCAs in polymers of block structures or statistics are known to the specialist and are detailed in the work of H.R. KRICHELDORF "a-Aminoacides-N-Carboxy Anhydrides and Related Heterocycles", Springer Verlag (1987).

EXAMPLE 1: SYNTHESIS OF A "STATISTICAL" POLYAMINOACIDO, EL POLI (LEU / GLU) 50/50.

STEP 1): COPOLYMERIZATION OF NCA-LEU AND NCA-GLU (OMT): POLY (LEU-CO-GLÜ (OMß)) 50/50 In a 1 liter reactor, equipped with a glass stirrer, with nitrogen and with outlet connected to a bubble producer, under nitrogen stream, 15.0 grams of N-carboxyanhydride of methyl glutamate (NCA-Glu (OMe): 0.08 moles) and 12.5 grams of N-carboxyanhydride of leucine (NCA-Leu) : 0.08 moles). Again 381 milliliters of dioxane are added and the reaction medium is brought to 40 ° C.

After the dissolution of the NCAs, 24 milliliters of water are introduced, followed by 0.22 milliliters of triethylamine (that is, 1 mole percent of the NCAs).

The monitoring of the polymerization is carried out by IR, observing the disappearance of the carbonyl bands at 1,860 and 1,790 cm "1. The duration of polymerization varies between 1.5 and 3 hours, depending on the composition of the monomers After the disappearance of the bands, the reaction medium is diluted with 380 milliliters of dioxane, then it is homogenized for 3 hours. At room temperature, the copolymer is recovered by precipitation in 5 liters of water, stirring well, the product is filtered and dried at 50 ° C in vacuo. 12 hours. The copolymer mass obtained is 18.4 grams, that is, a weight yield of 90 percent. NMR 1H (trifluoroacetic acid-d): 0.85 ppm (CH3-Leu, 6H * 0.5); 1.58 (CH2 and CHMe2Leu, 3H * 0.5); 4.62 (NCHCO-Leu, 1H * 0.5); 4.70 (NCHCO-Glu, 1H * 0.5). Reduced viscosity (0.5 g / dl in trifluoroacetic acid at 25 ° C = 2.2 dl / g.

STEP 2): POLYESTER METHYL ESTER HYDROLYSIS (LEU-CO-GLÜ (OM?)) 50/50: The copolymer obtained above (17.7 grams) is placed in a reactor, in which 354 milliliters of trifluoroacetic acid are added. The reaction medium is brought to 40 ° C under agitation. When the polymer is completely dissolved, 354 milliliters of water are added in small amounts. The reaction medium is kept under stirring for 48 hours. The polymer is recovered by precipitation in 5 liters of water. After the filtration, it is again put in suspension and stirred in the water for 0.5 hours, after which it is filtered and dried. The purification is carried out by dialysis in water. Yield 15.9 grams (95 percent). NMR 1H (trifluoroacetic acid-d): identical to the starting polymers with one exception, the signal with 3.75 (CH3-Glu) decreases strongly or is absent. In the present case, the percentage of residual esters is less than 1 percent relative to the monomers glutamate. Reduced viscosity (0.5 g / dl in trifluoroacetic acid) at 25 ° C = 0.95 dl / g.

EXAMPLE 2: SYNTHESIS OF A POLYAMINOACIDO "BLOCK", THE POLY (LEU / GLU) 50/50 DIBLOQUES. In a 1 liter reactor, 15.0 grams of NCA-Glu (OMe) (0.08 moles) and 180 milliliters of dioxane are introduced under stirring. After dissolution, 180 milliliters of toluene are added and the medium is brought to 6 ° C. The IR spectrum of the solution is made before adding 0.156 grams of benzylamine (1.58 mole percent / NCA). The reaction medium quickly becomes turbid and, after 40 minutes, the characteristic bands at 1,860 and 1,790 cm "1 disappear, after one hour a solution of 12.5 grams of NCA-Leu (0.08 mol) is introduced into a dioxane mixture. / toluene (15 milliliters each) Stirring is continued for 18 hours (this duration has not been optimized) As a result, the carbonyl bands disappear 100 milliliters of dioxane are added and the reaction medium is homogenized for 1 hour. The copolymer is precipitated in 3 liters of absolute ethanol under vigorous stirring, washed with 1 liter of ethanol, filtered and finally dried at 50 ° C under vacuum overnight The mass of product recovered is 19.5 grams (yield = 95.8) percent) NMR 1H (trifluoroacetic acid-d): 0.85 ppm (CH3-Leu, 6H * 0.5), 1.58 (CH2 and CHMe2 Leu, 3H * 0.5), 2.10 and 2.22 (CH2-Glu, 2H * 0.5 ), 2.58 (CH2-Glu, 2H * 0.5), 3.75 (CH3-Glu, 3H * 0.5), 4.62 (NCHCO-Leu, 1H * 0.5), 4.70 (NCHCO-Glu, 1H * 0.5). reduced content (0.5 g / dl in trifluoroacetic acid) at 25 ° C = 0.62 dl / g. The second step of hydrolysis of the methyl esters is identical to that described in Example 1, Step 2. Yield 95 percent. NMR 1H (trifluoroacetic acid-d): identical to the starting polymers with one exception, the signal of 3.75 (CH3-Glu) decreases strongly or is absent. In the present case, the percentage of residual esters is less than 1 percent of the monomers glutamate. Reduced viscosity (0.5 g / dl in trifluoroacetic acid) at 25 ° C = 0.55 dl / g.

EXAMPLE 3: SYNTHESIS OF A POLYAMINOACIDO "BLOCK", THE POLY (GLU-LEU / GLU) 29/57/14 TRIBLOQUES. In a 1 liter reactor, 7.5 grams of NCA-Glu (OMe) (0.04 moles) and 180 milliliters of dioxane are introduced under stirring. After dissolution, 180 milliliters of toluene are added and the medium is brought to 60 ° C. The IR spectrum of the solution is made before adding 0.156 grams of benzylamine. After the total disappearance of the monomer, a solution of 12.5 grams of NCA-Leu (0.08 mol) is introduced into a dioxane / toluene mixture (15 milliliters each). Stirring is continued for 18 hours. Subsequently, 7.5 grams of NCA-Glu (OMe) (0.04 moles) are again introduced and allowed to react for 12 hours. 100 milliliters of dioxane are added and the reaction medium is homogenized for 1 hour. The copolymer is precipitated in 3 liters of absolute ethanol under vigorous stirring, washed with 1 liter of ethanol, filtered and finally dried at 50 ° C under vacuum overnight. The mass of product recovered is 19.4 grams (yield = 95 percent). NMR 1H (trifluoroacetic acid-d): 0.85 ppm (CH3-Leu, 6H * 0.5); 1.58 (CH2 and CHMe2 Leu, 3H * 0.5), 2.10 and 2.22 (CH2-Glu 2H * 0.37); 2.58 22 (CH2-Glu, 2H * 0.37); 3.75 (CH3-Glu, 3H * 0.37); 4.62 (NCHCO-Leu, 1H * 0.5); 4.70 (NCHCO-Glu, 1H * 0.37). Reduced viscosity (0.5 g / dl in trifluoroacetic acid) at 25 ° C = 0.58 dl / g. The second step of hydrolysis of the methyl esters is identical to that described in Example 1, Step 2. NMR 1H (trifluoroacetic acid-d): identical to the starting polymers with one exception, the signal of 3.75 (CH3-Glu) it decreases strongly or is absent. In the present case, the percentage of residual esters is less than 1 percent relative to the monomers glutamate. Reduced viscosity (0.5 g / dl in trifluoroacetic acid) at 25 ° C = 0.38 dl / g.

II. FORMATION OF NANOPARTICLES OF POLYAMINOACIDES (NPV) WITH OR WITHOUT INCORPORATION OF ACTIVE PRINCIPLES. II.1 INFLUENCE OF THE CONCENTRATION IN AAN ON THE FORMATION OF PARTICLES.

EXAMPLE 4: TRAINING OF NANOPARTICLES OF POLY (LEU / GLU) 30/70, 50/50, 75/25 OF "STATISTICAL" STRUCTURE. 100 milligrams of statistical copolyamino acids of leucine and sodium glutamate, of composition Leu / Glu = 30/70 and of molar mass M "= 36,000 D, are dispersed in 100 milliliters of a molarity sodium chloride solution 10" 2 mol / 1 Whatever the pH of the solution between 4.5 and 12 which can be adjusted by the addition of hydrochloric acid or sodium hydroxide, the polymer spontaneously forms a colloidal dispersion of nanoparticles In acidic medium of pH less than 4.5, which corresponds to an ionization fraction £ equal to 0.05, the lyophilized polymer does not disperse in the solution and remains insoluble Table 1 below shows the observations made under the same dispersion conditions, with the statistical polyaminoacids of leucine and glutamate of sodium, of composition Leu / Glu = 50/50 and 75/25 and of molar mass K ^ equal to 60,000 D and 34,000 D, respectively.

EXAMPLE 5: TRAINING OF NANOPARTICLES OF POLY (LEU / GLU) 20/80, 40/60, 50/50 OF STRUCTURES "BLOCKS". 100 milligrams of copolyaminoacids blocks of leucine and sodium glutamate, of composition Leu / Glu = 50/50 and of molar mass "- 14,600 D, are dispersed in 100 milliliters of a sodium chloride solution of molarity 10 ~ 2 mol / 1. Whatever the pH of the solution between 3 and 12 can be adjusted by the addition of hydrochloric acid or sodium hydroxide, the polymer spontaneously forms a colloidal dispersion of nanoparticles that diffuse the light and give the solution a high turbidity. The polymer nanoparticles do not decant when the solution is left standing for several hours at room temperature between 15 ° C and 20 ° C. In an acidic Ph medium less than 3, the polymer does not disperse in the solution and remains insoluble. Under the same pH conditions between 3 and 12, poly (Leu / Glu) 20/80, 40/60 of "block" structures, of molar mass M ", respectively, equal to 11,000 D, 15,000 D, are dispersed and form colloidal suspensions. These colloidal suspensions diffuse more light when the proportion of leucine in the polymer is high. At a pH lower than 3, it is observed, in the same way as for poly (Leu / Glu) 50/50, that the polymers do not disperse and become insoluble.

EXAMPLE 6: SOLUBILITY OF THE POLI (LEU / GLU) 18/82 OF "STATISTICAL" STRUCTURE. This example demonstrates that the leucine and sodium glutamate copolymers of composition Leu / Glu = 18/82 do not form nanoparticles, since they are completely soluble in water, whatever the pH < 4.5. 10 milligrams of lyophilized poly Leu / Glu-18/82 are dispersed in 0.5 milliliters of a molarity 10"2 mol / l sodium chloride solution The polymer is completely dissolved and the solution is clear No nanoparticles are formed .

EXAMPLE 7: STABILITY OF COLOID SUSPENSIONS OF DIFFERENT POLY (LEU / GLU). 100 milligrams of statistical copolyamino acids of leucine and sodium glutamate of composition are dispersed Leu / Glu = 30/70, 50/50, 75/25 and of molar mass Mw, equal, respectively, to 36,000 D, 60,000 D and 34,000 D, in 10 milliliters, 5 milliliters and 2 milliliters of a hydroxide solution of molarity sodium 10"2 mol / l, thus forming 1%, 2%, and 5% w / v concentration solutions of each polyamino acid, then the dispersions are subjected to stability tests at room temperature (15 -25 ° C) for 4 months At the end of this period, the nanoparticles have not decanted and the diffusivity of the solutions has not changed.

EXAMPLE 8: FORMATION OF NANOPARTICLES OF POLY (LEU / GLU) 50/50 OF "STATISTICAL" STRUCTURE AND OF STRUCTURE "BLOCKS", IN A TAMPON ISOTONIC PHOSPHATE. 100 milligrams of poly polyamino acids are dispersed (Leu / Glu) 50/50, of "statistical" structure and of molar mass M ", equal to 60,000 D, in a solution of pH = 7.4 isotonic, containing 0.01 mol / l of phosphate buffer, 0.138 mol / l of sodium chloride and 0.0027 mol / l of potassium chloride (PBS, see catalog SIGMA P4417). The polymer spontaneously forms a colloidal dispersion of nanoparticles that diffuses light. The polymer nanoparticles do not decant when the solution is left standing for several hours at room temperature between 15 ° C and 20 ° C. Under the same conditions, when 100 milligrams of Poly (Leu / Glu) polyamino acids are dispersed 50/50 blocks and of molar mass M "equal to 14,600 D, in a PBS buffer solution, a stable suspension at room temperature of nanoparticles is obtained that diffuses the light with a high turbidity.

II.2 DIMENSIONS AND STRUCTURE OF NANOPARTICLES OF POLY (LEU / GLU) OF "STATISTICAL" STRUCTURE AND OF "BLOQUE" STRUCTURE. The polyamino acid nanoparticles form a colloidal solution. Measurements by static or quasi-elastic diffusion of light allow to measure the size and density of polymer in the nanoparticles. Table 2 below shows measurements made from the statistical polyaminoacids of compositions Leu / Glu = 30/70, 50/50 and molar masses "between 46,000 D and 21,000 D, as well as the polyamino acid" block "of Leu compositions. / Glu 20/80, 50/50 of molar masses between 11,000 D and 16,300 D. For such measurements, the polyamino acids are dispersed in a solution of pH = 7.4 isotonic, containing 0.1 mol / l of phosphate buffer, 0.138 mol / 1 of sodium chloride and 0.0027 mol / 1 of potassium chloride (see SIGMA catalog P4417).

The dimensions of the nanoparticles vary with the composition of the polyamino acids. For the same composition, these depend on the diblock or disordered structure of the polyamino acid chains. In addition, the distributions of the diameters of the polyamino acid nanoparticles are, according to the quasi-elastic light diffusion analysis, monomodal and enclosed around their mean value. The widths of the distributions obtained are comparable or lower than that of polydispersity polystyrene equal to 1.2. The polymer concentration in the nanoparticles is considerably lower and always lower than 6 percent w / v. This depends on the composition and the diblock or disordered structure of the polyamino acids. On the other hand, observation by electron microscopy (TEM with negative coloration) shows that the nanoparticles have a spherical or slightly elongated shape.

II.3 IMMUNOGENICITY OF NANOPARTICLES EXAMPLE 9: IMMUNOGENOUS POWER OF NANOPARTICLES OF POLY (LEU / GLU) 40/60, 50/50, 60/40 BLOCKS. The poly (Leu / Glu) 40/60, 50/50, 60/40 blocks, of molar masses equal to approximately 12.00 D, are dispersed in a solution of pH = 7.4 isotonic, which contains 0.01 mol / 1 of buffer phosphate, 0.138 mol / l of sodium chloride and 0.0027 mol / l of potassium chloride (see SIGMA catalog P4417). The concentration in polymer is equal to 2.5 mg / ml. The suspensions are turbid and filtered without particular difficulty on a polysulfone membrane of 0.2 micron porosity, in order to sterilize them. The animals used are rats (series of five rats per tested polymer) of non-blood-vessel strain OF1. The polymer suspensions are injected subcutaneously, at the height of 100 microliters of suspension (250 micrograms of polymer) per injection. A first injection is applied at time JO, performing a repetition at time J35. The survey is done at time J42, that is, 7 days after the second injection. The blood samples are left for 24 hours at room temperature, then centrifuged for 10 minutes at 3,000 tr / min. The sera are analyzed by ELISA assay. No anti-polymer antibody was detected in the sera, even at minimal dilutions in serum (1/10). This example demonstrates that poly (Leu / Glu) 40/60, 50/50, 60/40 block nanoparticles do not induce specific immune response.

II.4 ASSOCIATION OF NANOPARTICLES WITH COLORED MODEL PROTEINS. The encapsulation procedure is described having as models of hemoglobin, the cytochromes C of horse heart and of Saccharomyces cerevisea. The association between polymer nanoparticles and proteins is evidenced by analytical ultracentrifugation. The polymer and protein solutions are centrifuged at high speeds and the advance of the sedimentation fronts of the polymer and the proteins is followed by the measurement of the optical density at wavelengths at 250 nanometers and at 410 nanometers. The association between proteins and colloidal particles is characterized by the existence of a single sedimentation front corresponding to the superposition of the sedimentation fronts with the two wavelengths. In the opposite case, in the absence of association, the sedimentation fronts of the protein and the colloidal particles are different and do not overlap.

EXAMPLE 10: ASSOCIATION OF THE POLY (LEU / GLU) 30/70 WITH THE CYTOCHROME C. 10 milligrams of cytochrome or C are dissolved in 100 milliliters of a sodium phosphate buffer of pH equal to 7.2 and of molarity 0.01 mol / 1. . Subsequently, 100 milligrams of poly Leu / Glu 30/70, of molar mass M "= 36,000 D, are directly dispersed in this solution. Most of the cytochrome C sediments with the colloidal polymer particles at the time of centrifugation. The analysis of the optical density of the sedimentation fronts shows that more than 80 percent of the cytochrome is associated with colloidal particles.

EXAMPLE 12: ASSOCIATION OF THE POLY (LEU / GLU) 30/70 WITH THE HEMOGLOBIN. In this example, the colloidal suspension of the polyamino acid and the protein is prepared in two different ways, from the same buffer solution of Example 4, but modifying the order of dissolution. 1. Poly Leu / Glu-30/70, with molar mass M "= 90,000 D is dispersed in the hemoglobin solution according to the same conditions as those of Example 4. Ultracentrifugation analysis of the colloidal suspension demonstrates the association of hemoglobin and polyamino acid in the particles. 2. The poly Leu / Glu-30/70, with molar mass M "= 40,000 D, is dispersed in the buffer solution that does not contain hemoglobin. Subsequently, the colloidal suspension formed is mixed with the hemoglobin solution. In this case, a significant fraction of the hemoglobin, estimated at 80 percent, is not associated with the polyamino acid nanoparticles and the ultracentrifugation analysis shows two sedimentation fronts that correspond, respectively, to the polyamino acid nanoparticles and to the hemoglobin. The first stage of dissolution of the protein, before the dispersion of the polyamino acid, ensures, in the case of hemoglobin, a better encapsulation yield.

II.5 ASSOCIATION OF NANOPARTICLES WITH PROTEINS EXAMPLE 13: ASSOCIATION OF THE POLY (LEU / GLU) 30/70 IN THE PRESENCE OF OVOALBUMIN. Poly Leu / Glu-30/70, of molar mass M "= 90,000 D, is dispersed in a sodium chloride solution under the same conditions as those of Example 2, plus ovalbumin. The characteristics of the colloidal particles analyzed by diffusion of light are identical to those formed in the absence of protein. Therefore, the protein does not prevent the association of polyamino acids in nanoparticles, and this for protein concentrations that can reach 20 percent w / w of polyamino acid.

EXAMPLE 14: ASSOCIATION OF THE POLY (LEU / GLU) 50/50 BLOCKS WITH THE INSULIN. From a isotonic solution of pH = 7.4 containing 0.01 mol / l of phosphate buffer, 0.138 mol / l of sodium chloride and 0.0027 mol / l of chloride, a solution of recombinant human insulin is prepared (SIGMA, reference 10259) of concentration 1 mg / ml. In 5 milliliters of this insulin solution, 50 milligrams of poly (Leu / Glu) 50/50 blocks of molar mass equal to 12,400 D are dispersed. A very turbid and stable suspension is obtained. By ultrafiltrate on 300,000 D cut-off membrane (Millipore, Ultrafree-CI filter) the insulin associated with the nanoparticles is separated. This is measured, by difference with the amount of free insulin, the amount of insulin associated with the nanoparticles equal to 0.65 mg / ml.

EXAMPLE 15: ASSOCIATION OF POLI (LEU / GLU) 50/50 OF "STATISTICAL" STRUCTURE WITH INSULIN. The procedure is identical to those of Example 14, using poly (Leu / Glu) 50/50 structure "statistic" instead of poly (Leu / Glu) 50/50 blocks. The amount of insulin, associated with the nanoparticles, is equal to 0.60 mg / ml.

III. AN AGGREGATION BY ADDITION OF A SALT EXAMPLE 16: AGGREGATION BY ADDITION OF AMMONIUM SULFATE 100 milligrams of poly Leu / Glu-30/70, of molar mass M ^ = 36,000 D, are dispersed in 200 milliliters of citric acid buffer solution and of sodium phosphate of molarity 0.05 mol / 1 and of a pH equal to 5, slowly adding to it a concentrated solution of ammonium sulfate. The poured volume is sufficiently scarce before that of the solution of the dispersion, until the aggregate of the NPV in MPV. The MPV thus obtained have an average diameter equal to 8 microns.

III.2 SECOND AGGREGATION FOR pH DOWN The lateral carboxylic functions of the polyamino acids in the nanoparticles are partially ionized. Its neutralization, by the addition of an acid, causes the aggregation of the nanoparticles. The aggregation can be carried out with acids whose dissociation constant (PA) is lower than that of the lateral carboxylic functions of the polyamino acids.

EXAMPLE 17: ADDED BY ADDITION OF HYDROCHLORIC ACID The statistical polyaminoacids of composition Leu / Glu = 30/70, 50/50 and 75/25 and of molar masses M ", equal respectively to 36,000 D, 60,000 D and 34,000 D, are dispersed. in buffer solutions of citric acid and sodium phosphate of molarity 0.05 mol / 1 and of a pH equal to 5. The concentrations of the polyamino acids are 0.01 percent w / v for the polyamino acids of composition Leu / Glu-30/70 and 50/50 and 0.005 percent w / v for that of composition 75/25. The aggregation of the nanoparticles in colloidal suspension is carried out by the progressive addition of a 0.1 mol / 1 solution of hydrochloric acid, until the addition of the NPV in MPV. The results of the granulometry measurements of the MPV are grouped in Table 3 below.

TABLE 3 III.3 ADDED BY COMPLEXATION WITH A CATIONIC POLYMER EXAMPLE 18: AGGREGATE OF POLI LEU / GLU- 50/50 NANOPARTICLES BY COMPLEXATION WITH POLY D, L-LYSINE. The lateral carboxylic functions of the polyamino acids in the nanoparticles are partially ionized. Its complexation with a cationic polymer, such as polylysine, causes aggregation of the nanoparticles. 10 milligrams of poly Leu / Glu-50/50, of molar mass Vl ^ = 60,000 D, are dispersed in 100 milliliters of a sodium phosphate buffer of molarity 0.01 mol / 1 and of a pH equal to 6. The addition of 15 milligrams of poly D, L-Lysine hydrogen bromide, of molar mass M "= 15,000 D, allows to add the polymer nanoparticles into microparticles. The average diameter of the microparticles is between 10 and 20 microns, varying the pH between 2 and 9 by the addition of hydrochloric acid or sodium hydroxide.

III.4 ENCAPSULATION OF PROTEINS BY AGGREGATION OF NANOPARTICLES. EXAMPLE 19: ENCAPSULATION OF CYTOCHROME C BY COMPLEXATION WITH PLI D, L-LYSINE. In 100 milliliters of a molarity phosphate buffer of 0.01 mol / 1, of a pH equal to 6, and containing 10 milligrams of horse heart cytochrome C, disperse 10 milligrams of poly (Leu / Glu) 50/50, of molar mass M "= 60,000 D. The addition of 15 milligrams of hydrogen bromide of poly D, L Usina, of molar mass Mw = 15,000 D, allows to add nanoparticles of polymers in microparticles. By centrifugation, the microparticles settle; the red coloration of the bottom of the centrifuge indicates that almost all of the citrochrome sedimented with the nanoparticles, which shows that the cytochrome is encapsulated in the microparticles.

Claims (17)

1. Vectorization nanoparticles (NPV) of active principle (s), of the base type of polyamino acid (s), and characterized: because, in an average size, they comprise between 0.01 and 0.5 microns, preferably between 0.03 and 0.4 microns, because these NPV are obtained by contacting the polyamino acids (PAA) with an aqueous solution, because their polyamino acids are linear in a-peptidic chains, and comprise at least two recurrent amino acids AAN and AAI: * the corresponding AAN type to a hydrophobic neutral amino acid, * and the AAI type corresponding to an ionizable side chain amino acid, being at least a part of the recurring amino acids of type AAI in ionized form, being the recurring amino acids of each type, AAN and AAI, identical or different from each other, and because the molar mass by weight M "of the polyamino acids is greater than or equal to 4,000 D, preferably at 5,000 D, - because these PAAs are not water soluble at an acid pH and in a pH comprised between 3 and 12.

2. Particles according to claim 1, characterized: because their constitutive PAAs are PAA "blocks" and / or "statistical" PAA, and because: * for the PAA "blocks": # the molar ratio AAN / AAN + AAI is > 6 percent, preferably > 15 percent, # Mw > 5,500 D, preferably 6,500 D < Mw < 200,000 D and, more preferably still, 8,000 D < M "< 200,000 D, * for the "statistical" AAPs: # the molar ratio AAN / AAN + AAI is > 20 percent, preferably > 25 percent, * M "> 10,000 D, preferably 20,000 D < 500,000 D and, more preferably still, 200,000 D < M "< 150,000 D.

3. Particles according to claim 1 or 2, characterized: because the ANN (or AAN) is (are) chosen in the following list: Leu-lie-Val-Ala-Pro -Phe - and their mixtures, and because the AAI (or the AAI) is (are) chosen (s) by the Glu and / or the Asp.

4. Particles according to claim 1 or 2, characterized in that the polyamino acid comprises a recurring amino acid AAN and a recurring amino acid AAI.

5. Particles according to claim 1, characterized by an average concentration in polyamino acid ranging from 0.01 to 25 percent dry weight, preferably 0.05 to 10 percent dry weight.

6. Particles according to claim 1, characterized in that they are Vectorization Microparticles (MPV) of average size greater than 0.5 microns, preferably less than or equal to 20 microns.

7. Particles according to claim 6, characterized in that they are obtained from particles according to claim 5, and in that they preferably comprise at least one aggregation agent.

8. Particles according to claim 1, characterized in that they comprise at least one active principle.

9. Procedure for the preparation of particles based on polyamino acid (s) and which can be used as vectors of active principle (s), characterized: because amino acids (PAA) are applied: * linear chain-peptides, * which comprise at least two recurrent amino acids AAN and AAI: # the AAN type corresponding to a neutral hydrophobic amino acid, # and the AAI type corresponding to an ionizable side chain amino acid, with the recurring amino acids of each type being AAN and AAI identical or different from each other, * the molar ratio being AAN / AAI + AAN > 3 percent, preferably > 5 percent, * the molar mass by weight being U ^ of the polyamino acid (s) greater than or equal to 4,000 D, preferably 5,000 D, * said polyamino acids are not water soluble at an acid pH and at a pH comprised between 3 and 12, and because a dispersion of these polyamino acids is carried out in a liquid, preferably in an aqueous solution, and because a colloidal solution of particles is thus obtained.

10. Procedure according to the claim 9, characterized in that a polyamino acid comprising a recurring amino acid AAN and a recurring amino acid AAI is applied.

11. Process according to claim 9, characterized in that at least one active principle is dissolved in the liquid, preferably before the introduction of the polyamino acids in the medium, so as to obtain, after this introduction, a colloidal solution. of charged particles in active principle (s).

12. Procedure according to the claim 9, characterized in that it comprises at least one additional stage of aggregation of particles, by means of at least one aggregation agent, preferably constituted by a salt and / or an acid and / or a base and / or an optionally ionic polymer .

13. Process according to claim 12, characterized in that polymer concentrations in the solution expressed in percent by weight / volume, greater than or equal to 10 ~ 2, preferably comprised between 0.05 and 30 and, more preferably, between 0.05 and 5.

14. Particles according to claim 1 and / or obtained by the process according to claim 9, characterized in that the active principle is medicated and preferably chosen from; - proteins and / or peptides among which are preferred: hemoglobins, cytochromes, albumins, interferons, antigens, antibodies, calatonin, erythropoietin, insulin, growth hormones, factor IX, the interleukin or its mixtures. - polysaccharides, among which heparin, nucleic acids and, preferably, RNA and / or DNA oligonucleotides and their mixtures are preferred.

15. Particles according to claim 1 and / or obtained by the process according to claim 9, characterized in that the active principle is constituted by at least one vaccine.

16. Particles according to claim 1 and / or obtained by the process according to claim 9, characterized in that the PA is formed by at least one phytosanitary or cosmetic product.

17. Pharmaceutical, nutritional, phytosanitary or cosmetic specialty, characterized in that it comprises particles according to claim 1 and / or obtained by the process according to claim 9.