EP1355627A1 - Block-structure copolymer consisting of a saccharide segment bound to at least a biodegradable hydrophobic segment, and corresponding particles - Google Patents

Abstract

The invention concerns a block-structure copolymer consisting of a hydrophilic saccharide segment and at least a biodegradable hydrophobic segment of general formula (I), wherein: X represents a CN or CONHR radical; Y represents a COOR', CONHR'' radical with R, R' and R'' representing, independently of each other, a hydrogen atom, a linear or branched C1-C20 alkyl group, a linear or branched C1-C20 alkoxy group, an amino acid radical, a monohydroxylated or polyhydroxylated radical or a C5-C12 aryl or heteroaryl radical, said saccharide segment being bound either by one of its ends to a single segment of general formula (I), or by each of its two ends, to a segment of general formula (I), the two hydrophobic segments being identical or different. The invention also concerns particles based on said copolymer and a corresponding preparation method.

Description

 COPOLYMER WITH SEQUENCED STRUCTURE COMPOSED OF A SEGMENT

SACCHARIDIC LINKED TO AT LEAST ONE BIOERODABLE HYDROPHOBIC SEGMENT,

AND CORRESPONDING PARTICLES

The invention relates to a new family of bioerodible copolymers based on a polymer of alkyl cyanoacrylate type, or related and of poly- or oligosaccharides, particularly useful in the pharmaceutical, veterinary, food and cosmetic fields, in particular as vectors and / or excipients. It also proposes a process for preparing these copolymers.

Bioerodible polymers generally formulated in the form of liposomes, microemulsions, nanospheres, nanocapsules, microspheres, microcapsules, microparticles and nanoparticles constitute effective delivery systems for active ingredients. Among these systems, micro- and nanoparticles based on poly (alkyl cyanoacrylate) are particularly advantageous given their rapid bioerosion compared to other biodegradable polymers, such as poly (lactic acid) / poly (ε-caprolactone) for example. However, these particles based on poly (alkyl cyanoacrylate) are not entirely satisfactory. On the one hand, there is currently no effective preparation method for obtaining these systems with a controlled structure and / or composition of the polymers entering into their constitution. In addition, these particles have the disadvantage of being rapidly captured by the macrophages of the System of

Mononuclear phagocytes (PMS). Their lifespan, in vivo, is therefore very short.

The present invention aims precisely to overcome the aforementioned drawbacks and to propose a new material for particles whose polymer structure derives from the association with a polymer. related to poly (alkyl cyanoacrylate), of a segment of poly- or oligosaccharide nature, such as dextran for example.

Nanoparticles based on amphiphilic block copolymers comprising dextran and poly (alkyl cyanoacrylate) segments have already been described (S.J. Douglas et al.; Journal of Controlled Release (1986) 15-23). However, these copolymers which are derived from the anionic polymerization of cyanoacrylate monomers in the presence of dextran have grafted structures. In fact, the anionic polymerization of alkyl cyanocrylates in the presence of dextran leads to the grafting of several poly (cyanoacrylate) chains on the dextran polymer chain without it being moreover possible to control the number, size and localization of these poly (cyanoacrylate) chains. Finally, the particles thus formed have an interfacial layer of dextran on the surface, the overall structure of which leads to recognition by the complement system and by the macrophages of the SPM.

The present invention aims for its part to provide copolymers derived from cyanoacrylate or equivalent monomers and from oligo- or polysaccharides but having a completely different structure. Thus, in the context of the present invention, the copolymers are in a block form, as opposed to the graft forms described above. This sequenced form is in fact inaccessible by the anionic polymerization path discussed above. The present invention also aims to propose a synthetic route for preparing this type of block copolymer.

The inventors have thus demonstrated that it is possible to effectively polymerize, by the radical route, molecules with a high charge density, of cyanoacrylate type, in the presence of poly- or oligosaccharides, this despite the fact that the energy of activation required for this radical polymerization is much higher than that required by anionic polymerization. In fact, because of their high charge density, the cyanoacrylate or equivalent type monomers are naturally inclined to generate their anionic form when they are placed in the presence of a nucleophilic agent such as, for example, OH anions ^" and therefore to polymerize anionically.

However, the inventors have put. in evidence that it was possible, on the one hand, to effectively slow down the manifestation of this polymerization in particular by controlling the pH of the polymerization medium and, on the other hand, to favor polymerization by the radical route. Finally, i o against all odds, the radical polymerization route proposed by the inventors turns out to be faster than anionic polymerization.

Consequently, the first object of the present invention is a copolymer with a block structure composed of a hydrophilic segment of saccharide nature, at least one of the ends of which is linked to a

15 bioerodible hydrophobic segment of general formula (I):

20 in which:

- X represents a CN or CONHR radical, - Y represents a COOR 'or CONHR "radical, with R, R' and R" representing, independently of one another, an atom

Hydrogen, a linear or branched C ₂₀ to C ₂₀ alkyl group, a linear or branched C 1 to C ₂₀ alkoxy group, an amino acid radical, a mono- or poly-hydroxylated acid radical or an aryl radical or C ₅ to C ₁₂ heteroaryl, with said segment of saccharide nature being linked either by one of its

30 ends with a single segment of general formula (I), either by each from its two ends, to a segment of general formula (I), the two hydrophobic segments being identical or different.

In the segment of general formula (I), X preferably represents a CN radical. More preferably, Y represents a radical COOR 'with R' as defined above.

The repeating unit of isobutyl cyanoacrylate may be more particularly cited by way of illustration of a unit capable of composing a segment of general formula (I).

The term “sequenced structure” is intended to denote according to the invention a structure which derives from the establishment of a covalent bond between at least one of the ends of the segment of saccharide nature and one of the ends of the polymer chain of general formula (I). Thus, within the scope of the invention, copolymer structures comprising either a single segment of general formula (I) linked to one end of the segment of saccharide nature, or two segments of general formula (I), identical or different, linked respectively on either side of the saccharide segment. In contrast to the grafted structures mentioned above, the claimed copolymers do not have side branch (s) of saccharide nature on the hydrophobic segment, nor side branch (s) of hydrophobic nature on the saccharide segment .

The covalent bond, established between the two types of segment, is generally of C-C or C-O-C nature. Preferably, it is derived from the radical polymerization of at least one molecule of a compound of formula (II):

in which X and Y are as defined above, in the presence of a poly- or oligosaccharide. This radical polymerization is preferably carried out under the conditions set out below for the process claimed. In particular, it is carried out under conditions of pH and atmosphere unfavorable to the presence and or to the generation of anions in the reaction medium and in the presence of a sufficient amount of a suitable radical initiator Redox. The saccharide-type segment is derived from an oligo- or polysaccharide of natural or synthetic origin, modified or not.

Modified polysaccharide is understood to mean any polysaccharide which has undergone a change on its skeleton, such as for example the introduction of reactive functions, the grafting of chemical entities (molecules, aliphatic links, PEG chains, etc.). There are commercially available polysaccharides modified by grafting biotin, fluorescent compounds, etc. Other polysaccharides grafted with hydrophilic chains (eg PEG) have been described in the literature. It is also possible to envisage using, within the framework of the present invention, polysaccharides modified like those described in the reference Jozefowicz and Jozefonvicz, Biomaterials, 18, 1633-1644 (1997). Of course, this modification should not affect the polymerization of the monomer of general formula (II) in the presence of the modified oligo- or polysaccharide. According to a preferred variant of the invention, the oligo- or polysaccharide used according to the invention may already in itself have biological properties and / or activities. For example, it can confer anticoagulant, vaccinating, targeting or even mask properties to avoid capture by PMS macrophages. This is how it can be:

- oligo- or polysaccharides with antigenic properties such as those of bacterial or viral origin,

- oligo- or polysaccharides with biological activity such as, for example, heparin, heparan sulfate, dermatan sulfate, dextran sulfate and pentosan sulfate, dextran substituted with carboxylic groups and sulfates or sulfonates , polysaccharides sulfates extracted from algae (fucans and fucoidans), poly (sialic acids), sulfated hyaluronic acid which have anticoagulant, anti-inflammatory activities to varying degrees, and / or

- oligo- or polysaccharides involved in cell recognition and cell signaling processes such as, for example, poly (sialic acids), heparan sulfate, blood group antigens, polysaccharides and lipopolysaccharides of various strains bacterial, oligosaccharide chains of membrane and / or circulating glycoproteins, and oligosaccharide chains of glycolipids.

The copolymers derived from this type of polysaccharides are of course particularly advantageous in terms of therapeutic use, insofar as they naturally have their own biological activity and therefore can be used as such for this reason. The polysaccharides which are particularly suitable for the invention are or are derived from D-glucose (cellulose, starch, dextran, cyclodextrin), D-galactose, D-mannose, D-fructose (galactosane, manane, fructosan), fucose (fucan). The majority of these polysaccharides contain the elements carbon, oxygen and hydrogen. The polysaccharides according to the invention can also contain sulfur and / or nitrogen. They can thus derive from glycoprotein or glycolipid. Likewise, hyaluronic acid (composed of N-acetyl glucosamine and glucuronic acid units), poly (sialic acid) also called colominic acid or poly (N-acetyinaminic acid), chitosan, chitin, heparin or the orosomucoid contains nitrogen, while the agar, a polysaccharide extracted from seaweed, contains sulfur in the form of acid sulfate (> CH-O-SO ₃ H). Chondroitin-sulfuric acid and heparin simultaneously contain sulfur and nitrogen.

According to a preferred variant of the invention, the polysaccharide has a molecular weight greater than or equal to 6000 g / mole. In the particular case of dextran and amylose (C ₆ H ₁₀ O ₅ ) _n , n varies between 10 and 620 and preferably between 33 and 220. In the case of hyaluronic acid, the molar mass varies between 5 10 ³ and 5 10 ⁶ g / mole, preferably between 5 10 ⁴ and 2 10 ⁶ g / mole. In the case of chitosan, the molar mass varies between 6 10 ³ and 6 10 ⁵ g / mole, preferably between 6 10 ³ and 15 10 ⁴ g / mole.

By way of illustration of the polysaccharides which are more particularly suitable for the invention, mention may be made of polydextroses such as dextran, chitosan, pullulan, starch, amylose, cyclodextrins, hyaluronic acid, heparin, amylopectin, cellulose, pectin, alginate, curdlan, fucan, succinoglycan, chitin, xylan, xanthan, arabinan, carragheenane, poly (glucuronic acid), poly ( N-acetylneuraminic acid), poly (mannuronic acid), and their derivatives (such as, for example, dextran sulfate, amylose esters, cellulose acetate, pentosan sulfate, etc.).

In the particular case of a cyclic polysaccharide like cyclodextrins, its covalent coupling with a compound of general formula (II), during radical polymerization, has the effect of causing an opening of the cycle and therefore of driving the polysaccharide to adopt a linear structure in accordance with the invention.

More particularly preferred are dextran, heparin, poly (N-acetylneuraminic acid), amylose, chitosan, pectin and hyaluronic acid, and their derivatives. Advantageously, the copolymers have a controlled oligo- or polysaccharide content.

The claimed copolymer can be in a soluble form or in the form of a precipitate, micelles or particles. According to an advantageous aspect of the invention, it takes the form of particles. They can be micro- or nanoparticles. In the particular case of particles and micelles, it is likely that the copolymer has a structure organized as follows: chains of the same nature, that is to say saccharide or hydrophobic, are grouped together, either to constitute the structure heart of the micelle or particle, or the brush crown around this heart structure. Their distribution between the core structure and the crown will of course depend on the nature, aqueous or organic, of the solvent in which the particles or micelles are dispersed. The term “brush crown” is intended to denote a structure in which the segments constituting the crown are linked by one of their ends to the segments constituting the heart. Their free ends constitute the periphery of the crown. Thus, in an aqueous medium, the hydrophobic segments are grouped so as to constitute the heart and the segments of a saccharide nature are arranged in a brush crown all around this heart. In an organic type solvent, this organization between the two types of segment is reversed: the heart is hydrophilic in nature and therefore consists of the saccharidic segments and the brush crown is hydrophobic in nature and therefore constituted by the segments of formula general (I). In the case of the grafted structure copolymers described above, this brush crown structure cannot exist in an aqueous medium, insofar as several hydrophobic segments are linked by covalent bond to a single chain of saccharide nature.

A second aspect of the invention relates to particles consisting of a copolymer in accordance with the invention.

By way of representative of the particles claimed, ^" there may be more particularly mentioned those composed of a copolymer derived from the polymerization of:

- isohexyl cyanoacrylate, isobutyl cyanoacrylate, N-butyl cyanoacrylate, N-propyl cyanoacrylate, ethyl cyanoacrylate or 2-methoxyethyl cyanoacrylate in the presence of dextran, - isobutyl cyanoacrylate in the presence of heparin, chitosan, pectin, hyaluronic acid, dextran sulfate or γ-cyclodextrin.

The claimed particles can have a size between 1 nm and 1 mm and preferably between 60 nm and 100 μm.

Generally, the particles having a size between 1 and 1000 nm are then called nanoparticles. Particles varying in size from 1 to several thousand microns refer to microparticles. These particles may in certain cases be in an aggregated or micellar form. Thus, the particles resulting from the polymerization of isobutyl cyanoacrylate in the presence of γ-cyclodextrin have an aggregated appearance.

These particles can have a biological activity either because of the nature of the polysaccharide which constitutes them, or because they incorporate in addition a biological or pharmaceutical active material.

As biological active materials, mention may more particularly be made of peptides, proteins, carbohydrates, nucleic acids, lipids, polysaccharides or their mixtures. It can also be synthetic organic or inorganic molecules which, administered in vivo to an animal or to a patient, are capable of inducing a biological effect and / or of manifesting a therapeutic activity. It can thus be antigens, enzymes, hormones, receptors, peptides, vitamins, minerals and / or steroids.

Mention may be made, as representative of the drugs which may be incorporated into these particles, of anti-inflammatory compounds, anesthetics, chemotherapeutic agents, immunotoxins, immunosuppressive agents, steroids, antibiotics, antivirals, antifungals, antiparasitics, immunizing substances, immunomodulators and analgesics. Likewise, it is possible to envisage combining with these active materials compounds intended to intervene at the level of their release profile.

For example, it is possible to add, in the composition of the particles, PEG or polyester chains (modified or not), and thus obtain so-called composite particles.

It is also possible to incorporate into the particles, compounds for diagnostic purposes. It can thus be substances detectable by X-rays, fluorescence, ultrasound, nuclear magnetic resonance or radioactivity. The particles can thus include magnetic particles, radio-opaque materials (such as, for example, air or barium) or fluorescent compounds. For example, fluorescent compounds such as rhodamine or Nile red can be included in particles with a hydrophobic core. Alternatively, gamma emitters (for example Indium or Technetium) can be incorporated. Hydrophilic fluorescent compounds can also be loaded into the particles, but with a lower yield compared to hydrophobic compounds, because of their reduced affinity with the matrix.

Finally, these particles can be combined with peptides / proteins capable of helping them to diffuse through biological membranes such as the TAT peptide, or compounds such as the 2OT protein (Zonula Occludens Toxin) and zonulin or equivalents, or any other absorption promoter.

In this case, this type of association can be achieved by chemical functionalization of the polysaccharide surface of the particles. It is thus possible to envisage covalently fixing, at the level of functions present on the skeleton of a saccharide nature, specific ligands, such as targeting agents, markers or more generally any compound capable of conferring on said particles an ability to react with a external species, as by example a function on a support or a biological entity present in a considered environment.

Commercial magnetic particles with controlled surface properties can also be incorporated into the particle matrix or covalently attached to one of their constituents.

The active material can be incorporated into these particles during their formation process or, on the contrary, be loaded at the level of the particles once they are obtained. It is thus possible to charge them by adsorption or by covalent grafting.

The particles according to the invention can be administered in different ways, for example by the oral, parenteral, ocular, pulmonary, nasal, vaginal, cutaneous, oral, etc. routes. The non-invasive oral route is the route of choice. The present invention also relates to the use of the particles as a vector of pharmaceutical, cosmetic, agrifood or veterinary active principles.

A third aspect of the present invention relates to a process for the preparation of the claimed copolymer. More specifically, the present invention relates to a process which is useful for the preparation of block copolymers composed of a hydrophilic segment of saccharide nature, at least one of the ends of which is linked to a hydrophobic segment, characterized in that it comprises polymerization by radical route of at least one molecule of a compound of general formula (11):

in which :

- X represents a CN or CONHR radical, - Y represents a COOR 'or CONHR "radical with R, R 'and R "representing independently of one another, a hydrogen atom, an alkyl group in C ₁ -C ₂₀ linear or branched alkoxy, C, to C ₂₀ linear or branched , an amino acid radical, a mono- or poly-hydroxylated acid radical or a C ₅ to C ₁₂ aryl or heteroaryl radical, said radical polymerization being carried out in the presence of at least one molecule of a poly- or oligosaccharide , under pH and atmosphere conditions unfavorable to the presence and / or generation of anions in the reaction medium and in the presence of a sufficient amount of a suitable radical redox initiator.

Preferably, X represents a CN radical and / or more preferably Y represents a COOR radical.

As stated previously, it turns out to be possible to favor radical polymerization, to the detriment of naturally preponderant anionic polymerization, in particular carrying out the polymerization reaction under pH conditions unfavorable to the generation of free anions, such as for example ions OH ^" .

To do this, the pH of the reaction medium is preferably adjusted to a value less than 2 and more preferably less than 1.5. Surprisingly, it appeared that adjusting the pH to such a value was not detrimental to the rate of polymerization. Against all expectations and as is apparent from the examples present below, the radical polymerization initiated under these pH conditions is carried out on the contrary at a speed greater than that of an anionic polymerization. This is illustrated in particular in FIGS. 1 and 2.

According to a preferred variant of the invention, the reaction is also carried out under inert atmosphere conditions.

The solvent is advantageously chosen so that, while maintaining conditions favorable to radical polymerization and more particularly to the formation of the hydrophobic segment of formula (I), the solubility of the oligo or polysaccharide is complete in the medium defined by this solvent.

Insofar as it is desired to favor the formation of the copolymer directly in the form of particles or micelles, the solvent is also chosen to be weakly or non-solubilizing with respect to the copolymer.

Preferably, the poly- or oligosaccharide molecule is chosen from dextran, heparin, poly (N-acetylneuraminic acid), amylose, chitosan, pectin and hyaluronic acid, and their derivatives. It is also of course chosen to remain inert with respect to the polymerization.

Advantageously, such a solvent is preferably chosen from aqueous, hydroalcoholic or hydroacetonic solvents.

The solvent chosen is acidified with an organic or inorganic acid and preferably a nitric acid to obtain an adequate pH during the course of the radical polymerization.

As regards the respective amounts of oligo- or polysaccharide and monomer of general formula (II), they can vary widely. The claimed process precisely has the advantage of allowing control of the structure of the copolymer which it is desired to prepare. The quantities of reagents introduced also depend on their respective molecular weights and their degrees of solubility in the reaction medium.

As regards the Redox initiator, these are generally mixtures of oxidizing and reducing agents, organic or inorganic, generating radicals during the electronic transfer step. This generation of radicals has the advantage of requiring a low activation energy unlike conventional radical initiators, which makes it possible to initiate radical polymerizations at relatively low temperatures (0-50 ° C). The Redox initiator used preferably comprises at least one metal salt chosen from the salts of Ce ⁴⁺ , V ⁵⁺ , Cr ⁶⁺ , Mn ³⁺ . According to a preferred variant of the invention, it is Ce ⁴⁺ . It is generally introduced in the form of cerium and ammonium nitrate. The concentration of radical initiator is also capable of influencing the course of the radical polymerization. Thus, the composition of the copolymer and the length of the respective blocks of the polysaccharide and of the polymer of general formula (I) can be adjusted as a function of the initiator concentration. Its adjustment is within the competence of a person skilled in the art.

As regards the reaction temperature, it is adjusted to a value compatible with the initiation of the polymerization.

Generally, this temperature is between 0 and 50 ° C. As regards the order of introduction of the various reagents, the oligo- or polysaccharide is preferably dissolved in the chosen solvent, then the radical initiator Redox is added. The monomer of formula (H) is then introduced into the mixture.

At the end of the process, the copolymer can be obtained in a soluble form or in the form of micelles, powders or particles. Preferably, it is obtained directly in the form of particles. The particles can be loaded with active ingredients either after their preparation or during their preparation.

When the copolymer is obtained in the form of a powder, it is of course possible to formulate this powder in the form of particles, using suitable transformation techniques. As an illustration of these techniques, one can more particularly mention the techniques of emulsification-evaporation of solvent, emulsification-diffusion of solvent or nanoprecipitation.

The particles meet the specificities set out above. According to a variant of the invention, the polymerization of the compound of general formula (II) is carried out in the presence of the active material to be loaded.

At the end of the polymerization, the pH of the reaction medium can be neutralized if necessary. Preferably, this is adjusted to a value that remains less than or equal to 7.5. The copolymer is recovered by conventional techniques.

According to a preferred variant of the invention, the metal salt is complexed before the isolation of the copolymer resulting from the reaction of the radical initiator. This complexation, which is within the competence of a person skilled in the art, makes it possible to eliminate these metal salts.

The examples and figures appearing below are presented by way of illustration and without limitation of the present invention.

FIGURES

Figure 1: Kinetics of radical polymerizations according to the invention of isobutyl cyanoacrylate in the presence of dextran, chitosan or pectin.

Figure 2: Reference kinetics of anionic polymerizations of isobutyl cyanoacrylate in the presence of dextran or chitosan.

Figure 3: Electronic paramagnetic resonance spectrum (EPR) of copolymers in accordance with the invention of dextran-poly (isobutyl cyanoacrylate) labeled with 4-aminoTEMPO.

MATERIAL AND METHOD

- The size of the polymer particles (average hydrodynamic diameter) is determined using a nanosizer (Coulter N4 PLUS®) by quasi-elastic diffusion of laser radiation. - The surface charge of the particles is determined using a

Zetasizer 4 Malvern®. To do this, the dextran-poly (isobutyl cyanoacrylate) and heparin-poly (isobutyl cyanoacrylate) suspensions are diluted 1/200 ^e and 1/30 ^{e respectively} in potassium chloride at 1 mmol / l.

EXAMPLE 1:

In a 2 cm diameter glass tube, 0.1375 g of dextran 70,000 g / mol are dissolved in 8 ml HNO ₃ (0.2 mol / l), with magnetic stirring at 40 ° C and with a gentle bubbling at argon. After 10 minutes, 2 ml of acid solution of cerium ions (8.10 ^"2 mol / l of cerium IV ammonium nitrate in HNO ₃ at 0; 2 mol / l) then 0.5 ml of isobutyl cyanoacrylate are added. 10 minutes, bubbling with argon is stopped and the glass tube is plugged. After at least 40 min, stirring is stopped and the glass tube is cooled under tap water. The pH is adjusted with NaOH ( 1 N) so that after adding 1.25 ml of tri sodium citrate dihydrate (1.02 mol / l) it comes directly to a value of 7 ± 0.5 Finally, the suspension is stored in the refrigerator.

At this stage, a suspension of stable colloidal polymer particles is obtained. The copolymer particles can then be purified.

The purification of the copolymer particles is carried out as follows:

Dialysis tubes (Spectra / Por ^® CE MWCO: 100,000) are regenerated for 30 minutes with osmosis water, and the colloidal suspensions passed through a vortex and then introduced into the tubes.

Two successive 1 h 30 dialyses are carried out against 5 liters of osmosis water which are followed by another overnight dialysis against 5 liters of osmosis water. The suspensions of copolymer particles thus purified and contained in the dialysis tubes are recovered and then stored at (+ 4 ° C) or optionally dried by lyophilization.

The lyophilization of the copolymer particles is carried out as follows:

Lyophilizations are carried out without the addition of cryoprotective.

The suspensions of colloidal particles are aliquoted in pillars and then frozen (-18 ° C). Lyophilization (Bioblock Scientific Christ alpha 1-4) is carried out for 48 hours. The lyophilisates (white powders) are stored in the refrigerator.

Particle reconstruction:

To reconstitute the dispersion particles from the lyophilizate, a specific mass lyophilisate is dispersed in a known volume of water MilliQ ^® so that the mass ratio of freeze-dried / water volume is 1%. The suspension is homogenized using a vortex at maximum speed and then by ultrasound for a few minutes using a sonicator (Branson 5200 ^® ).

EXAMPLE 2:

The same protocol as that described in Example 1 is reproduced using, instead of dextran 70,000 g / mol, one of the following polysaccharides:

- 0.1375 g of heparin; - 0.0230 g of chitosan;

- 0.0230 g of pectin;

- a soluble quantity of hyaluronic acid in 8 ml HNO ₃ (0.2 mol / l);

- 0.1375 g of dextran sulfate of molecular mass (6-8000, 10000, 40000, 50000 or 500000 g / mol); - 0.1375 g of γ-cyclodextrin; or

- 0.1375 g of dextran 15-20000 g / mol.

EXAMPLE 3 The protocol of Example 1 is reproduced by substituting for isobutyl cyanoacrylate, one of the following monomers:

- isohexyl cyanoacrylate,

- N-butyl cyanoacrylate,.

- N-propyl cyanoacrylate, - ethyl cyanoacrylate, or

- 2-methoxyethyl cyanoacrylate.

EXAMPLE 4:

PHYSICAL CHARACTERIZATION OF THE PARTICLES: The copolymers obtained in examples 1, 2 and 3 are characterized, in terms of size, stability and load.

Size of the suspensions:

The suspensions were previously diluted with illiCΛ water

Table 1 below summarizes the particle sizes of the various particulate suspensions produced.

The stability of the suspensions obtained was evaluated as a function of the size of the particles over time. The results obtained are shown in Table 2 below. Table 1

*: mass of polysaccharide solubilized in 8 ml of 0.2 mol / l nitric acid. **: mass soluble in 8 ml of 0.2 mol / l nitric acid. The standard deviations represent the repeatability of the measurements.

Table 2

T ₀ : day of preparation.

The standard deviations represent the repeatability of the measurements.

Zeta potential:

Table 3 below reports the results obtained.

Table 3

It is noted that the Zeta potential obtained with the heparin-poly particles (isobutyl cyanoacrylate) is more distant from neutrality than that obtained with the dextran-poIy particles (isobutyl cyanoacrylate).

These results therefore reproduce the difference in natural charge between heparin and dextran. Obviously, the polysaccharide present in each of the two types of particles is located on the surface of these.

EXAMPLE 5: CHARACTERISATIQN OF THE COPOLYMER CONTENT OF

SUSPENSIONS AND COMPOSITION OF COPOLYMERS:

The polymer content of the suspensions is determined by evaluating the weight of the dry residue obtained after lyophilization of a known quantity of suspension purified by dialysis. To do this, an aliquot of purified suspension prepared according to Example 1 or 2 is weighed with precision in a pill box and then frozen at (-18 ° C) before lyophilization for 48 hours in a Christ Alpha 1-4 lyophilizer (Bioblock Scientific). The mass of lyophilisate is weighed and then brought back to the mass of initial suspension. The suspension of dextran-poly copolymer (isobutyl cyanoacrylate) obtained according to Example 1 contains 3.1 ± 0.4% of copolymer (mass / mass).

The suspension of heparin-poly copolymer (isobutyl cyanoacrylate) obtained according to Example 2 contains 2.4 ± 0.7% of copolymer (mass / mass).

The composition of the copolymers is evaluated by elementary analysis of the powders obtained by lyophilization of the purified suspensions as indicated above. The dextran-poly (isobutyl cyanoacrylate) copolymer obtained according to Example 1 contains 20%

(mass / mass) of dextran.

EXAMPLE 6: KINETICS OF POLYMERIZATION

Material and apparatus:

- “PC2000 Plug-in” type spectrometer (Ocean Optics Europe) inserted into a PC type computer, an HL-2000-LL light source (Ocean Optics Europe), Fiber optics (200 and 100 μm) (Top sensor Systems FC-UV, Ocean Optics Europe) and OOI Base V 1.5 Software (Ocean Optics Europe).

- Glass tube with round bottom, 2 cm in diameter to carry out the polymerization.

- Teflon ® ring with holes at 0 °, 90 ° and 180 °. The size of the holes is adjusted to serve as a support for the optical fibers and the size of the Teflon® ring is adjusted to the glass tube in which the polymerization will be carried out. The optical fibers are mounted on the ring in the 0 ° and 180 ° position for absorbance measurements. In this test, the kinetics of radical polymerization of isobutyl cyanoacrylate in the presence of dextran, chitosan or pectin were followed.

The polymerization is carried out according to the protocol described in examples 1 to 3 in the 2 cm diameter glass tube placed in a water bath at 40 ° C. and on which is mounted the Teflon® ring supporting the optical fibers connected to the spectrometer and the light source. Argon bubbling is placed so as not to disturb the acquisition of the measurements. The background noise of the spectrometer is recorded before the introduction of the acid solution of cerium IV ions (8.10 ^"2 mol / l of cerium ammonium nitrate in HNO ₃ at 0.2 mol / l). The reference is recorded after l addition of the acid solution of cerium IV ions (8.10 ^"2 mol / l of cerium ammonium nitrate in HNO ₃ at 0.2 mol / l). The recording of the polymerization kinetics is started as soon as the 0.5 ml of monomer is added. It is carried out by the almost instantaneous acquisition of an absorbance spectrum over a wide wavelength range (400 - 800 nm) every 30 seconds for 50 min. The absorbances measured at the wavelength of 650 nm are used to draw absorbance curves as a function of time, thus reflecting the kinetics of polymerization.

The results are presented in Figure 1.

In FIG. 2, the kinetics of an anionic polymerization carried out with the same monomer are reported for comparison in the presence of dextran or chitosan but in the absence of the redox initiator responsible for the radical polymerization.

It is noted that the start of the anionic polymerization is delayed compared to the radical polymerization.

EXAMPLE 7 This example illustrates the synthesis of isobutyl dextran-polycyanoacrylate particles on a larger scale. In a 50 ml ground glass flask with a flat bottom, 0.6875 g of dextran 70,000 g / mol are dissolved in 40 ml of 0.2 mol / l nitric acid with magnetic stirring, at 40 ° C. and under gentle bubbling of argon for 10 min. An acid solution of cerium ions (10 ml) (8.10 ^'2 mol / l of cerium IV ammonium nitrate in nitric acid 0.2 mol / l) then 2.5 ml of isobutyl cyanoacrylate are then added and maintained with vigorous stirring and argon bubbling for another 10 min. The argon bubbling is stopped and the balloon is blocked. The reaction is continued with stirring at 40 ° C for 50 min. The reaction is stopped and the flask is cooled under tap water. The pH is adjusted with NaOH (1N) so that after the addition of 6.25 ml of tri sodium citrate dihydrate (1.02 mol / l) it comes directly to a value of 7 ± 0.5. The average hydrodynamic diameter of the copolymer particles obtained is 291 ± 1 nm.

EXAMPLE 8:

This example illustrates the synthesis of isobutyl dextran-polycyanoacrylate particles according to simplified experimental conditions.

In a 20 ml screw flask, 0.1375 g of dextran 70,000 g / mol are dissolved in 8 ml HNO ₃ (0.2 mol / l), with magnetic stirring at 20 ° C. After 10 minutes, 2 ml of acid solution of cerium ions (8.10 ^"2 mol / l of cerium IV ammonium nitrate in HNO ₃ at 0.2 mol / l) and then 0.5 ml of isobutyl cyanoacrylate are added. Stirring is stopped for 60 minutes, the pH is adjusted with NaOH (1N) so that after the addition of 1.25 ml of tri sodium citrate dihydrate (1.02 mol / l) it directly reaches a value of 7 ± 0.5 The average hydrodynamic diameter of the copolymer particles obtained is 393 ± 5 nm. EXAMPLE 9:

ASSESSMENT OF THE RESIDUAL ANTICOAGULANT BIOLOGICAL ACTIVITY OF THE HEPARIN OF THE HEPAR1 COPOLYMER- ISOBUTYL POLYfCYANOACRYLATE:

The anticoagulant activity of the heparin of the heparin-poly (isobutyl cyanoacrylate) copolymer is evaluated by measuring the activated partial thromboplastin time (TCA) or anti-Ila activity and by measuring the anti-Xa activity produced by the particles consisting of said copolymer synthesized according to Example 2.

Measurement of anti-lla activity:

Normal frozen plasma is thawed in a water bath at 37 ° C and then placed in an ice bin. The APTT reagent (Organon Teknica

Corporation, Fresnes, France) is regenerated with 3 ml of sterile water. A solution of CaCI ₂ 1/40 mol / l is prepared in Owren-Koller buffer

(TOK) (Stago Diagnosis).

Sample preparation:

A calibration range of the method is carried out with the same heparin as that used for the synthesis of the copolymer. A stock solution at 1700 IU / ml is prepared in the TOK buffer and then diluted in this same buffer to give solutions at 0.17; 0.85; 1, 7; 4.25 and 8.5 IU / ml. 100 μl of each of the dilutions are themselves diluted in 900 μl of normal plasma. The suspensions copolymer particles are diluted in the TOK at ^1/100 and 1/200 ^Θ. 100 μl of each of the dilutions of the suspension are themselves diluted in 900 μl of normal plasma.

A coagulation control consists of 100 μl of TOK and 900 μl of normal plasma. Measurement of coagulation times:

A bead is placed in each of the tanks of the ST4 coagulometer (Diagnostica Stago) then 100 μl of one of the samples prepared in the previous step and 100 μl of the APTT solution are introduced into the various tanks. After 300 seconds of incubation at 37 ° C, 100 μl of the calcium chloride solution are added. The coagulometer measures the coagulation times of the different samples in seconds. The results obtained for the heparin standard solutions make it possible to establish a calibration curve giving the activity of the heparin solution expressed in U.l./ml as a function of the coagulation time expressed in seconds. The heparin activity associated with the copolymer particles is evaluated on the calibration curve from the coagulation times measured for the suspensions.

Thus, the suspension containing particles of heparin-poly copolymers (isobutyl cyanoacrylate) prepared according to Example 2 have an anti-lla activity of 329 + 28 U.l./ml.

Measurement of anti-Xa activity:

The copolymer particle suspension was diluted to 1/50 ^th, 1/100 ^th and 1/200 ⁸ in TOK buffer then to ^1/10 in the normal plasma thawed as described above. The coagulation times are automatically evaluated on an ST1 coagulomer

(Diagnostica Stago) automatically.

The anti-Xa activity of the particle suspension of heparin-poly copolymers (isobutyl cyanoacrylate) is 408 ± 50 IU / ml. EXAMPLE 10:

GRAFT OF A MARKER ON PARTICLES OF COPOLYMERS:

A suspension not purified by dialysis of particles of copolymers is prepared according to Example 2 with dextran 15-20000 g / mol. The suspension is filtered on a filter 1, 2 .mu.m (Millipore ^® SLA PO 2550) and then purified by dialysis 2 2 hours against 11 of osmosed water followed by dialysis for 2 hours against 11 of phosphate buffer (Sigma ref. P 3813) (dialysis membrane: Spectra / Por ^® CE MWCO: 100,000 regenerated 30 min in osmosis water).

For grafting, 0.0270 g of 1, 1'-carbonyldiimidazole (Sigma ref. C-7625) and 0.0113 g of 4-amino TEMPO (Aldrich) are introduced into a 20 ml screw flask fitted with a plug. These products are dissolved in 0.5 ml of phosphate buffer with stirring. To this mixture, 3 ml of the purified suspension of copolymer particles are added. The whole is kept under magnetic stirring at room temperature for 48 hours. After the reaction, excess reagents and reaction byproducts are removed by dialysis. The suspension was placed in dialysis tubing (Spectra / Por ^® CE MWCO: 100000) previously regenerated with 30 min of reverse osmosis water and then dialyzed 3 times against 1 L of phosphate buffer for 2 hours. The suspensions of grafted particles are recovered and can be stored at (+ 4 ° C).

The grafting of the marker can be demonstrated by electron paramagnetic resonance spectroscopy (EPR). To do this, the suspension obtained is placed in a measurement cell of a Varian E-4 EPR spectrometer. The spectrum obtained presented in FIG. 3 indicates that the 4-amino TEMPO has indeed been grafted onto the dextran chains of the copolymer forming the particles and that it is animated by slow movements for 81% and by rapid movements for 19% according to Kivelson's simulation (Kivelson DJ, Journal Chem. Phys; 1960; 33; 1107).

Claims

1. Copolymer with a sequenced structure composed of a hydrophilic segment of saccharide nature and at least one bioerodible hydrophobic segment of general formula (I):

in which :

- X represents a CN or CONHR radical,

- Y represents a COOR 'or CONHR "radical with R, R' and R" representing, independently of one another, a hydrogen atom, a linear or branched C ₂₀ -C ₂₀ alkyl group, a group linear or branched C ₂₀ to C ₂₀ alkoxy, an amino acid radical, a mono- or poly-hydroxylated acid radical or a C ₅ to C ₁₂ aryl or heteroaryl radical, with said segment of saccharide nature being linked either by one of its ends to a single segment of general formula (I), or by each of its two ends, to a segment of general formula (l), the two hydrophobic segments being identical or different.

2. Copolymer according to claim 1, characterized in that X represents, in general formula (I), a CN radical.

3. Copolymer according to claim 1 or 2, characterized in that Y represents in general formula (I) COOR 'with R' as defined in claim 1.

4. Copolymer according to one of the preceding claims, characterized in that the saccharide-type segment derives from a natural or synthetic oligo- or polysaccharide, modified or not.

5. Copolymer according to one of claims 1 to 4, characterized in that the oligo- or polysaccharide has biological properties and / or activities.

6. Copolymer according to one of claims 1 to 5, characterized in that the oligo- or polysaccharide is chosen from polydextroses such as dextran, chitosan, pullulan, starch, amylose, cyclodextrins, hyaluronic acid, heparin, amylopectin, cellulose, pectin, alginate, curdlan, fucan, succinoglycan, chitin, xylan, xanthan, arabinan, carragheenane, poly (glucuronic acid), poly (N-acetylneuraminic acid), poly (mannuronic acid) and their derivatives.

7. Copolymer according to claim 6, characterized in that it is dextran, heparin, poly (N-acetylneuraminic acid), 0 amylose, chitosan, pectin, hyaluronic acid, or one of their derivatives.

8. Copolymer according to one of claims 1 to 7, characterized in that it is obtained by radical polymerization of at least one molecule 5 of a compound of general formula (II):

in which X and Y are as defined in claim 1, 2 or 3, in the presence of a poly- or oligosaccharide.

9. Copolymer according to claim 8, characterized in that the radical polymerization is carried out under pH and atmosphere conditions unfavorable to the presence and / or to the generation of anions in the reaction medium and in the presence of an amount sufficient in a suitable Redox radical initiator.

10. Copolymer according to one of the preceding claims, characterized in that it is in the form of particles.

11. Particle characterized in that it consists of a copolymer according to one of claims 1 to 9.

12. Particle according to claim 11, characterized in that it is composed of a copolymer deriving from the polymerization of: - isohexyl cyanoacrylate, isobutyl cyanoacrylate, N-butyl cyanoacrylate, N-propyl cyanoacrylate, cyanoacrylate ethyl or 2-methoxyethyl cyanoacrylate in the presence of dextran, or

- isobutyl cyanoacrylate in the presence of heparin, pectin chitosan, hyaluronic acid, dextran sulfate or γ-cyclodextrin.

13. Particle according to claim 11 or 12, characterized in that it has a size between 1 nm and 1 mm.

14. Particle according to one of claims 11 to 13, characterized in that it incorporates a biological or pharmaceutical material.

15. Use of particles according to one of claims 1 1 to 14, as a vector of pharmaceutical, agrifood, cosmetic or veterinary cosmetic principles.

16. Process useful for the preparation of block copolymers composed of a hydrophilic segment of saccharide nature of which at least one of the ends is linked to a hydrophobic segment, characterized in that it comprises the polymerization by radical route of at least a molecule of a compound of general formula (II):

in which: - X represents a CN or CONHR radical, - Y represents a COOR 'or CONHR "radical with R, R' and R" representing, independently of one another, a hydrogen atom, a group C ₁ -C ₂₀ linear or branched alkoxy, C, to C ₂₀ linear or branched, an amino acid radical, a mono- or poly- hydroxy radical or an aryl or heteroaryl radical, C ₅ -C ₁₂ , said radical polymerization being carried out in the presence of at least one molecule of a poly- or oligosaccharide, under pH and atmosphere conditions unfavorable to the presence and / or to the generation of anions in the reaction medium and in the presence of a sufficient amount of a suitable radical redox initiator.

17. Method according to claim 16, characterized in that in the derivative of formula (II), X represents a CN radical and / or Y represents a COOR 'radical with R' as defined in claim 16.

18. The method of claim 16 or 17, characterized in that the radical polymerization is carried out at a pH of less than 2 and preferably less than 1.5.

19. Method according to any one of claims 16 to 18, characterized in that the radical polymerization is carried out in a solvent in which the oligo- or polysaccharide is in soluble form and the expected copolymer, weakly or not soluble.

20. Method according to one of claims 16 to 19, characterized in that the poly- or oligosaccharide molecule is chosen from dextran, heparin, poly (N-acetylneuraminic acid), amylose, chitosan, pectin and hyaluronic acid, and their derivatives.

21. Method according to one of claims 16 to 20, characterized in that the radical initiator Redox comprises at least one metal salt chosen from the salts of Ce ⁴⁺ , V ⁵⁺ , Cr ⁶⁺ , Mn ³⁺ and preferably a metallic salt of Ce ⁴⁺ .

22. Method according to one of claims 16 to 21, characterized in that the poly- or oligosaccharide is dissolved in the solvent, the radical initiator Redox is added, then the monomer of general formula (II).

23. Method according to one of claims 16 to 22, characterized in that the polymerization is carried out in the presence of an active material to be loaded into said particles.

24. Method according to one of claims 16 to 23, characterized in that the copolymer is isolated from the reaction medium after neutralization of the pH of the reaction medium.

25. Method according to one of claims 16 to 24, characterized in that one proceeds before the isolation of the copolymer to a complexation of the metal salt resulting from the reaction of the radical initiator.

26. Method according to one of claims 16 to 25, characterized in that the copolymer is obtained in the form of particles, aggregates and / or micelles.