Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Definitions

• the present invention relates to new materials based on biodegradable polymers and polysaccharides, vectors derived from these materials preferably in the form of particles, and their uses as biological vectors for active materials.
• Vectorization is an operation aimed at modulating and if possible completely controlling the distribution of a substance, by associating it with an appropriate system called a vector.
• a vector In the vectorization field, three main functions are to be ensured:
• the general principle of vectorization is also to make the distribution of the active substance as independent as possible from the properties of the active substance itself and to subject it to that of suitable vectors chosen according to the objective envisaged.
• the in vivo fate of the vector is conditioned by its size, its physicochemical characteristics and, in particular, its surface properties which determine the interactions with the components of the living environment.
• First generation vectors are systems designed to release an active ingredient within the target. It is necessary in this case to use a particular mode of administration. These vectors of relatively large size (greater than a few tens of microns) are either solid systems (microspheres) or hollow systems (microcapsules), containing an active substance, for example anticancer, in the dissolved or dispersed state in the constituent material of these systems.
• the materials which can be used are of variable nature (wax, ethylcellulose, polylactic acid, copolymers of lactic and glycolic acids) biodegradable or not.
• Second generation vectors are vectors capable, without any particular mode of administration, of transporting an active principle to the intended target. More precisely, these are vectors whose size is less than a micrometer and whose distribution in the organism is totally a function of their unique physico-chemical properties.
• vesicular vectors of the liposome type which are vectors constituted by one or more internal cavities containing an aqueous phase
• the nanocapsules which are vesicular vectors formed by an oily cavity surrounded by a wall of a polymeric nature.
• nanospheres which consist of a polymer matrix which can encapsulate active ingredients.
• nanoparticles includes nanospheres as well as nanocapsules.
• the active ingredients are generally incorporated at the level of the nanoparticles either during the polymerization process of the monomers from which the nanoparticles are derived, or by adsorption on the surface of the already formed nanoparticles, or during the manufacture of the particles from the preformed polymers.
• the present invention relates very particularly to the field of vectors of the nano- and micro-particle type and their applications.
• Different types of nano- and micro-particles are already proposed in the literature. Conventionally, they derive from a material obtained by direct polymerization of monomers (for example cyanoacrylates), by crosslinking, or else they are produced from preformed polymers: poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly ( ⁇ -caprolactone) ( PCL), and their copolymers, such as for example poly (lactic acid-co-glycolic acid) (PLGA), etc.
• a new type of particle has been obtained from a material derived from the catalytic polymerization of monomers (such as for example lactide or caprolactone), on the skeleton of a polysaccharide.
• monomers such as for example lactide or caprolactone
• this type of material has the main drawback of not being able to guarantee a reproducible composition.
• all the hydroxyl functions present on the backbone of the polysaccharide considered are capable of initiating the polymerization of the monomers.
• a very large number of chains of variable size derived from the monomer are thus formed on the skeleton, which "mask" this skeleton.
• the present invention has for first object a new composite material with controlled structure deriving from the coupling of biodegradable polymer chains directly on the backbone of polysaccharides. Its second object relates to a vector based on this material, preferably in the form of particles, and more preferably in the form of nanoparticles.
• the invention also aims in a third object, the use of this vector, preferably of particles, in particular as biological vehicles.
• the first aspect of the invention relates to a material with a controlled chemical structure composed of at least one biodegradable polymer and of a polysaccharide with a linear, branched or crosslinked backbone, characterized in that it derives from the controlled functionalization d 'at least one molecule of said biodegradable polymer or one of its derivatives by covalent grafting directly at the level of its polymeric structure, of at least one molecule of said polysaccharide.
• the material developed according to the present invention has the first advantage of having a controlled chemical structure and therefore of being therefore perfectly reproducible. Its chemical composition is clearly identified.
• the claimed material preferably consists of at least 90% by weight and more preferably entirely of a copolymer derived from the controlled functionalization of at least one molecule of a biodegradable polymer or of one of its derivatives by covalent grafting directly at the level of its polymeric structure of at least one molecule of a polysaccharide with a linear, branched or crosslinked backbone.
• the claimed material does not contain a starting molecule, that is to say of said biodegradable polymer or of said polysaccharide.
• the material claimed is therefore different from a mixture of polymeric nature in which the expected copolymer would be present but where the starting polymers would also remain, in very variable quantities.
• a mixture of polymeric nature 0 cannot be used as such to prepare nano- or microparticles.
• the claimed material has a polydispersity less than or equal to 2 and preferably less than 1.5.
• the claimed material is obtained by coupling directly, at the level of the polysaccharide molecule, with one or more identical or different molecules of biodegradable polymer.
• This covalent bond between the two types of molecule can be of various natures.
• the covalent bond o established between the two molecules is of the ester or amide type. More preferably, it derives from the reaction between a carboxylic function, if necessary activated, present on the biodegradable polymer and a hydroxyl or amine function present on the polysaccharide.
• the preferred activated functions of the acid are the N-hydroxysuccinimide ester, the acid chloride and the imidazolide derived from carbonyl diimidazole.
• This reactive function, preferably carboxylic can either be naturally present on the skeleton of the biodegradable polymer or have been introduced there before at the level of its skeleton, so as to allow its subsequent coupling with a polysaccharide molecule.
• This activation of a function present on one of the molecules, preferably a carboxylic function on the biodegradable polymer, is particularly advantageous when it is desired to prevent the manifestation of a parasitic secondary reaction, such as for example an intramolecular reaction.
• a parasitic secondary reaction such as for example an intramolecular reaction.
• the carboxylic function present on the biodegradable polymer of so as to favor the kinetics of its coupling reaction with the hydroxyl function of the polysaccharide to the detriment of that of an intramolecular reaction at the level of the polysaccharide molecule.
• the reproducibility and homogeneity of the corresponding material are thus ensured.
• the material according to the invention also has the advantage of having satisfactory biodegradability due to the nature of the polymers which constitute it.
• biodegradable is intended to denote any polymer which dissolves or degrades in a period acceptable for the application for which it is intended, usually in in vivo therapy. Generally, this period should be less than 5 years and more preferably one year when a corresponding physiological solution is exposed with a pH of 6 to 8 and at a temperature between 25 ° C and 37 ° C.
• the chains of biodegradable polymers according to the invention are or are derived from synthetic or natural biodegradable polymers.
• polyesters PLA, PGA, PCL, and their copolymers, such as for example PLGA. Indeed, their biodegradability and biocompatibility have been widely established.
• Other synthetic polymers are also being investigated. These are polyanhydrides, poly (alkylcyanoacrylates), polyorthoesters, polyphosphazenes, polyamino acids, polyamidoamines, polymethylidenemalonate, polysiloxane, polyesters such as polyhydroxybutyrate or poly (malic acid), as well as their copolymers and derivatives.
• Natural biodegradable polymers proteins such as albumin or gelatin, or polysaccharides such as alginate, dextran or chitosan may also be suitable.
• biodegradable polymers are particularly interesting because their bioerosion is observed quickly.
• these polymers are not always suitable for being coupled with one or more polysaccharides because they have almost no reactive groups, especially in the case of biodegradable polyesters (PLA, PCL, ...), and / or because these reactive groups only exist at the chain end. Consequently, the coupling of these polymers with a polysaccharide implies a prior functionalization of their chains with reactive groups while controlling the nature of the groups naturally present at the chain end.
• - n and m represent independently of each other, either 0 or 1,
• - R represents a C 1 -C 20 alkyl group, a polymer different from the biodegradable polymer [for example poly (ethylene glycol)
• PEG poly(ethylene glycol)
• Pluronic® polymer a copolymer containing PEG blocks or ethylene oxide units, such as for example a Pluronic® polymer]
• a protected reactive function present on the polymer eg BOC-NH-
• a function carboxyl activated or not or a hydroxyl function
• - R 2 represents a hydroxyl function or a carboxylic function activated or not.
• Polyesters are especially preferred as biodegradable polymers according to the invention: poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly ( ⁇ -caprolactone) (PCL), and their copolymers, such as for example poly (lactic acid-co-glycolic acid) (PLGA), synthetic polymers such as polyanhydrides, poly (alkylcyanoacrylates), polyorthoesters, polyphosphazenes, polyamides (eg polycaprolactam), polyamino acids, polyamidoamines, polymethylidene malonate, poly (alkylene d-tartrate ), polycarbonates, polysiloxane, polyesters such as polyhydroxybutyrate or polyhydroxyvalerate, or poly (malic acid), as well as the copolymers of these materials and their derivatives.
• PLA poly (lactic acid)
• PGA poly (glycolic acid)
• PCL poly ( ⁇ -caprolactone)
• PA poly (lactic acid-co-g
• the material according to the invention is particularly advantageous in terms of bioadhesion and targeting properties for the particles which are derived therefrom at the level of organs and / or cells. It is in particular through the choice of the associated polysaccharide, and in particular its composition and its structural organization at the particle level, that this second aspect is more precisely achieved.
• the polysaccharide (s) used according to the invention are polysaccharides with a linear, branched or crosslinked structure, modified or not.
• Modified polysaccharide is understood to mean any polysaccharide which has undergone a change in its skeleton, such as for example the introduction of reactive functions, the grafting of chemical entities (molecules, aliphatic links, PEG chains, etc.). Of course, this modification must relate to few of the hydroxyl or amino groups present on the skeleton, so as to leave the vast majority of them free to then allow the coupling of biodegradable polymers. Thus, 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.
• PEG polysaccharides grafted with hydrophilic chains
• polysaccharides which are very particularly suitable for the invention are or are derived from D-glucose (cellulose, starch, dextran), D-galactose, D-mannose, D-fructose (galactosan, manane, fructosan). 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.
• hyaluronic acid (composed of N-acetyl glucosamine and glucuronic acid units), chitosan, chitin, heparin or ovomucoid contain nitrogen, while agar, polysaccharide extracted from seaweed , contains sulfur in the form of acid sulfate (> CH-O-SOgH). Chondroitin-sulfuric acid contains sulfur and nitrogen simultaneously.
• the polysaccharide has a molecular weight greater than or equal to 6000 g / mole.
• n varies between 10 and 620 and preferably between 33 and 220.
• 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.
• 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 g / mole.
• polydextroses such as dextran, chitosan, pullulan, starch, amilose, hyaluronic acid, heparin, amilopectin, cellulose, pectin, alginate, curdlan, fucan, succinoglycan, chitin, xylan, xanthan, arab
• the material according to the invention in the form of a copolymer, can include the biodegradable polymer and the polysaccharide in a mass ratio varying from 1: 20 to 20: 1 and preferably from 2: 9 to 2: 1.
• the copolymers constituting the claimed material can be in the form of di-block copolymers, have a comb structure or a crosslinked structure.
• the preferred nature of the backbone is a polysaccharide, and the preferred nature of the grafts is a biodegradable polymer.
• Di-block or comb copolymers can be obtained by varying the polysaccharide: biodegradable polymer molar ratio during synthesis.
• the crosslinked structure copolymers can be obtained from biodegradable polymers comprising at least two reactive functions.
• the second aspect of the present invention relates to a process for preparing the claimed material.
• this process comprises bringing together at least one molecule of a biodegradable polymer or one of its derivatives carrying at least one reactive function F1 with at least one molecule of a polysaccharide with a linear, branched or crosslinked backbone and carrying at least one reactive function F2 capable of reacting with the function F1, under conditions suitable for the reaction between the functions F1 and F2 to establish a covalent bond between the said molecules and in that the said material is recovered.
• the claimed preparation process does not require the use of a catalyst like conventional methods.
• This specificity of the claimed process is therefore particularly advantageous in terms of safety and biodegradability in the resulting material.
• it is a quantitative reaction, that is to say at least one F1 function present on the polysaccharide molecules reacts with an F2 function present on a biodegradable polymer molecule.
• the reaction is carried out under conditions such that the manifestation of any parasitic reaction is prevented, in particular the involvement of one of the functions F1 or F2 in another reaction than the expected coupling reaction. It is thus intended to avoid the intramolecular reactions mentioned above.
• the reactive function present on the biodegradable polymer is an acid function or an activated acid function and the reactive function on the polysaccharide is a hydroxyl or amine function.
• the polysaccharide and the biodegradable polymer or derivative are brought together in a mass ratio varying from 1: 20 to 20: 1.
• the coupling reaction can be carried out by activation for example with dicyclohexylcarbodiimide (DCC) or carbonyldiimidazole (DCI).
• DCC dicyclohexylcarbodiimide
• DCI carbonyldiimidazole
• biodegradable polysaccharides and polymers meet the definitions proposed above.
• they can be derived from molecules of polysaccharides or biodegradable polymers, natural and which have been modified so as to be functionalized in accordance with the present invention.
• a third aspect of the invention relates to vectors made of a material according to the invention. These vectors are preferably particles having a size between 50 nm and 500 ⁇ m and preferably between 80 nm and 100 ⁇ m.
• the size of these can be fixed.
• the particles have a size between 1 and 1000 nm and are then called nanoparticles.
• Particles varying in size from 1 to several thousand microns refer to microparticles.
• the claimed nanoparticles or microparticles can be prepared according to methods already described in the literature, such as for example the solvent emulsion / evaporation technique [R. Gurny et al. "Development of biodegradable and injectable latices for controlled release of potent drugs” Drug Dev. Ind. Pharm., Vol 7, p. 1-25 1981)]; the nanoprecipitation technique using a water-miscible solvent (FR 2 608 988 and EP 274 691). There are also variants of these methods.
• the so-called “double-emulsion” technique which is advantageous for the encapsulation of hydrophilic active principles, consists in dissolving these in an aqueous phase, in forming an emulsion of the water / oil type with an organic phase containing the polymer. , then to form an emulsion of the water / oil / water type using a new aqueous phase containing a surfactant. After evaporation of the organic solvent, nano- or micro-spheres are recovered.
• a particularly advantageous new method has also been developed by the inventors which comprises:
• the polymers and copolymers constituting the claimed material comprise, as biodegradable polymer, a polycaprolactone derivative, and more preferably a polycaprolactone derivative with a molecular weight of less than 5000 g / mol.
• the material according to the present invention has the major advantage of having surfactant properties, by its amphiphilic nature. These properties can therefore be exploited advantageously during the preparation of particles, for example, so as to avoid the use of surfactants, systematically used in the above-mentioned processes. Indeed, these are not always biocompatible and are difficult to remove at the end of the process.
• Another advantage of the material according to the present invention is to offer the possibility of modulating the properties which intervene in the process of manufacturing particles through the choice: - of the mass ratio of biodegradable polymer polysaccharide and / or
• copolymers hydrosoluble or insoluble in water having hydrophilic-lipophilic balances which can vary between 2 and 18 (thus making it possible to stabilize water / oil or oil / water emulsions).
• a material derived from at least one polyester molecule linked by an ester or amide type bond to at least one polysaccharide molecule chosen from dextran , chitosan, hyaluronic acid and amilose are particles composed of a material derived from a block of polycaprolactone or of poly (lactic acid) linked by an ester or amide bond to at least one polysaccharide molecule chosen from dextran, chitosan, hyaluronic acid and amilose.
• particle structures obtainable from the material according to the invention and the abovementioned methods can be variable.
• the polysaccharide can be disposed either exclusively at the level of the aqueous inclusions, or at the level of these inclusions and of the surface of the particles. It can also protect the encapsulated active principles (proteins, peptides, etc.) from interactions, often denaturing, with the hydrophobic biodegradable polymer and the organic solvent;
• hydrophilic core structure polysaccharide
• hydrophobic ring biodegradable polymer
• micellar structure obtained thanks to the self-association of a material in accordance with the invention in the aqueous phase, and - a so-called gel structure formed by crosslinking of the polysaccharides with biodegradable polymers comprising at least two reactive functions.
• the particles preferably degrade over a period of between one hour and several weeks.
• the particles according to the invention can contain an active substance which can be hydrophilic, hydrophobic or amphiphilic and biologically active.
• biological active materials mention may more particularly be made of peptides, proteins, carbohydrates, nucleic acids, lipids, polysaccharides or their mixtures. They may 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 manifesting therapeutic activity. It can thus be antigens, enzymes, hormones, receptors, peptides, vitamins, minerals and / or steroids.
• the particles can thus include magnetic particles, radio-opaque materials (such as air or barium) or fluorescent compounds.
• fluorescent compounds such as rhodamine or Nile red can be included in particles with a hydrophobic core.
• gamma emitters for example Indium or Technetium
• Hydrophilic fluorescent compounds can also be encapsulated in the particles, but with a lower yield compared to hydrophobic compounds, due to the lower affinity with the matrix.
• 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.
• the particles in accordance with the invention can comprise up to 95% by weight of an active material.
• the active ingredient can thus be present in an amount varying from 0.001 to 990 mg / g of particle and preferably from 0.1 to 500 mg / g. It should be noted that in the case of the encapsulation of certain macromolecular compounds (DNA, oligonucleotides, proteins, peptides, etc.) even lower charges may be sufficient.
• the particles according to the invention can be administered in different ways, for example by the oral, parenteral, ocular, pulmonary, nasal, vaginal, cutaneous, buccal routes, etc.
• the non-invasive oral route is a preferred route.
• particles administered orally can undergo different processes: translocation (capture and then passage of the digestive epithelium by intact particles), bioadhesion (immobilization of particles on the surface of the mucosa by a membership mechanism) and transit.
• translocation capture and then passage of the digestive epithelium by intact particles
• bioadhesion immobilization of particles on the surface of the mucosa by a membership mechanism
• transit transit.
• the surface properties play a major role.
• the particles according to the invention have numerous hydroxyl functions on the surface, proves to be particularly advantageous for binding a biologically active molecule to it, a molecule intended for targeting or which can be detected. It is thus possible to envisage functionalizing the surface of these particles so as to modify their surface properties and / or target them more specifically towards certain tissues or organs.
• the particles thus functionalized can be maintained at the target level by the use of a magnetic field, during medical imaging or while an active compound is released.
• targeting molecule type ligands such as receptors, lectins, antibodies or fragments thereof can be attached to the surface of the particles. This type of functionalization falls within the competence of a person skilled in the art.
• the coupling of these ligands or molecules on the surface of the particles can be carried out in different ways. It can be carried out covalently by attaching the ligand to the polysaccharide covering the particles or non-covalently, that is to say by affinity. Thus, certain lectins could be attached by specific affinity to the polysaccharides located on the surface of particles according to the present invention, thereby enhancing the cell recognition properties of these particles. It may also be advantageous to graft the ligand via a spacer arm, to allow it to reach its target in an optimal conformation. Alternatively, the ligand can be carried by another polymer used in the composition of the particles.
• the invention also relates to the use of the vectors and preferably of the particles obtained according to the invention for encapsulating one or more active materials as defined above.
• Another aspect of the invention also relates to pharmaceutical or diagnostic compositions comprising vectors and preferably particles according to the invention, where appropriate associated with at least one pharmaceutically acceptable and compatible vehicle.
• the particles can be administered in enteric capsules, or incorporated into gels, implants or tablets. They can also be prepared directly in an oil (such as Migliol®) and this suspension administered in a capsule or injected at a specific site (for example tumor). These particles are in particular useful as stealth vectors, that is to say capable of escaping the immune defense system of the organism and / or as bioadhesive vectors.
• Figure 1 Representation using an optical microscope of R-PCL-COOH particles manufactured according to Example 13 (polymer synthesized according to Example 1).
• Figure 2 Distribution of hydrodynamic diameters of R-PCL-COOH particles.
• the acid and ⁇ -caprolactone were introduced into a flask surmounted by a condenser ascending. After purging the reagents, the flask was introduced into an oil bath thermostatically controlled at 225 ° C. The reaction continued for 3 h 30 min under an inert atmosphere (argon). It was stopped by immersion of the balloon in an ice bath. The solid obtained was dissolved hot in 15 ml THF, then was precipitated at room temperature with cold methanol.
• Mn number-average molar masses
• Mw number-average molar masses
• CES steric exclusion chromatography
• a number average molar mass equal to 3200 g / mole was determined by titration with a 10 "2 M KOH / EtOH solution of the polymer samples of approximately 100 mg dissolved in an acetone-water mixture.
• the bifunctionalized polymer HOOC-PCL-COOH was synthesized according to the procedure of Example 1.
• the succinic acid (99.9%, AIdrich) used as initiator was dried under vacuum at 110 ° C for 24 hours.
• the monomer ( ⁇ -caprolactone) was purified by distillation on calcium hydride.
• the polymerization from 0.2 g of succinic acid and 4 g of ⁇ -caprolactone made it possible to obtain after 3 hours of reaction 3.2 g of polymer (yield by weight 76% after four successive precipitations).
• the assay of the terminal COOH groups with KOH / EtOH 10 "2 M made it possible to determine an acidity corresponding to a molar mass of 3500 g / mole.
• Mn is equal to 4060 g / mole and Mw to 4810 g / mole, the polydispersity index is 1, 2.
• the monomer (D, L-lactide) was purified by two recrystallizations from ethyl acetate followed by sublimation.
• the catalyst (tin octanoate) was purified by distillation under very high vacuum.
• the capric acid used as initiator was purified by recrystallization from ethyl acetate, then anhydrous by azeotropic distillation with benzene.
• the capric acid (0.12 g) and the D, L-lactide (3.5 g) were introduced into a two-necked tube fitted with an ascending cooler connected to a vacuum / argon ramp.
• the reaction flask was inert, then 7 ml of anhydrous toluene were added through the septum.
• 0.284 g of catalyst was introduced and the reaction was immediately started by immersion of the flask in an oil bath at 120 ° C. After 4 hours, the reaction was stopped, the toluene was evaporated, and the polymer called R-PLA-COOH was dissolved in dichloromethane and precipitated with ethanol. After four successive precipitations, a constant acidity was obtained in the polymer, which was then dried.
• the molar mass Mw determined by CES is 22 Kg / mole.
• the assay of the terminal groups with KOH / EtOH 10 "2 M made it possible to determine an acidity corresponding to a molar mass of 21 Kg / mole.
• PCL or PLA polymers monofunctionalized at the chain end with an alcohol group were synthesized according to the protocol of Example 3, but by substituting for the acid initiator, an initiator alcohol, for example C 7 H 15 OH.
• the acid initiator the poly (ethylene glycol) comprising at one end of a chain a methoxy group and at the other a carboxylic acid group (MeO-PEG-COOH) (Shearwater Polymers, 5000 g / mole) was dried before the reaction.
• the lactide was purified by two recrystallizations (ethyl acetate) and by sublimation.
• the mass ratio of MeO-PEG-COOH: lactide reagents was 1: 9 and the MeO-PEG-COOH: catalyst molar ratio was 1: 1.
• the polymerization continued for 2 h under an inert atmosphere at reflux of toluene (solvent). After evaporation of the toluene, the copolymer is purified by two successive precipitations.
• the mass Mw determined by CES is 42 kg / mole. EXAMPLE 6.
• the acid function of the R-PCL-COOH polymers is transformed in the activated ester by reacting it with N-hydroxy succinimide (NHSI), in the presence of dicyclohexyl carbodiimide (DCC), in a DMF: CH 2 mixture.
• the DCC was added in slight molar excess (1, 1) relative to the R-PCL-COOH chains and the NHSI in excess relative to the -COOH functions.
• the reagents were dissolved in a minimum volume of solvent, with slight heating. The reaction takes place at 50 ° C for 24 hours under an inert atmosphere.
• the coupling reaction takes place for 144 hours at 70 ° C under argon.
• the transesterification reaction takes place with the release of NHSI.
• the final product is washed with water to remove the NHSI and water-soluble copolymers, then with dichloromethane to extract traces of unreacted polyester.
• a comb-type Dex-PCL copolymer is obtained with a yield of 40%, comprising a skeleton of dextran (Dex) (molar mass 40,000 g / mole) and side links of PCL linked by ester bridges.
• the copolymer is purified at the end of the reaction. Its overall composition is determined by elementary microanalysis and by NMR. The copolymer contains 33% by weight of PCL.
• R-PCL-COOH (Example 1) are anhydrated by azeotropic distillation, then dried under vacuum at 40-50 ° C, overnight, directly in the 50 ml reaction flask topped with an ascending cooler and connected to an empty ramp / argon. 5 ml of dry THF are then added to the flask. After dissolution of the acid, 0.243 g of carbonyl diimidazole (CDI) is added to the flask, which dissolves quickly. The inert mixture is brought to reflux of THF. CO 2 is observed. After 3 hours, the THF is evaporated.
• CDI carbonyl diimidazole
• the Dex-PCL copolymers were dissolved in dimethyl acetamide (DMAC) at concentrations of 5 mg / ml. The volumes injected were 100 ⁇ l. The eluent was DMAC containing 0.4% LiBr, at a flow rate of 0.5 ml / min. The molar masses were determined by the method of universal calibration. Some examples are shown in Table 1.
• Dex-PCL7 is derived from the presence of 5% dextran and 95% PCL.
• Dex-PCL5 is derived from the presence of 20% dextran and 80% PCL.
• Dex-PCL3 is derived from the presence of dextran at 33% and PCL at 67%.
• Mn number-average molar mass
• Rgw average radius of gyration in weight dn / dc: variation of the specific refractive index with the concentration.
• the three copolymers have a low polydispersity and weight-average molar masses of between 11,000 and 19,000 g / mole.
• 0.2g amilose (Fluka, extracted from potatoes) are dissolved in 8ml DMSO.
• the result is a cloudy solution, to which is added 0.2 g of R-PCL-ester of NHSI (Example 6) dissolved in 3 ml DMSO.
• This mixture is incubated at 70 ° C for 144 h. After evaporation of the solvents, the solid is taken up with 200 ml of water and 200 ml of chloroform in a separating funnel.
• the intermediate phase containing the amphiphilic polymer is recovered and extracted once again, then dried. This treatment is a variant of the purification method of Example 7.
• the yield by weight after the second extraction is 38% (wt).
• Chitosan-polycaprolactone copolymer is obtained according to the protocol of Example 9. The synthesis was carried out from crude chitosan (Fluka, 150 000 g / mol) and the yield for obtaining the copolymer was 22% by 'weight. According to elementary microanalysis, the copolymer contains 67% by weight of PCL. It is of the comb type, with a skeleton of chitosan and side links of PCL mainly linked by amide bonds.
• Hyaluronic acid (Accros, molar mass greater than 10 6 g / mole) in the form of sodium carboxylate is dissolved in MilliQ water, and converted into the free acid form using a cation super-exchange resin , and freeze-dried.
• the product thus obtained is fairly soluble in DMSO and allows the coupling with the NHSI ester of R-PCL-COOH, according to the protocol of Examples 7 and 9.
• the hyaluronic acid-PCL comb type copolymer is recovered in the aqueous phase. There is no intermediate phase.
• this copolymer contains 18% by weight of PCL.
• R-PCL-COOH nanoparticles A well-defined mass of R-PCL-COOH synthesized according to Example 1 is dissolved in acetone to obtain a concentration of 20 mg / ml. A volume of water equal to twice the volume of acetone is poured dropwise. Spontaneously, the polymer forms nanospheres with an average diameter of 210 nm (measured after the evaporation of the solvent), in the absence of surfactant.
• a well defined mass of Dex-PCL copolymer synthesized according to Example 7 is introduced into dichloromethane to obtain a concentration of 10 mg / ml.
• the polymer is dispersed and swollen by the solvent, but it does not dissolve.
• a volume of water two to twenty times greater than the volume of dichloromethane is added.
• a coarse emulsion is first formed, then refined using ultrasound.
• the amphiphilic copolymer stabilizes the emulsion, thus avoiding the need to add surfactants. After evaporation of the organic solvent, nanoparticles are obtained.
• the average particle diameter is determined by light scattering (PCS).
• PCS light scattering
• Particles were formed according to the protocol of Example 14, except that instead of water, an acetate buffer solution pH4.8 saturated with chitosan was used. Spherical particles were thus obtained.
• the tritiated PLA was encapsulated as a radioactive marker in Dex-PCL nanoparticles (Example 7) to allow precise determination of the localization of the particles (inside or on the surface of the cells or in the culture medium). This marking was found to be perfectly stable in the culture medium, thus authorizing these studies.
• Caco2 cells were grown in 24-well plates, with medium change (1.5 ml / DMEM well 4.5 g / l glucose, 15% fetal vow serum) every 1 or 2 days until confluence.
• the medium is removed, 1.5 ml of Hank's medium are added, the mixture is waited for 2 hours and then the nanosphere suspensions containing well-defined quantities of particles are added (in a total volume 100 ⁇ l).
• the activity per well in the culture medium was fixed at 0.1 ⁇ Ci.
• the supernatant was removed, the cells were washed twice with PBS, then lysed for 1 h with 1 ml of 0.1 M NaOH. radioactivity was counted in the supernatant, the washings and the cell lysate.
• the quantity of Dex-PCL nanoparticles associated with Caco2 cells is double compared to those in polyester (PLA, Phusis, Mw 40,000 g / mole) manufactured by the nanoprecipitation technique (Example 10) in the presence of Pluronic®. Thus, 2.5% and 1.1% respectively of the nanoparticles are associated with the cells.
• a suspension of radiolabelled nanoparticles made from Dex-PCL (Example 7) is brought into contact with a solution of pea lectin (Lens culinaris) in excess relative to the particles, so as to saturate the surface of the latter. in affinity adsorbed lectin.
• the quantity of nanoparticles associated with Caco2 cells is significantly increased compared to those not covered with lectin. Thus, 3.5% of the nanoparticles introduced into each well are associated with the cells, compared to 2.5% in the absence of lectin.
• the capacity of nanoparticles coated with dextran (made from Dex-PCL, example 7) to avoid capture by phagocyte cells (J774) was compared with those of the same size (approximately 200 nm) and coated with PEG 5000 g / mole (made from PEG-PLA synthesized according to Example 4, from Me-O-PEG-OH 5000 g / mole and lactide, with a molar mass of the PLA block of 50,000 g / mole).
• the J774 cells were cultured in 24-well plates, in DMEM medium containing 4.5 g / 1 glucose and 10% fetal wish serum.

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