WO1999001213A9

WO1999001213A9 - Microbeads prepared from cross-linked polysaccharide hydrogel capable of containing molecules of biological interest or cells

Info

Publication number: WO1999001213A9
Application number: PCT/FR1998/001414
Authority: WO
Inventors: Karim Ioualalen; Rosanne Raynal
Original assignee: Karim Ioualalen; Rosanne Raynal
Priority date: 1997-07-03
Filing date: 1998-07-02
Publication date: 1999-04-15
Also published as: FR2765496A1; FR2765496B1; WO1999001213A1; EP0930937A1

Abstract

The invention concerns a novel type of capsule resulting from the covalent cross-linking of a biocompatible natural polymer, for example starch. The encapsulating method used provides the capsule with very high physico-chemical stability and enables encapsulation of fragile water-soluble or lipid soluble compound, and of cells. This novel technology can be used for stable encapsulation of cells of therapeutic interest, designed to be implanted.

Description

Microbeads prepared from crosslinked polysaccharide hydrogel which may contain molecules of biological interest or cells

The use of cells. d ^"cell organelles or even many fragile compounds such as essential oils, flavorings, vitamins, can be considered only by ^the use of techniques called Bioencapsulation. We can thus protect, isolate or change the behavior of encapsulated elements screw with respect to the medium in which they are used or formulated.

Bioencapsulation allows:

- protect cells, organelles or molecules against thermal or mechanical stress, against shearing and pressure variations. - to protect against chemical stress such as ^oxidation or the action of acids and bases

- to protect against ^the action of the immune system in the case of microcapsules implanted.

- to modulate the release parameters of the encapsulated compounds.

- modulate water stress.

- to improve conservation and storage.

Encapsulation strategies

The different technologies used all follow the same scheme comprising two stages. The first consists in dispersing the product or the preparation to be encapsulated into fine droplets. The second sees the realization of the encapsulation proper by the solidification of droplets either on the surface or in the mass

The dispersion is a key step in the success of a good encapsulation because it conditions at the same time the efficiency of the encapsulation, the size of the beads, the homogeneity of size and shape.

The two routes well known to those skilled in the art remain extrusion and emulsification as described by Levy and Poncelet, 1994, Biofutur, March 16-21.

• Extrusion:

Extrusion consists in dispersing the solution to be encapsulated by dropping it drop by drop in a medium where the gelling of the drops or the formation of a surface membrane is carried out (Levy and Poncelet, 1994, Biofutur, Mars, 16-21) . The drip is obtained using a needle. However, depending on the size of the desired capsules, this technique can be modulated by the application of a coaxial air stream, a potential difference or a vibration. If it is thus possible to better control the size range, it It seems impossible to envisage industrial production for capsules smaller than 500 microns at a time for reasons of yields and size dispersion.

An interesting development is the technique of coextrusion of the solution to be encapsulated and the solution of polymers forming the membrane of the microcapsule. This technique makes it possible to obtain attractive encapsulation rates but it is difficult to master and requires specific equipment.

• Dispersion by emulsification

It is the simplest technique for dispersing the solution to be encapsulated. It consists in dispersing this aqueous solution in a hydrophobic phase by stirring, generally in the presence of surfactant. This simple technique can be industrialized on a very large scale. It is mainly used for the production of capsules of sizes between 10 and 500 microns. Outside this domain, the size distributions are no longer homogeneous.

Whatever the dispersion method used, it is followed by the solidification step. This step leads to the. synthesis of the microcapsule containing the structure to be encapsulated, by the establishment of a more or less resistant membrane of varied nature or by complete solidification forming a full bead by polymerization or gelation.

The notion of encapsulation must be understood here in the broad sense, that is to say that it corresponds to inclusion in a generally polymeric structure allowing:

- protection - ease of separation

- conditioning as a microreactor

- the modulation of the mode of release of molecules

The various Bioencapsulation techniques have made it possible to obtain interesting results in very varied fields. We can distinguish the first applications in the field of carbonless paper but also the stabilization of mycorrhizae and root fragment encapsulated in alginate beads (Strullu et al, 1991, World J. Mictobiol. Biotechnol. 7, 292-297).

Excellent results have been obtained in the cryσconservation protocols for plant tips by always coating with alginate beads (Scottez C. et al 1993, Ciyobiology, 29, 691). Finally the production of ethanol and the maturation of beer or wine (FR 2736924A1) have benefited from these techniques for immobilizing microorganisms in thermoplastic capsules of alginate beads or in a granular derivative of cellulose (DEAE- cellulose) used by Finnish KERAVA breweries. In these latter cases, recovery by decantation is obtained without the heavy ultrafiltration techniques and the problem of flocculation is prevented. At the same time, companies use these technologies for the production of monoclonal antibodies from alginate beads (Damon biotech) and for the immunoprotection of xenografts by encapsulation in hollow fibers by Cytotherapeutics (Emerich et al, 1996, Cell Transplantation, 5, 589-596).

We can therefore define the main criteria for ideal encapsulation. The capsule must be biocompatible, must not modify physiological balances, must not induce side effects, must be biodegradable after having fulfilled its role and above all be stable in its conditions of use. In addition, it must have a high encapsulation capacity and allow effective modulation of the properties of permeability to nutrients, to the compounds produced or encapsulated, as well as to the various compounds or elements of the medium of use or implantation of the capsules. Finally, the encapsulation technique must be easily industrializable.

The capsule can therefore have the following criteria:

- a particular structure obtained by establishing covalent bonds, brings good chemical, thermal and mechanical stability. It must be obtained without using the difficult conventional techniques of interfacial polymerization (Levy et al, 1982, 1.Pharm.Sci., 71,759-762), thermal gelling (Poncelet et ai 1993, Fundamentals of animal cell encapsulation and immobilization, CRC Press Boca Raton, 297-314) or ionic (Kierstan et al, 1977, Biotechnol. Bioeng., 19.387), of cσvalent film-coating (Dupuy et al, 1988, J. Biomed. Mater. Res., 22, 1061-1070 ) or ionic (Lim et al., 1980, Science, 210, 908), interfacial precipitation (Stevenson et al., 1988, Biomater Artif. Cells Artif. Organs, 16 (4), 747-769), or coacervation or surface gelation by solvent change (Balladur et al, Ann. de Gastroenterologie et d'Hépatologie, 30 (6), 265-269).

- the compound must be compatible with the stability of the compounds to be encapsulated

- a significant capacity for change as well for biological compounds, cells or organelles, as for hydrophobic molecules

- significant and modular incorporation stability - good dispersibility in aqueous media

- easy size control

These specifications are only approached by a few encapsulation techniques. Among these techniques, the most used is the gelation of alginate. It has made it possible to provide answers to certain problems such as the conservation of seed / micro-organism complexes (Strullu et al, 1994, Biofutur, Mars, 35-36), the conservation of multidimensional activity during fermentation, production ethanol and winemaking. In addition, the size of the pores is not stable and the exchanges are not controlled. Those skilled in the art know that gelled capsules in mass, obtained by extrusion of the alginate then precipitation in a CaCl ₂ bath, as described by Kierstan and Bucke, 1977, Biotechnol. Bioeng .. 19, 387, are extremely fragile and sensitive to the presence of agent complexing the Ca ++ cation, such as phosphate, citrate or tartrates present in the wines. They then release their content. An alternative to alginate / Ca-H- gelation is provided with heat-sensitive gels such as carrageenans or agarose. Thus κ-carrageenan gels when the temperature is brought back below 45 ° C and remains stable whatever the ionic species present. However, it only allows the encapsulation of compounds tolerating temperatures above 45 ° C, which excludes heat-sensitive cells and molecules.

One of the evolutions of alginate gelation is the technique of film coating by ionic bonds (Lim F. and Sun A.M., 1980, Sciences, 210, 908). It consists in depositing a polymer of charge opposite to the alginate on the surface of the preformed alginate ball, by simple dispersion in a bath. The most widely used polymer is polylysine. A permeability membrane defined by the operating conditions is thus obtained. By this new technique, it was possible to prepare capsules containing cells while controlling the permeability of the membrane. It then becomes possible to confine metabolites produced by the cells in the capsule produced, in order to facilitate their manipulation, their recovery. This is the technology chosen by Damon Biotech (GB A2094750, WO A8904637) as described by Posillico, 1984, Biotechnology, 4, 114-117. Other teams om realized by following the technique of ionic coating, the encapsulation of cells in order to obtain immunoisolation by the membrane and thus allow the transplantation of xenografts, among others, of isolates of Langerhans (Zekorm et al, 1992 , Acta Diabetol., 29, 41-45) and hepatocytes. This work has shown the validity of the concept of immunoprotection by Bioencapsulation. However, the stability of these capsules is still too low. In vivo, we quickly witness the infiltration of monocytes, macrophages then degradation of the capsule, resulting in the loss of the implant in a few weeks (Clayton et al, Diabetes Research, 1990, 14, 127-132). In addition, even in sites with weak reactions such as the central nervous system or the peritoneum, pericapsular fibrosis is quickly observed.

To compensate for the low chemical stability of the capsules obtained by ionic coating, other techniques have been developed, called interfacial polymerization. All these techniques are based on the same principle. An aqueous suspension of proteins or polymers containing the product to be encapsulated, is dispersed in an organic phase to which is added the crosslinking agent, generally a polychloride of acid which reacts with the amino groups at the interface, to give a membrane nylon type (FR 2 642329A1). These techniques, based on the use of collagen, have enabled the encapsulation of enzymatic complexes as well as hydrophobic compounds. However, those skilled in the art know that it remains too aggressive for cell encapsulation. On the other hand, the protein nature of the membrane considerably limits the possibilities of therapeutic application or administration, because of the risks of immune response.

Macroencapsulation on hollow fiber brings very good results in terms of cell survival and stability in vivo (Aebischer et al, 1991, Exp. Neurol., 111, 269). However, these techniques have certain drawbacks such as:

- a large size requiring a surgical act in the case of allograft and xenograft implants. - a large membrane thickness which causes diffusion problems and does not allow a rapid response to a physiological stimuli. Insulin secretion by islets of encapsulated Langerhans is thus shifted.

- a low density of encapsulated cells implying a long fiber length.

To overcome these difficulties, some teams offer microencapsulation techniques using hydrogels from polymers used in hollow fibers such as racrylonitrile and sodium methallysulfonate (FR 2696755 Al), or photoporymers ethylene glycol muttiacrylate (WO 9631199), or (your polyacrylamides.

These latter techniques are still difficult to master. In addition, they use solvents or photoinitiators producing free radicals, which all cause toxicity problems.

The present invention relates to a new type of microcapsule intended for the internal incorporation of fragile compounds and characterized in that:

- the physico-chemical stability can be modulated

- the capsule is biocompatible and does not cause the body to respond when it is implanted

- the size is controllable - the porosity can be modulated

The microcapsules according to the invention are characterized in that they consist of a hydrogel prepared by covalent reύculation of biocompatible polymers preferably chosen from polysaccharides.

More particularly the present invention relates to a new type of microcapsule useful in particular for the transport of compounds for biological use.

These microcapsules have a very important stability, a defined size which can be modulated according to the applications. They are suitable for the encapsulation of various synthetic, semi-synthetic, recombinant or natural molecules, bacterial cells, yeasts, mammalian cells, oleous compounds, flavors and powders. These microcapsules can be used to allow or increase solubility and aqueous dispersibility, to provide protection against responses of the immune system during implants, to facilitate the handling and recovery of ferments during vinification and fermentation processes in general. These microcapsules can also be used to obtain a modulation of the modes of release of the molecules over time, to improve the physicochemical stability over time of sensitive molecules, to protect the encapsulated compounds against mechanical and thermal aggressions, to ensure transport molecules within complex eukaryotic or prokaryotic biological systems intended to ensure chemical, photochemical, immunological enzyme reactions for pharmaceutical, cosmetological, diagnostic, study and research, fermentation applications. The present invention relates to a new type of biocompatible polymer matrix intended for the encapsulation of various compounds and characterized in that the crosslinking phase of the polymer can be modulated to allow the internal incorporation of water-soluble or hydrophobic molecules. This homogeneous encapsulation is introduced after the first step of crosslinking the polymer and before complete gelation.

If these crosslinking reactions, for example with epichlorohydrin, are well known to those skilled in the art, the method according to the invention provides an advantageous modification in a preferred embodiment by carrying out:

- the dispersion of the polymer in an alkaline aqueous phase - the addition of the crosslinking agent, for example epichlorohydrin to the alkaline dispersion of polymers with stirring

- After a reaction time allowing complete dispersion of the crosslinking agent and before the start of gelation, the compound to be encapsulated is added while maintaining agitation

- the reaction medium is then dispersed in a hydrophobic organic phase until the capsules have completely polymerized

After washing, microcapsules are thus obtained which allow the encapsulation of numerous compounds. In another embodiment, the reaction medium containing the encapsulation product is not dispersed in the hydrophobic organic phase. It is left to stand until solidified in the form of a gel. The microcapsules containing the product to be encapsulated are then obtained by mechanical grinding. The microcapsules obtained are not spherical, but this technique makes it possible to obtain very small sizes of microcapsules without the use of surfactants.

According to the present invention, it has been discovered completely unexpectedly that this technique allows the incorporation of cells while maintaining excellent viability and functionality, both for bacterial cells, yeasts and cells of marmifers, for example glands adrenal or dopaminergic lines PC 12.

More particularly, the present invention relates to a microcapsule characterized in that it comprises in order:

- a hydrogel matrix based on carbohydrates or crosslinked polyols, hydrophilic, non-liquid and biocompatible - inclusions of the compounds to be encapsulated which are dispersed throughout the matrix

The matrix can be prepared by various methods well known to those skilled in the art. In particular when it is a porysaccharide, preferably biodegradable, linear or branched, for example starch and derivatives, cellulose, dextran, polysaccharides naturally derived by ionic functions, for example chitosan, hyaiuronic acids, alginates, carrageenans, hydrogel or polysaccharide matrix, is obtained by crosslinking, by methods well known to man of the 'art. The crosslinking processes can be carried out by the use of bifunctional agents capable of reacting with the hydroxyl groups of the polysaccharides such as epichlorhydrin, epibromohydrin or difunctional agents such as bis-epoxides, dialdehydes, dichlorides of dicarboxylic acids, diisothiocyanates, mixed anhydrides of dicarboxylic acids. The microcapsules are formed during the last step of the process. A first variant of this step consists in mechanically grinding large blocks of matrices obtained by mass polymerization. The second consists in producing the matrix in the form of capsules by the polymerization technique in dispersion in a liquid immiscible with the reaction phase. In the dispersion technique, the use of surfactants such as Tween or Span is dependent on the size sought. The dispersion in the hydrophobic phase is carried out by stirring in a reactor using a blade or by stirring on a stirring table with circular movement. The physico-chemical and mechanical stability as well as the porosity of the microcapsule thus prepared are a function of the crosslinking operating conditions such as the initial dilution of the polymer and / or the amount of crosslinking agent. These microcapsules can be used for the administration of molecules by the oral, per lingual, nasal, vaginal, rectal, cutaneous, ocular but also pulmonary and parenteral routes. They can also be used for topical applications of keratinous tissues such as the integuments. They can be used as a protective agent during long-term storage, in dry or wet form, of molecules of cell organelles or clusters of cells or fragments of tissues or seeds or embryos of apex. These new active ingredient vehicles are capable of encapsulating a large number of compounds. Hydrophobic compounds such as mineral oils, for example paraffin or silicone, organic oils, for example, olive, calendula, sweet almond, salmon, cod liver, evening primrose, essential oils, flavorings , dyes, mineral powders such as talc, titanium dioxide, zinc oxide, silicates, foundations, insoluble dyes and pigments, oily dispersions of plant extracts, vitamins alone or in dispersion oily, cells for example hepatocytes, islets of Langerhans, cells of pancreas, cells of medullo-adrenal, cells of dopaminergic lines PC 12, genetically modified cells, yeasts for example saccharσmyces boulardi, saccharomycεs cerevisiae, bacteria, for example leuconostoc oenos and molecules with therapeutic activity. For example: - peptides and their derivatives, glucagon, somatostatin, calcitonin, interferon and interleukins, LHRH, erythropoietin, bradykinin antagonists, polypeptides as well as recombinants from biotechnology

- antibodies

- proteoglycans - anticancer drugs

- antibiotics

- antivirals, in particular oligonucleotide analogs and reverse transcriptase inhibitors

- anti-proteases

- insecticides and antifungals - oligonucleoids

- local anesthetics and anesthetics such as benzocaine

- vasoconstrictors

- cardiotonics such as digitoxin and digitalis and its derivatives

- vasodilators - diuretics and antidiuretics - prostaglandins

- neuroleptics

- antidepressants

- hormones and derivatives - steroidal and nonsteroidal anti-inflammatory drugs

- antihistamines

- anti-allergic agents

- antiseptics

- diagnostic agents - amino acids and mineral salts - enzymes

- molecules with UV absorption activity.

Most molecules with biological activity can be incorporated directly as an oil mixture or dispersion. The fields of use of these innovative capsules are very wide as well for pharmaceutical, cosmetic and hygienic applications, as well as biotechnological and agro-food applications. The present invention and its numerous advantages will be better understood by referring to the following specific examples, given by way of example and which in no way limit the said invention. All the parts indicated in the examples are parts by volume and all the percentages are percentages by weight.

Examples

Example 1 according to the invention: Preparation of 400-micron Silicone Oil Microcapsules

In a 500 ml beaker, 20 grams of 10,000 molecular weight starch are dispersed in 52 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system equipped with a blade, 8 ml of 8N NaOH solution are added. When the solution is homogeneous, 1 ml of epichlorohydrin is added and then after 15 minutes of stirring at 65 rpm, 20 grams of Hûls silicone oil are added. The mixture is dispersed at 100 rpm for 8 minutes and then left to stand for 5 minutes. Finally, it is dispersed with stirring at 100 rpm in 200 ml of thick Giffrer paraffin oil, then placed under slow stirring on a circular stirring table of 40 mm orbit at 145 rpm for 3 hours. The capsules are recovered by decantation and then taken up in 200 ml of water and neutralized with 2N HCl. The microcapsules are then washed by decantation with 200 ml of water. 400 micron silicone oil capsules are obtained

Example 2 according to the invention: Preparation of 40 micron vitamin A propionate microcapsules

In a 500 ml beaker, 20 grams of 10,000 molecular weight starch are dispersed in 52 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system equipped with a blade, 8 ml of 8N NaOH solution are added. When the solution is homogeneous, 1 ml of epichlorohydrin is added, then after 15 minutes of stirring at 65 revolutions / min, 20 grams of vitamin A propionate are added. The mixture is dispersed at 100 revolutions min for 8 minutes and then left at rest 5 minutes. Finally it is mixed with 200 mg of Tween 80 and dispersed with stirring at 100 revolutions min in 200 ml of thick paraffin oil Giffrer, then placed with slow stirring on a circular stirring table of 40 mm orbit at 145 revolutions / min for 3 hours. The capsules are recovered by decantation and then taken up in 200 ml of water and neutralized with 2N HCl. The microcapsules are then washed by decantation with 200 ml of water. 40 micron vitamin A ester capsules are obtained

Example 3 according to the invention: Preparation of microcapsules of vitamin E / vitamin C / sweet almond oil mixture

In a 150 ml beaker, 2 grams of vitamin C finely dispersed in 18 ml of sweet almond oil mixture containing 10% vitamin E by weight are finely dispersed by extrusion using a syringe.

In a 500 ml beaker, 20 grams of 10,000 molecular weight starch are dispersed in 52 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system equipped with a blade, 8 ml of 8N NaOH solution are added. When the solution is homogeneous, 1.1 ml of epichlorohydrin is added, then after 15 minutes of stirring at 65 rpm, the oily mixture of vitamins is added. The mixture is dispersed at 100 rpm for 8 minutes then left to stand for 3 hours. The gel obtained containing inclusions of the vitamin mixture is coarsely ground, taken up in 200 ml of water and neutralized with 2N HCl. The ground material is then homogenized using a turbine at 1000 rpm until obtaining microcapsules of 20 microns. The vitamin microcapsules are then washed with water by centrifugation.

Example 4 according to the invention: Preparation of microcapsules for mixing titanium dioxide powders and magnesium silicate

In a 150 ml beaker, 2 grams of titanium dioxide and 1 gram of magnesium silicate finely dispersed in 20 ml of water are finely dispersed by extrusion using a syringe.

In a 500 ml beaker, 20 grams of 10,000 molecular weight starch are dispersed in 52 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system equipped with a blade, 8 ml of 8N NaOH solution are added. When the solution is homogeneous, 1.1 ml of epichlorohydrin is added, then after 15 minutes of stirring at 65 rpm, the mixture of titanium dioxide and magnesium silicate is added. The mixture is dispersed at 100 rpm for 8 minutes and then left to stand for 3 hours. The gel obtained, containing inclusions of the mixture of titanium oxide and magnesium silicate, is coarsely ground, taken up in 200 ml of water and neutralized with 2N HCl. The ground material is then homogenized using a turbine at 1000 rpm until microcapsules of 20 microns are obtained. The microcapsules of are then washed with water by centrifugation.

Example 5 according to the invention: Preparation of microcapsules for mixing titanium dioxide and zinc oxide powders

In a 150 ml beaker, 2 grams of titanium dioxide and 1 gram of zinc oxide finely dispersed in 20 ml of water are finely dispersed by extrusion using a syringe.

In a 500 ml beaker, 20 grams of 10,000 molecular weight starch are dispersed in 52 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system equipped with a blade, 8 ml of 8N NaOH solution are added. When the solution is homogeneous, 1.1 ml of epichlorohydrin is added, then after 15 minutes of stirring at 65 rpm, the mixture of titanium dioxide and zinc oxide is added. The mixture is dispersed at 100 rpm for 8 minutes. Finally, it is dispersed with stirring at 100 rpm in 200 ml of paraffin oil φaisse Giffrer, then placed under slow stirring on a circular stirring table of 40 mm orbit at 145 rpm for 3 hours. The capsules are recovered by decantation and then taken up in 200 ml of water and neutralized with 2N HCl. The microcapsules are then washed by decantation with 200 ml of water. 400 micron capsules containing the powder mixture are obtained. Example 6 according to the invention: Preparation of microcapsules of rose essential oil of 2000 microns

In a 30 liter ml reactor, 500 grams of 10,000 molecular weight starch are dispersed in 1000 ml of water. After 3 hours of stirring at 36 rpm using a Heidolph RZR 21 system fitted with a blade, 250 ml of 5N NaOH solution are added. When the solution is homogeneous, 25 ml of epichlorohydrin are added, then after 15 minutes of stirring at 65 rpm, 400 ml of rose essential oil are slowly added. The mixture is kept stirred at 100 rpm for 8 minutes. Finally, the mixture is dispersed with stirring at 100 revolutions / min using a three-blade stainless steel propeller 200 mm in diameter in 15 liters of thick Giffrer paraffin oil. The emulsion is kept dispersed by slow stirring using an anchor-type blade at a speed of between 30 and 50 rpm. After 3 hours of stirring, the capsules are recovered by decantation and then taken up in 16 liters of water and neutralized with 2N HCl. The microcapsules are then washed 3 times by decantation with 8 liters of water. We obtain essential oil capsules of 2000 microns

Example 7 according to the invention: Preparation of microcapsules of essential oil of rose of 40 microns

In a 30 liter mL reactor is dispersed 500 grams of 10,000 molecular weight starch in 1000 ml of water. After 3 hours of stirring at 36 rpm using a Heidolph RZR 21 system fitted with a blade, 250 ml of 5N NaOH solution are added. When the solution is homogeneous, 25 ml of epichlorohydrin are added, then after 15 minutes of stirring at 65 rpm, 400 ml of rose essential oil are slowly added. The mixture is stirred at 100 rpm for 8 minutes. Finally, the mixture is dispersed by stirring at 100 rpm using a 200 mm diameter three-blade stainless steel propeller in 15 liters of paraffin oil thick Giffrer containing 15 grams of t een. The emulsion is kept dispersed by slow stirring using an anchor-type blade at a speed between 30 and 50 revolutions / min. After 3 hours of stirring, the capsules are recovered by decantation and then taken up in 16 others of water and neutralized with 2N HCl. The microcapsules are then washed 3 times by decantation with 8 liters of water. 40 micron essential oil capsules are obtained.

Example 8 according to the invention: Preparation of encapsulated calf adrenal medulla cells

In a 500 ml beaker, 5 grams of 10,000 molecular weight starch are dispersed in 10 ml of water. After 3 hours of stirring at 50 revolutions min using a Heidolph RZR system fitted with a two-bladed propeller, 2.5 ml of 4N NaOH solution is added. When the solution is homogeneous, 0.980 ml of epichlorohydrin is added and then after 15 minutes of stirring at 65 rpm, 2 ml of suspension of adrenal medullary cells are added in DMEM culture medium. The mixture is dispersed at 100 rpm for 3 minutes and then left to stand for 5 minutes. Finally, it is dispersed with stirring at 100 rpm in 150 ml of thick Giffrer paraffin oil, then placed under slow stirring on a circular stirring table of 40 mm orbit at 145 rpm for 3 hours at 36 °. vs. The capsules are recovered by decantation and then taken up in 200 ml of culture centre . The microcapsules are then washed by decantation with 200 ml of medium until neutralization. 250 micron capsules containing the cells are obtained.

Example 9 according to the invention: Preparation of encapsulated Boulardii saccharomvce yeasts

In a 500 ml beaker, 5 grams of 10,000 molecular weight starch are dispersed in 10 ml of water. After 3 hours of stirring at 50 revolutions min using a Heidolph RZR system fitted with a two-bladed propeller, 2.5 ml of 4N NaOH solution is added. When the solution is homogeneous, 0.25 ml of epichlorohydrin is added, then after 15 minutes of stirring at 65 rpm, 2 ml of thick yeast suspension are added. The mixture is dispersed at 100 rpm for 3 minutes and then left to stand for 5 minutes. Finally it is dispersed with stirring at 100 rpm in 150 ml of thick paraffin oil Giffrer, then placed under slow stirring anime circular stirring table of orbit 40 mm at 145 rpm for 3 hours at 36 ° C . The capsules are recovered by decantation then taken up in 200 ml of water and neutralized with 2N HCl. The microcapsules are then washed 3 times by decantation with 200 ml of water. Capsules of 200 to 400 microns containing yeasts are obtained.

Example 10 according to the invention: Preparation of encapsulated Leuconostoc Oenos bacteria

In a 500 ml beaker, 5 grams of 10,000 molecular weight starch are dispersed in 7.5 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system fitted with a two-bladed propeller, 2.5 ml of 4N NaOH solution is added. When the solution is homogeneous, 0.25 ml of epichlorohydrin is added and then after 15 minutes of stirring at 65 rpm, 2 ml of thick suspension of bacteria are added. The mixture is dispersed at 100 rpm for 3 minutes and then left to stand for 5 minutes. Finally, it is dispersed with stirring at 100 rpm in 150 ml of thick Giffrer paraffin oil, then placed under slow stirring on a circular stirring table of 40 mm orbit at 145 rpm for 3 hours at 36 °. vs. The capsules are recovered by decantation then taken up in 200 ml of water and neutralized with 2N HCl. The microcapsules are then washed 3 times by decantation with 200 ml of water. Capsules of 200 to 400 microns containing bacteria are obtained.

Example 11 according to the invention: Preparation of large-pore capsules containing leuconostoc oenos

In a 500 ml beaker, 5 grams of 10,000 molecular weight starch are dispersed in 15 ml of water. After 3 hours of stirring at 50 rpm using a Heidolph RZR system fitted with a two-bladed propeller, 3.5 ml of 6N NaOH solution is added. When the solution is homogeneous, 0.75 ml of epichlorohydrin is added and then after 15 minutes of stirring at 65 rpm, 2 ml of thick suspension of bacteria are added. The mixture is dispersed at 100 rpm for 3 minutes and then left to stand for 5 minutes. Finally it is dispersed under 13 stirring at 100 revolutions / min in 150 ml of thick paraffin oil Gifiirer, then placed with slow stirring on a circular stirring table of 40 mm orbit at 145 revolutions / min for 3 hours at 36 ° C. The capsules are recovered by decantation then taken up in 200 ml of water and neutralized with 2N HCl. The microcapsules are then washed 3 times by decantation with 200 ml of water. Capsules of 400 microns containing the bacteria are obtained and characterized by a large porosity close to 500,000 daltons allowing the passage of nutrients and fermentation products.

Claims

1 - Microcapsules characterized in that they consist of a polysaccharide hydrogel crosslinked by covalent bonds and in which inclusions of encapsulated products are dispersed

2 - A method of manufacturing capsules according to claim 1, characterized in that it comprises the following successive stages: a) - the dispersion of the polymer in an alkaline aqueous phase b) - the addition of the crosslinking agent, for example epichlorohydrin to the alkaline dispersion of polymers with stirring c) - the addition of the compound to be encapsulated with stirring, after a reaction time allowing complete dispersion of the crosslinking agent and before the start of gelling d) - complete crosslinking and dispersion of the polymer leading to the formation of capsules e) - recovery of the capsules by decantation or filtration are neutralized and washed with water

3 - Process according to claims 2, characterized in that step d) is carried out, leaving the reaction medium to stand for 3 hours until complete crosslinking and solidification in the form of gel which is then neutralized and ground mechanically to form the microcapsules containing the product to be encapsulated.

4 - Process according to claim 2, characterized in that step d) is carried out by dispersing the reaction medium in the form of droplets in a hydrophobic organic phase with stirring until complete crosslinking of the capsules.

5 - Method according to claim 4, characterized in that the hydrophobic phase is a thick paraffin oil

6 - Process according to any one of claims from 2 to 5, characterized in that the polymer is a starch with a molecular weight of 10,000.

7 - Process according to any one of claims from 2 to 6, characterized in that one or more compounds to be encapsulated are introduced into the polymer solution, in the form of powder, solution, suspension, emulsion .

8 - Capsule according to claim 1 or as obtained by the methods according to any one of claims 2 to 7, characterized in that the encapsulated compound or compounds are substances of biological, food, cosmetic, therapeutic interest.

9 - Capsule according to claims 8, characterized in that the encapsulated compound or compounds are substances chosen from: - antibodies

- proteoglycans

- Anti-cancer drugs

- antibiotics - antivirals, in particular oligonucleotide analogs and reverse transcriptase inhibitors

- antiproteases

- insecticides and antifungals

- oligonucleotides, DNA and genome elements - local anesthetics and anesthetics such as benzocaine

- vasoconstrictors

- cardiotonics such as digitoxin and digitalis and its derivatives

- vasodilators

- diuretics and antidiuretics - prostaglandins

- neuroleptics

- antidepressants

- hormones and derivatives

- steroidal and nonsteroidal anti-inflammatory drugs - antihistamines

- anti-allergic agents

- antiseptics

- diagnostic agents

- vitamins - amino acids and mineral salts

- enzymes

- hydroxy acids and essential oils

- molecules with absorption activity of UV radiation or hydration of the epideπne 10- Capsule according to claim 8 characterized in that the encapsulated compounds are yeasts, bacteria, mammalian cells, plant apices

11 - Capsule according to claim 10, characterized in that the cells are chosen from hepatocytes, islets of Langerhans, pancreatic cells, adrenal medulla cells, cells of dopaminergic lines PC 12, genetically modified cells and characterized in that the capsule is implantable and the crosslinked hydrogel forming it is permeable to insulin, to the nutrients necessary for the cells, to dopamine, to metenkφhalines, to the products of the metabolism of hepatocytes and impermeable to the cellular and molecular elements of the system immune.

12 - Capsule according to any one of Claims 8 to 10 or as obtained by the methods according to any one of Claims 2 to 6, characterized in that the encapsulated substance is an oily phase containing one or more substance biological, food, cosmetic, therapeutic interest, in the form of a solution, dispersion or emulsion.