FR2811590A1 - HOLLOW POLYMERIC PARTICLE FOR THE ENCAPSULATION OF SUBSTANCES, AUTOMATED MANUFACTURING METHODS AND DEVICE - Google Patents

Abstract

The invention concerns a method for filling hollow polymeric particles with chemical, biochemical of biological particles of interest, including living cells, the hollow particles obtained by said method and capable of receiving such substances of interest, the filled particles obtained by said method, and a device for automatic implementation of said method comprising means for fixing on a support a hollow particle, means for piercing the wall of said particle and means for injecting a substance of interest into said particle. Said particles filled with chemical, biochemical or biological substances have numerous applications in various technical fields: therapeutics, cosmetics, diagnosis, food industry, phytosanitary, hygiene, prevention and treatment of degradation of natural environment.

Description

I

HOLLOW POLYMERIC PARTICLE

  FOR THE ENCAPSULATION OF SUBSTANCES,

  AUTOMATED MANUFACTURING METHODS AND DEVICE

  The present invention relates to hollow particles intended for the encapsulation of chemical or biological substances, a method of manufacturing said hollow particles and a method of encapsulation of substances in said hollow particles which can be carried out using

of an automated device.

  A drug can be characterized by the nature of its active ingredient and its dosage form.

  The latter has an important effect on the therapeutic action of the drug by modifying the intensity and kinetics of activity of the active ingredient. Traditional dosage forms such as tablets, oral or injectable solutions, creams, etc., make it possible to control the passage of the active principle in the body's blood circulation. But once in the bloodstream, the active molecule is no longer controlled: its distribution is kinetically and quantitatively dependent on factors such as the degree of affinity of the molecule for each tissue or organ or the blood flow supplying the target organs. The active molecules are unable to discern the specific sites for which they are intended, causing the appearance of side effects and limiting their use in certain therapies. For the past twenty years, advances in pharmaceutical and bio-pharmaceutical research have made it possible to offer several solutions to better control the fate of active molecules in the body. One of these solutions consists in encapsulating the active principles in particles of microscopic or macroscopic size. This strategy has proven to be

  most interesting and promising present.

  Various delivery systems using micro or macroparticles have been proposed. They are based on the principle of encapsulation of active molecules in generally polymeric matrices (R. Arshady, 1999, "Microspheres, microcapsules and

  liposomes ", MML Series, R. Arshaby Ed.).

  One method consists in trapping the active principle in a polymer network by carrying out a

  polymerization reaction in the presence of the active principle We then speak of pre-loading.

  The methods for preparing such polymeric particles are well known to those skilled in the art. They use chemical, thermal or electrochemical reactions, the operating conditions of which are dictated by the need to carry out the polymerization of the carrier matrix. However, most of the active ingredients have significant chemical and thermal instability and are incompatible with the polymerization conditions, which cause a reduction in the functionality of the active molecule or even its premature degradation. In the case of cell encapsulation, it is at least

  % of the population that is destroyed at the end of the process.

  This incompatibility between the substances to be encapsulated and the reaction conditions of the polymerization also constitutes an obstacle to the encapsulation of molecules of non-therapeutic interest such as molecules sensitive to oxidation such as phenolic derivatives, molecules such as vitamins or plant extracts sensitive to heat, salinity and pH conditions. This is why, at present their use is little developed in technical fields such as the preparation of products.

  food, cosmetics, hygiene, environmental protection or others.

  Another encapsulation method consists in introducing by diffusion the molecule of interest into a preexisting polymer matrix. We then speak of post-loading. This

  less aggressive method for organic molecules, is however of reduced application.

  Indeed, the size of the objects which can diffuse in the meshes of the matrix is limited which eliminates a priori the encapsulation of macromolecules and of all biological objects such as cells or organelles. On the other hand, a sufficient affinity must exist between the polymer matrix and the substance to be encapsulated so that the latter remains fixed in the polymer network and is not eliminated with the solvent. However, this affinity must be gentle enough not to denature the substance of interest and also to allow its release at a rate suitable for the intended use. Such constraints are most often incompatible with each other and encapsulation by post-loading remains of interest limited to specific cases. In all cases, the active substance is trapped in the mass of the polymer network. This state of affairs is in itself an obstacle to the encapsulation of living and viable cells in

  particularly because of the de facto limitation of exchanges between the cell and the external environment.

  However, cells are the natural reactors of choice for producing bio-molecules of interest. They can be considered as open systems which are capable of producing metabolites with varied biological properties. In recent years, numerous studies have been directed towards a better understanding of cellular metabolism and towards research into the means of using animal or plant cells and unicellular organisms, possibly genetically modified, for the production of metabolites of interest (Lysaght M. and Aebisher P., 1999, "Encapsulated cells", Pour la science, N 199). The intervention of the cellular machinery and its retro-controls to adjust the rates of released biomolecules, explains the advantage of -the use of whole living cells to deliver biological assets. But for that the producing cells

  must be in a supportive environment.

  The step of incorporating a substance of interest into a particulate polymer matrix is therefore extremely restrictive and delicate and constitutes at present the factor limiting the development of the microencapsulation technique and its wide application.

  in many technical fields and primarily in the medical field.

  The present invention proposes to overcome this problem by creating hollow polymer particles, of which a cavity can be filled at will with chemical or biological substance. A method of preparing such hollow particles is claimed as well as a method of filling said hollow particles and a device allowing the implementation of said filling process and which has in particular the essential advantage of being able to automate the implementation of this process while having a relatively simple structure Such particles make it possible to encapsulate entities of various sizes, starting from small entities such as salts in solution to solid elements in suspension such as cells including microorganisms, while preserving the integrity and functionality of the entities thus formulated. In particular, the proposed method makes it possible to encapsulate cells

  living excreting molecules of interest.

DESCRIPTION

  The present invention relates to a hollow polymer particle of 0.1 mm to 5 mm in diameter, and preferably from 0.2 mm to 2 mm in diameter. The hollow particle according to

  the invention comprises at least one cavity with a diameter greater than 0.01 mm.

  According to the method of preparation of the hollow particle, the method of which is claimed and described below, the cavity initially contains a gas, for example air or nitrogen. Subsequently, the cavity can be filled with other gaseous substances, liquid or solid, or with a mixture of substances such as a solute or a suspension, including a cell suspension. This operation is carried out by piercing the wall of the particle and by injecting into the cavity a substance of interest. The complete method of loading the cavity of the hollow particles according to the invention with a substance of interest is also an object of the present invention.

and will be described later.

  The present invention also relates to a device for filling with a substance

  of interest of the particle cavity, making it possible to implement the process defined above

  above, characterized in that it comprises: - means for applying a first force to said particle-so as to maintain it on a first face of a support plate arranged to generate a reaction force to said first force, - means for making a breakthrough in the wall of said particle so that said

  breakthrough opens into said cavity when said particle is held on said plate-

  support by said first force, and - means for introducing, by said breakthrough, a determined quantity of the substance of interest into said cavity. Other characteristics and advantages of the present invention will become apparent during the course of the

  following description given with reference to the accompanying drawings by way of illustration but not at all

limited

  Figure 1 shows the block diagram of the basic principle of the device according to the invention.

  Figures 2, 3, 4 show in schematic form, an embodiment of the device

according to the invention in three parts.

  FIG. 5 represents, in schematic form in top view, the embodiment of the

  device according to sfigures 2 to 4.

  FIG. 6 shows, under an optical microscope, the piercing of the wall of a hollow polysaccharide particle. The pressure exerted by the needle deforms the flexible wall of the

  particle just before entering it. The cavity appears as a white disc in the center.

  Figure 7 shows under a light microscope a particle filled with PC 12 animal cells. The cells form a darker and irregular mass in the cavity of the particle

appearing in white.

  It is specified that, in all of the above figures, attached to this description, the

  same references designate the same elements, whatever the figure on which they appear and whatever the form of representation of these elements. Similarly, if elements are not specifically referenced in one of the figures, their references

  can be easily found by referring to another figure.

  It is also specified that, when, according to the definition of the invention, the object of the invention comprises "at least one" element having a given function, the embodiment described

  may contain more than one of these elements.

  Similarly, if the embodiment of the object according to the invention as illustrated comprises

  several elements of identical function and if, in the description, it is not specified that

  the object according to this invention must necessarily include a particular number of these elements, the object of the invention could be defined as comprising "at least one" of these

elements.

  The wall of a hollow particle according to the invention is a polymer matrix, the composition of which is chosen as a function of the physicochemical properties which it is desired to obtain. In fact, because the chemical or biological substances to be encapsulated are introduced in an independent step subsequent to the manufacture of the hollow particles according to the invention, the operating conditions for preparing the polymer matrix

  can be chosen from the many techniques available to those skilled in the art.

  The nature of the starting monomer (s) only matters insofar as the synthesized polymer meets a specification adapted to the intended use of the hollow particle. The basic matrix can be modified and adapted to the use which will be made of it, by chemical, thermal or other treatments, without the choice of the protocols used being

  limited due to constraints linked to the fragility of the substances to be encapsulated.

  Only the properties of the hollow particles ultimately obtained are to be taken into account, such as, for example, biocompatibility, non-toxicity, biodegradability, selective diffusion through the mesh of the polymer network, specific affinity for certain tissues or organs, the compatibility between the polymer matrix and the encapsulated substance, the

  stability in the storage medium, or other parameters.

  For example, for therapeutic, cosmetic or food applications, the polymeric wall of the hollow particle according to the invention will preferably be biocompatible and non-toxic. Biocompatibility can be ensured as much vis-à-vis the implantation medium as vis-à-vis the active substances or encapsulated biological objects. By biocompatibility

  it is understood that the particle does not disturb the implantation medium.

  The implantation medium can be a physiological medium, as is the case during the administration of a drug by oral, subcutaneous, intramuscular route, etc., or it can be a solution such as a medium cell culture, a food product such as an alcoholic beverage, a dairy product, a cosmetic product or any other medium. The starting constituents and the operating conditions will be chosen so as to obtain a biocompatible polymer matrix with a given medium. Such starting constituents are for example natural polysaccharide polymers such as chitosan, acids

  hyaluronic, alginates, carrageenans.

  It may be desired that the hollow particle according to the invention is also biodegradable. The degradation rate determining the dose of active material released per unit of time, it is according to the intended use of the particle that the choice of polymer is made. For example, the particles according to the invention can be used to transport substances to a target and quickly release them once at their destination. For this, the polymer network can be weakly crosslinked, allowing rapid enzymatic hydrolysis, and allowing the release of the active compounds in a few minutes or a few hours after.

particle administration.

  On the contrary, it may be beneficial to gradually release the transported substance, or even to fix it for long periods, as in the case of immobilized cells, at a target or in a support medium. The polymer network can then be highly crosslinked or be grafted by specific radicals so as not to be rapidly degraded by cellular enzymes and to thus protect for several days and

  even several months the assets contained in the particle or immobilized cells.

  The rate of degradation of the polymer is regulated by the nature of the starting monomer and by the choice of operating conditions. Those skilled in the art have a complete specialized literature to modulate this parameter. The polymer matrix can also undergo chemical or physical treatments which give the particle particular properties. For example, the particle can be made unrecognizable by the reticuloendothelial system by grafting into the polymer network polyethylene glycol molecules known to be stealthy. It is also possible to mask cationic or anionic radicals by coupling with molecules of opposite charge. Molecules or radicals conferring particular physicochemical properties in the mass can also be grafted to the polymer matrix in order to modify the biodegradability, the pigmentation, the permeability to radiation, or to confer on the polymeric wall bioadhesive or mucoadhesive properties, or others

properties yet.

  The particles according to the invention can also be endowed with selective diffusion properties of the molecules. The matrix structure of the wall of said particles is analogous to a three-dimensional mesh network. The mesh size is a function of the density of the polymer network itself determined by various well-known parameters, in particular the nature of the starting monomer and of the crosslinking agent, and the reaction time. The rate of

  crosslinking is then inversely proportional to the size of the meshes.

  A polymer matrix composing the wall of a hollow particle intended for the encapsulation of cells will for example be chosen for its high crosslinking rate. Indeed the small molecules like glucose (molar mass of 180g / mol) or inulin (molar mass of the order of 5000g / mol) pass through the meshes of the matrix, while larger molecules such as l human albumin (molar mass of approximately 69,000 g / mol) does not cross the polymer network. The nutrients necessary for the life of

  cells can therefore diffuse from the implantation medium of the particles to the cells.

  Metabolites excreted by encapsulated cells can also diffuse through the polymeric wall. In contrast, cells are protected from macromolecules such as

  antibodies or enzymes that can damage them.

  Thus the hollow particles according to the invention are particularly suitable for the encapsulation of living cells because they protect the cells while letting diffuse the nutrients necessary for the long-term viability of the latter, but also the metabolites of interest, for example having a therapeutic, cosmetic, aromatic, or any other effect

interesting effect.

  In the same way and for the same reasons, the hollow particles according to the invention can be used for the transport and the maintenance in a chosen medium of organelles, such as for example chloroplasts or mitochondria, or of active cellular fractions such as

enzymes or ribosomes.

  Finally, it is possible to cover the particles according to the invention with a film which gives them particular surface properties, such as a specific affinity for a tissue, bioadhesion or mucoadhesion properties, or other properties which are also useful. . The coating can be carried out by soaking in a solution of the compound to be deposited on the particle, or by spraying. Spraying can take place before or after

  injecting substances of interest into the particle.

  The wall of a particle according to the invention is therefore a polymer forming a network called a matrix. This matrix can consist of basic elements, called monomeric compounds, which can be a single monomer (polymer in the strict sense) or two or more different monomers (copolymer) or one or more macromolecules themselves having a polymeric structure and generally assembled by bridging using a crosslinking agent. The polymer matrix can be constituted by any known polymer meeting the specifications defined according to the requirements set out above. They can be of natural, synthetic or semi-synthetic origin. For the preparation of the particles according to the invention, use is preferably made of polysaccharides, such as for example starch, starch or cellulose chemically or enzymatically modified. By way of example, common polymers can also be used, such as polyesters, polyamides, polyacrylates, glycolic polylactics, or even peptides, polypeptides and proteins, peptide gels, gelatins, alginate gels. Their weight

molecular is variable.

  In order to adapt its properties to the intended use of the hollow particle, the polymer matrix

  constituting the wall can undergo different treatments.

  In particular, the polymer matrix can be modified, that is to say that chemical groups such as functional compounds or ionic compounds can be attached thereto,

  or compounds modifying the properties of the polymer such as a pigment for example.

  All these compounds can be chemically coupled to the matrix on the surface or in the polymer network by low energy bonds (for example hydrogen bonds), medium energy (for example ionic bonds) or high energy (for example bonds) covalent). They can also be physically trapped in the network. The modification can be carried out before, during or after the crosslinking reaction. In some cases, polymer matrix modification reactions can be performed

  after loading the substance of interest into the hollow particle.

  The techniques for grafting molecules and functions are well known to those skilled in the art.

  art and conventionally used in chemistry.

  In general, the polymeric matrices described in patent application WO 98/13030 under the title "Biodegradable ionic matrices of modular internal polarity with grafted polymer" are such as to meet the characteristics sought for.

  polymer matrices forming the particles according to the invention.

  The particles according to the invention have a diameter of 0.1 mm at 5 nm, preferably from 0.2 mm to 2 mm. The thickness of the wall varies between 0.05 mm and 0.5 mm. They comprise one or more cavities, at least one of which has a section greater than 0.01 mm, filled with the gas which is used for their manufacture. The presence of such a cavity is checked at the end of their

  preparation, for example optically, or by any other means. -

  A method is proposed for the manufacture of hollow particles according to the invention consisting in: - dissolving in a reactor at least one monomeric compound in the presence of a crosslinking agent, - blowing a gaseous fluid into the reactor while stirring until '' to obtain a viscous mixture saturated with micro-bubbles, - fractionate the viscous mixture into isolated polymeric particles, - place the isolated polymeric particles in a dispersing medium until complete gelation,

  - recover the polymerized particles.

  The monomeric compound is chosen according to the criteria set out above. It is understood that it may be a single monomer or two different monomers resulting in the formation respectively of a polymer in the strict sense or of a copolymer, or even one or more macromolecules leading to the formation of 'united cross-linked polymer matrix. After solubilization of the monomer, the crosslinking agent is added with vigorous stirring

to get total dispersion.

  The crosslinking agent is chosen from bifunctional molecules, well known to those skilled in the art, such as epichlorohydrin, diepoxides, dialdehydes, dicarboxylates, diisothiocyanates, carbodiimides, etc. The monomers can themselves serve as crosslinking agent if they have several reactive sites. Adding an initiator of

polymerization will sometimes be useful.

  The gaseous fluid can be a pure gas such as nitrogen, a gaseous mixture such as air, or any gaseous mixture inert with respect to the polymeric wall and the substance of interest. The bubbling thus produced can possibly take the place at the same time of means of agitation of the

reaction mixture.

  The addition of the crosslinking agent and the gas bubbling can be carried out concomitantly, so that from the initiation of the bridging reaction between the

  monomers, the reaction mixture whose viscosity increases, traps the micro-bubbles.

  Fractionation of the viscous mixture into isolated particles is carried out by one of the techniques available to those skilled in the art: for example mechanical agitation in emulsion, grinding or extrusion. The particles must be kept in a dispersing medium until complete gelation of the polymer network containing the gas bubbles. To carry out the fractionation into isolated particles, the viscous reaction mixture can be transferred to a hydrophobic medium and an emulsion is created by mechanical stirring until the polymer has completely solidified. The transfer is carried out before the polymer network is frozen, this time varying mainly as a function of the strength and the amount of crosslinking agent used. A hydrophobic medium commonly used is paraffin oil or

silicone oil.

  Another fractionation method consists in passing the viscous reaction mixture through a nozzle extruder, and in collecting the particles at the outlet of the nozzle in a bath.

hydrophobic with stirring.

  In a variant of the method for manufacturing hollow particles according to the invention, grafted hollow particles are prepared. To do this, a chemical radical carrying an ionic function or charge or giving the polymer matrix an advantageous property as set out above is reacted with the reaction mixture. The addition of such a radical can be done as soon as the monomer is dissolved, or during the polymerization reaction, or even by impregnation at the end of the process for manufacturing the

  particle, depending on the nature of the grafted compound and the desired result.

  The particles thus obtained are hollow. They are provided with one or more cavities

  filled with the gas used for bubbling.

  They can then be recovered by sieving, by filtration, by centrifugation or by any

  other convenient means, and possibly calibrated by sieving on calibrated grids.

  They can be, if necessary, sterilized or added with a preservative avoiding the development of microbial flora, and stored without risk of degradation for several months, even several years. Storage can be done in dry form or in a solution suitable for the use which is planned later. Their transport poses no problem. The hollow particles according to the invention, prepared by the process described above or by any other process, are used for the encapsulation of chemical or biological substances of all kinds. They can therefore be filled with a gaseous, liquid or solid phase

  pure or incorporating a chemical or biological entity, constituting the substance of interest.

  The gas phase can be any chemical compound in gaseous form due to temperature or due to a thermodynamic reaction. The liquid phase may be an aqueous or alcoholic or oily solution or their mixture in the form of an emulsion or microemulsion of the oil in water or water in oil type. The solid substances can for example be powders of variable particle size or greases, possibly in

  suspension or emulsified in the liquid phase.

  A chemical or biological entity can be any simple or complex, synthetic or natural entity having a chemical or biological activity or simply a property of interest for a given application. It can be an organic or mineral molecule, or also a cell, a micro-organism, a cell fraction, biologically

active or not.

  Such a molecule can be for example: - an amino acid, a peptide in particular glucagon, somatostatin, calcitonin, interferons and interleukins, LHRH (Lutesine Hormone Releasing Hormone), erythropoietin, antagonists of bradykinin, insulin, or a peptide derivative or a polypeptide, a protein, an antibody, an enzyme, a proteoglycan; - a vitamin; - an oligonucleotide, an RNA, DNA molecule or a fraction thereof; a hormone or hormone derivative; - a therapeutic active ingredient such as an antibiotic, an antiviral, an anticancer, a vasoconstrictor, a cardiotonic, a vasodilator, a diuretic or an antidiuretic, a neuroleptic, an antidepressant, an anti-inflammatory, an antihistamine, an antifungal, a anti-allergic agent, an anesthetic, a diagnostic agent; - an antiprotease, an antiseptic, an antioxidant, a hydrating agent; - a liposome; - an essential oil, a plant extract - an insecticide, a fungicide; - a flavor, a color, possibly of food quality; - a molecule absorbing radiation, or having chromophoric, fluorophoric, or radioactive properties; A biological entity can be a cell, biologically active or not, or a cell fraction biologically active or not. A cell incorporated into a particle according to the invention can be any natural or genetically modified cell, of plant origin or

  animal including human, or bacteria, virus, yeast or any other micro-

  organization. A cell fraction can be for example a membrane extract, an extract

  enzymatic, an organelle such as mitochondria or ribosomes.

  II The cells or active cell fractions trapped in the particles and protected by them are capable of excreting in the cavity of the particle, molecules resulting from the metabolism of said cells or cell fractions. Such molecules of interest can then diffuse through a polymeric wall of adequate mesh and produce their effects.

for long periods.

  Hollow polymer particles filled with chemical or biological substances as described above have many applications in a wide variety of technical fields. They can be used as a product with a defined activity. They then constitute, for example, a product for therapeutic use which it is possible to administer either by oral, per lingual, vaginal, rectal route, and because of the reduced size of the particles, also by nasal, cutaneous, ocular, pulmonary and even parenterally, that is, both intrathecal as well as intravenous or intramuscular. They can also be used in the composition of a cosmetic, phytosanitary, textile, hygiene, prevention or treatment product for degradation of the natural environment. They can also be used as one of the elements involved in a process for preparing a product, in particular thanks to the activity of living cells and the excretion of metabolites. It may be the production of a therapeutic product, or a cosmetic product, but also a food product, for example fermented or flavored using the substances delivered by the hollow particles according to the invention. Particles according to

  the invention can also be used in a diagnostic test.

  The present invention also relates to a device for implementing the method described above, to fill, with a substance of interest described above, the cavity 2

  made in a particle like the one described above.

  Figure 1 shows the block diagram of the basic principle of such a device. This device comprises means 10 for applying a first force to the particle so as to maintain it on a first face 11 of a support plate 12 arranged to generate a reaction force to this first force. In the block diagram according to FIG. 1, the particle maintained on the first face 11 of a support plate 12 is shown in broken lines and the device in this first configuration is represented in A. The device further comprises means 13 for producing a breakthrough in wall 1 of

  so that it opens into cavity 2 when the particle is held on the plate-

  support 12 by the first force. The device in this second configuration is represented at B. The device also comprises means 14 for introducing, by this breakthrough, a determined quantity of a substance of interest into the cavity 2, the particle being always maintained

  on the first face 11 1 of the support plate 12.

  When the cavity 2 is filled with the determined quantity of the substance, the particle thus produced is recovered, for example, in a receptacle 27 as will be described below. The device in this third configuration is shown in C. In an advantageous embodiment, the means 10 for applying a first force to the particle so as to hold it on a first face of the support plate consist of a through orifice 15 made in the support plate 12 and means 16 for creating a pressure difference on either side of the support plate, the highest pressure being applied on the side of the first face 11 of this plate. In addition, the support plate is made of a relatively waterproof material and the maximum section of the orifice 15 is

  less than the minimum section of the particle.

  In an advantageous embodiment, the device further comprises means 29 for bringing the particle to the orifice 15, these latter means 29 comprising a reservoir 17 comprising at least one opening 18, this reservoir being capable of containing the particle so that, between the reservoir and the particle, a relatively large pressure drop can be created, and means 19 for positioning the support plate and the reservoir relative to each other at the first determined location A so that the opening 18 is located opposite the first face 11 of the support plate 12 and substantially in the axis of the orifice so that, under the action of the pressure difference, the particle comes to be positioned at

the entrance to this opening 15.

  Figures 2 to 5 show, in schematic form, an advantageous embodiment of the device according to the invention for filling, with a substance of interest of the cavities 2 made in the particles, automatically and according to the

  technique known to technicians under the terminology "in masked time".

  With such a device, it is possible to simultaneously perform the following three operations: placing a first plurality of particles on the orifices 15 of a first support plate, filling the cavities with a second plurality of particles which had been placed on a second support plate before the establishment of the first plurality, and the recovery of a third plurality of particles which had been placed on a third support plate before the establishment of the second plurality on the second support plate and the cavities 2 of which had been filled before the filling of

this second plurality.

  This device illustrated in Figures 2 to 5 and applying the basic structure described with reference to Figure 1, comprises at least three support plates 12-1, 12-2, 12-3, means 40 for moving in the space these three support plates so that they can successively take three first, second and third determined positions respectively referenced A, B, C, namely: the first position A in which a particle is fixed on the first face 11 of a support plate at the entrance of an orifice, the second position B in which the cavity 2 of the particle is filled with a determined quantity of the substance of interest, and the third position C in which the

  particle with its cavity 2 filled with the substance is recovered in a receptacle 27.

  Of course, each of the three support plates 12-1, 12-2 and 12-3 has a plurality

  orifices 15 making it possible to fix, at the same time, a plurality of particles.

  In an advantageous embodiment, the means 40 for moving in space the three support plates are constituted by means for carrying out a simultaneous rotation R of these three support plates around an axis of rotation W so that they pass respectively and successively by the three first, second and third positions described above, and means for carrying out a translation T of the three support plates

  between two positions on the axis of rotation W. Advantageously, these three plates-

  supports are located substantially in the same plane perpendicular to the axis of rotation W,

  one hundred and twenty degrees from each other.

  As for the means 40 for obtaining the rotation and translation of the support plates, they are well known in themselves. They consist for example of two motors and a rack, one of the motors driving the rotation of the rack about its axis,

  the other motor driving the translation of this rack along its axis.

  When the support plates have a plurality of orifices 15, the means 29 for bringing a particle in front of each orifice, consist of the reservoir 17, FIG. 2, which has an opening 18 of a cross section greater than each plate- support 12, the reservoir being able to contain a large quantity of particles to form at least one homogeneous layer 51 of these particles, so that a support plate 12 can come

  in contact with this layer. In this case, the relatively large pressure drop defined above

  before with reference to FIG. 1, part A, between a particle and the reservoir 17 is that which is created between the particles themselves, and possibly all of the particles and the reservoir 17. Consequently, for optimal operation of the device, care must be taken to ensure that the reservoir 17 contains a sufficient quantity of particles to form at least

  a layer 51 of continuous and homogeneous particles.

  As for the means 13 for making a breakthrough in the wall 1 of a particle so that this breakthrough opens into the cavity 2 when the particle is held on the first face of the support plate 12 by the first force, they can be formed in different ways, for example by a beam of laser radiation or the like. However, in a currently preferred embodiment as illustrated in FIG. 3, they are constituted by at least one first needle 21 and means for moving this first needle and the support plate relative to one another, while now the first force 10 on the particle so that it remains fixed relative to the support plate 12, so that the

  first needle 21 is able to penetrate into the cavity 2 by crossing the wall 1.

  In this embodiment according to Figures 2 to 5, the means for moving the first needle and the support plate relative to each other and the means 14 for introducing, by the breakthrough, a determined amount of the substance interest in the cavity 2, form a combination of means which are constituted by an injection device, for example a first syringe 22 capable of containing a sufficient quantity of the substance of interest, and means 23 for binding the first needle and the first syringe so that the substance contained in the syringe can be ejected and enter the cavity through the capillary of the first needle. These means 23 well known in themselves are for example constituted

  by a male-female conical fitting.

  This combination of means further comprises means for bringing the support plate 12-1, 2, 3, and the first needle 21 mounted in cooperation with the first syringe 22 to a second determined location B, means 24 for moving the first syringe relative to the support plate when it is positioned in this second determined location B, and means 25 for controlling the injection of the substance by the first needle 21 into the cavity 2 by controlling the first syringe 22, while maintaining the first force 10

on the particle.

  These means 25 schematically illustrated in FIG. 3 can consist of

  different ways, for example by a cylinder of any type acting on the plunger of the syringe.

  In an advantageous embodiment, the device may further comprise, to promote the filling of the cavities 2 of the particles, means 33 for aspirating the gaseous phase contained in the cavity of each body substantially simultaneously with the introduction of the product and depending on the amount of substance introduced. These means 33 comprise for example at least a second needle 34 and a second syringe 35, means 36 for connecting the second needle and the second syringe, means for moving the second needle linked to the second syringe so that the second needle comes pass through the wall 1 of the particle to open into the cavity 2 when the support plate 12 is located at the second determined location B, and means 37 for controlling the second syringe 35 so

let her be aspiring.

  As mentioned above, each support plate has a plurality of orifices 15 and can therefore hold a plurality of particles. It would therefore be possible to use a plurality of needle-syringe pairs 21-22 and 34-35 to simultaneously fill the cavities of the particles maintained on the same support plate. This achievement would

  however relatively complicated to implement and relatively expensive.

  Also, advantageously, the means for moving the first needle 21 linked to the first syringe 22 and the second needle 34 linked to the second syringe 35 relative to the support plate consist of at least one plate 41, means 42 to link the two syringes 22, 35 to the plate 41 so that the two needles 21, 34 are located on two concurrent straight lines, means 50 for moving the plate along two axes in X and Y, and means 48, 49 for move the two needles 21, 34 respectively on the two concurrent straight lines. These means 48, 49 will for example advantageously consist of controllable cylinders or the like respectively linked to the two syringes to allow the translation of each needle linked to its syringe. Advantageously, the device can also further comprise means schematically represented at 43, for determining the presence and position of the first and second needles 21, 34. These means are well known in themselves and can consist of sensors operating according to the principle of triangulation by laser beams or the like. These sensors make it possible to verify on the one hand that the needles are correctly positioned relative to the particle whose wall they must pierce, and on the other hand the integrity of each needle and possibly order the automatic replacement of

  the needle which would have been detected as being defective, for example broken.

  The device further comprises a receptacle 27, means for moving this receptacle and the support plate 12 relative to one another at a third determined location C, after the desired quantity of substance of interest has been introduced. in the cavity 2 and while maintaining the first force 10 on the particle, that is to say the pressure difference, and means for, on the one hand, canceling this first force and, on the other hand, applying a second force 28 on the particle to detach it from the support plate 12 and so that it is able to

  be collected in receptacle 27.

  Advantageously also, the device further comprises means 30 for creating mechanical forces in a direction substantially tangential to the first face 11 of the support plate 12, so as to be able to detach all the particles which would have remained stuck on the first face 11 of the support plate 12. These means 30 are constituted for example by jets of gas or liquid under pressure. Of course, in the embodiment of the device as illustrated in FIGS. 2 to 5, the reservoir 17, the plate 41 and the receptacle 27 will be respectively positioned in determined locations for example on a base shown diagrammatically at E and the three support plates will be mounted in rotation and in translation relative to this base E so that they cooperate successively with the reservoir 17, the plate 41 and the receptacle 27 as described below, this base E serving

  therefore referential to all displacements defined in this description.

  It is moreover very obvious that such a device is essentially intended for use in an environment in which atmospheric pressure and a gravitational force prevail. In this case, the means for creating a pressure difference are, as illustrated in FIGS. 2 to 5, constituted by means 45 for creating a sealed enclosure 46 with the second face 47 of the support plate 12 onto which the orifices open out. 15, and means 48 for producing the vacuum in this enclosure 46, for example by means of a vacuum pump or the like. In this case also, the second force 28 is constituted by the gravitational force itself, the axis of rotation W being in the vertical position and the first face 11 of the support plates being at a

  lower level compared to their second face 47.

  The device described above in its embodiment illustrated in FIGS. 2 to 5 operates and is used as follows: It is first of all specified that the entire device as illustrated in FIGS. 2 to 5 comprises a management unit making it possible to control the operations described below, in particular the translation T, the rotation R of the three support plates 12-1, 12-2 and 12-3, the control 25, 37 of the syringes 22, 35, the control of the movement 48, 49 of these syringes, the control 50 of the plate 41, the control of the means 48 for producing a vacuum. This having been specified, the device is originally in a position as illustrated in FIG. 5, in which the first support plate 12-1 is opposite the opening of the reservoir 17, the second support plate 12- 2 is opposite plate 41 and the third

  support plate 12-3 is opposite receptacle 27.

  In this initial state, no particles are maintained on the inlets of the orifices 15, but the reservoir 17 contains a large layer 51 of particles whose cavities 2 do not contain

no substance of interest.

  A first order is then given so that the axis W undergoes a translation T so that the first face 11 of the first support plate 12-1 comes into contact with the layer 51, then the means 48 for achieving a relative vacuum in enclosure 46 are controlled. A pressure difference is thus created between the space adjacent to the first face of the first support plate and the interior of the enclosure 46. As the layer 51 of particles is homogeneous, the particles which are opposite or near the entrances of the orifices 15 are sucked in and stick to these inlets to take a position like that which

  is schematically illustrated in FIG. 1.

  While maintaining the vacuum in the enclosure 46, the axis W undergoes a reverse translation of the previous one to move the support plate 12-1 away from the opening 18 of the reservoir 17, then a rotation of one hundred and twenty degrees from this shaft W is controlled, in the direction dextrorsum with reference to FIG. 5, so that the support plate 12-1 assumes the position occupied by the second support plate 12-2 before this rotation. In this same rotation, the third support plate 12-3 is positioned opposite the opening 18 of the reservoir 17, it being noted that this third support plate 12-3 does not carry any particles on its first face 11. In this second position of the device, the shaft W is controlled so that the third support plate 12-3 comes into contact with the layer 51 of particles, the means 48 are controlled to create a relative vacuum in the enclosure 46 and to vacuum the particles that get

  flatten on the inputs of the orifices 15, as described above for the first plate-

support 12-1.

  In the translational movement of the shaft W to bring the third support plate 12-

  3 in contact with the layer 51, the first plate 12-1 is positioned relative to the plate 41 carrying the syringes 22, 35 in order to be able to fill the cavities 2

as described below.

  For this, the plate 41 is controlled in X and in Y to bring the ends of the needles 21, 34 opposite each of the particles which are held on the inlets of the orifices 15. For each position of the two needles, the two syringes 22 , 35 are controlled in translation by the means 48, 49 so that the two needles cross 0 the wall 1 of the particle and open into the cavity 2 of this particle. In this situation, the syringe 22, which is presumed to contain the substance to be injected into the cavity 2, is controlled to perform this injection. Simultaneously, the syringe 35 is controlled in the opposite direction to carry out a suction of the atmosphere contained in the cavity. The injection carried out by the first syringe 22 and the aspiration carried out by the second syringe 35 are

  stopped when the second syringe draws up the injected substance.

  At this stage, the two syringes are controlled to remove the two needles from the particle, the holes made in the wall 1 being plugged in by the natural elasticity of the material.

constituting this wall.

  The plate 41 is controlled in X and in Y so that the operations described above are carried out successively for all the particles maintained on the first plate.

  support 12-1. The shaft W then undergoes translation to move the third plate away-

  support 12-3 of the layer 51, then a second rotation of one hundred and twenty degrees, still in the dextrorsum direction, for, simultaneously, bringing the third support plate 12-3 opposite the plate 41 and bringing the first support plate 12-1 next to receptacle 27, in

  now always the depression in the enclosures 46 of these first and third plates-

  supports. In this second rotation R, the second support plate 12-2 is positioned opposite the opening 18 of the reservoir 17. The shaft W undergoes a new translation to bring the first face 11 of this second support plate into contact of layer 51 of body 1 and its means 48 are activated to produce a vacuum in its enclosure 46 and

  sucking bodies 1 on the inlets of orifices 15, as described above.

  In this phase, the first support plate 12-1 being located opposite the receptacle 27, its means 48 are deactivated to remove the vacuum in its enclosure 46 and bring the interior of this enclosure to atmospheric pressure. Under the action of the gravitational force, the particles whose cavities have been filled with substance of interest fall into the receptacle to be recovered there. Possibly, the compressed gas jets

  allow to detach the particles which would have remained fixed on the support plate.

  During this operation relating to the first support plate 12-1, the cavities 2 of the particles maintained on the third support plate 12-3 are filled with substance of interest,

  as described above for the particles carried by the first support plate 12-1.

  A third rotation of one hundred and twenty degrees of the shaft W, still in the dextrorsum direction, is controlled to bring the first support plate 12-1 opposite the opening 18 of the reservoir 17, and all the operations described above. can repeat until exhausted

  total particles in the tank 17.

  In the description given above, it can be seen that the device makes it possible to fill the cavities 2

  particles fully automatically using the so-called "masked time" technique since three main operations can be performed simultaneously. In the mode of

  embodiment described above, the device has three support plates.

  It is however quite obvious that it can comprise more than three support plates, for example a number N multiple of three, the N support plates then being uniformly distributed around the shaft W, at 360 / N one with respect to the other, and mounted in association with N / 3 assemblies each essentially comprising a reservoir 17, a plate 41 with

  its two needle syringes as described above and a receptacle 27.

  This realization does not pose difficulties for a person skilled in the art knowing the structure

  of the device according to the embodiment described above.

  The following examples illustrate specific cases allowing a better understanding of the present invention and its numerous advantages. They cannot in any way limit the

scope.

EXAMPLE 1

  Preparation of hollow polysaccharide particles filled with air, according to the technique

of water in oil emulsion.

  In a 1 liter reactor, 50 g of starch having a molecular weight of approximately 000 are dispersed in 150 ml of water. The mixing is carried out at room temperature with mechanical stirring using a stirring motor at 100 rpm for 2 hours. The starch solution is then added with 300 ml of 6N sodium hydroxide solution and mixed for 2 hours at

rpm (solution S 1).

  The homogeneous mixture obtained is subjected to intensive bubbling: the reactor is connected to an air compressor under pressure of 1 bar. The bubbling is continued until the solution in gas bubbles is saturated. 4 ml of epichlorohydrin are then added to initiate the polymerization reaction while continuing aeration and with mechanical stirring at 150 rpm. After a few minutes, a low viscosity gel containing air bubbles is obtained, which is poured into 500 ml of paraffin oil and which is maintained under strong mechanical stirring carried out using a mixer (household mixer type) in a bath thermostatically controlled at 40 C for 2 hours. The particles formed are collected on a sieve in

  stainless steel mesh greater than 100 microns and are washed with reverse osmosis water.

  They have a high Tr crosslinking rate with Tr = 17%, and are noted P (17). Weakly crosslinked particles (Tr = 8.5%) are obtained by the same protocol by reacting 50 g

  starch with 2 ml of epichlorohydrin, and are called P (8.5). The polysaccharide hollow particles obtained are ready to be

used directly

for encapsulation or stored.

EXAMPLE 2

  Preparation of polysaccharide hollow particles filled with nitrogen, according to the technique of

extrusion.

  A solution of starch SI is prepared as above. The mixture is then subjected to intensive bubbling by connecting the reactor to a nitrogen reserve at 0.5 bar. The bubbling is continued until the solution in gas bubbles is saturated. 4 ml of epichlorohydrin are then added to initiate the polymerization reaction while continuing the aeration and with mechanical stirring at 150 rpm. After a few minutes, a low viscosity gel containing nitrogen bubbles is obtained. The gel is then passed through a nozzle extruder. The gelatinous particles formed at the outlet of the nozzle are collected in a reactor containing 500 ml of silicone oil and kept under low mechanical stirring (50 rpm) in a bath thermostatically controlled at 40 C for 2 hours. The particles formed are collected on a stainless steel screen with a mesh size greater than 100 microns and are washed with water.

* Osmosis.

  The polysaccharide hollow particles obtained are ready to be used directly

for encapsulation or stored.

EXAMPLE # 3

  Preparation of positively grafted polysaccharide hollow particles, filled with air

  by the water-in-oil emulsion technique.

  In a 0.5 liter reactor, 10 g of starch having a molecular weight of approximately 10,000 is dispersed in 30 ml of water. The mixing is carried out at ambient temperature with mechanical stirring using a motor. agitation at 100 rpm for 2 hours. The starch solution is then added with 60 ml of 6N sodium hydroxide solution and mixed until complete homogenization. 3.2 ml of an aqueous solution of chloride (2.3

  epoxypropyl) trimethylammonium 75%.

  The mixture obtained is subjected to intensive bubbling: the reactor is connected to a reserve of compressed air at 1 bar. The bubbling is continued until the solution in gas bubbles is saturated. 1 ml of epichlorohydrin is then added to initiate the polymerization reaction while continuing the aeration and with strong mechanical stirring at room temperature. After a few minutes, a low viscosity gel containing air bubbles is obtained, which is dispersed in 250 ml of paraffin oil and which is maintained under strong mechanical stirring carried out using a mixer (household mixer type) in a bath thermostatically controlled at 40 C for 2 hours. The particles formed are collected on a stainless steel sieve of mesh between 100 and 500 microns and are washed several times with osmosis water. Cationic hollow particles now have a specific affinity for anionic compounds. They are also bioadhesive towards biological tissues such as the mucosa

intestinal.

EXAMPLE # 4

  Preparation of cationic hollow particles treated with carboxymethyl cellulose.

  Carboxymethyl cellulose is an anionic polymer with bioadhesive properties. Hollow particles are prepared according to Example N 3, 50 g of which are dispersed in 250 ml of 2.5% aqueous carboxymethyl cellulose (Sigma) solution. The suspension is stirred slowly for 5 hours at room temperature, then the particles are recovered on a stainless steel mesh screen of between 100 and 500 microns and washed several times with water.

RO.

EXAMPLE NO.5

  Preparation of pigmented polysaccharide hollow particles.

  A starch solution Si is prepared as above. The mixture is then subjected to intensive bubbling by connecting the reactor to a nitrogen reserve at 0.8 bar. The bubbling is continued until the solution in gas bubbles is saturated. 2 ml of epichlorohydrin are then added to initiate the polymerization reaction while continuing aeration and with mechanical stirring at 150 rpm. After 2 min, 1 g of titanium dioxide is added to the reaction mixture while continuing the stirring. After 5 min, the mixture is dispersed in 1 l of paraffin oil at room temperature and vigorously stirred (household mixer)

during 3 hours.

  The particles formed are collected on a stainless steel screen with a mesh size greater than 100

  microns and are washed with reverse osmosis water.

  The particles obtained are white and opaque. They are ready to use

  directly for the encapsulation of light-sensitive or stored substances.

EXAMPLE NO 6

  Preparation of hollow polysaccharide particles grafted negatively and impregnated with vitamin E. Tocopherol acetate, also called vitamin E, is a hydrophobic molecule with antioxidant powers. The anionic charges grafted to the polymer matrix allow, despite the hydrophobic nature of tocopherol acetate, to fix it in the network where it is physically trapped. Its antioxidant properties are used to protect

  substances of interest with sensitivity to oxidation.

  In a 0.5 liter reactor, 30 g of starch having a molecular weight of approximately 10,000 are dispersed in 100 ml of an aqueous solution containing 5 g of trisodium trimetaphosphate. The mixing is carried out at ambient temperature with mechanical stirring using a stirring motor at 150 rpm for 1 hour. Then 10 ml of aqueous sodium carbonate solution at 0.25 g / ml are added with stirring until complete homogenization. The mixture obtained is subjected to intensive bubbling: the reactor is connected to a reserve of compressed air at 1 bar. The bubbling is continued until the solution in gas bubbles is saturated. The mixture obtained is then incubated at 40 ° C. for 1 hour. 10 ml of vitamin E are then added with vigorous stirring in the form of tocopherol acetate. After 2 minutes, the mixture saturated with air bubbles and containing tocopherol acetate is dispersed in a liter of paraffin oil thermostatically controlled at 40 ° C. with strong mechanical stirring carried out using a mixer (mixer type). household) for 2 hours. The particles formed are collected on a stainless steel sieve of mesh greater than 100 microns and are washed several times.

times with reverse osmosis water.

  The HPLC chromatography assay of tocopherol acetate impregnated in

  particles indicates an acetate loading of 8% by weight.

EXAMPLE # 7

  Preparation of anionic polysaccharide particles, impregnated with vitamin E and

  covered with a film of chitosan.

  Chitosan makes it possible to bring positive charges to the hollow particle.

  The particles prepared according to example N 6 above are taken up and washed with osmosis water. Then 10 g of particles are poured very slowly into 250 ml of a solution containing 0.5% of chitosan and 1% of acetic acid, with magnetic stirring. Stirring is continued for 3 hours, then the particles are collected on a stainless steel sieve (opening 100

  microns) and washed with a 0.1% acetic acid solution and then with reverse osmosis water.

  The hollow particles obtained carry anionic charges and impregnated with anti

  3 5 oxidizing in the mass of the polymer matrix and are covered on the surface with cationic charges.

EXAMPLE # 8

  Preparation of hollow particles by coreticulation of starch and chondroitin sulphate In a 1 liter reactor, 45 g of starch having a molecular weight of approximately 000 are dispersed in 150 ml of water. The mixing is carried out at room temperature with mechanical stirring using a motor set at 100 rpm, for 1 hour. 5 g of chondroitin sulfate are added and stirring is continued for 1 hour. 300 ml of 6N sodium hydroxide are added and stirring is continued for 2 hours. The homogeneous mixture is then subjected to intensive bubbling by connecting the reactor to an air reserve at 1 bar. The bubbling is continued until the solution in gas bubbles is saturated. 3 ml of epichlorohydrin are then added while continuing aeration and with mechanical stirring at 150 rpm. After 5 min, the mixture saturated with air bubbles is dispersed in one liter of paraffin oil at 40 C. After 2 hours of stirring, the hollow particles consisting of the starch / chondroitin sulfate copolymer

  are collected on a stainless steel sieve (opening 100 microns) and washed with reverse osmosis water.

EXAMPLE NO 9

  Hollow polysaccharide particles loaded with retolol and control assay.

  Retinol is a molecule sensitive to oxidation and to light. Hollow particles

  white pigmented with titanium dioxide protect the encapsulated retinol.

  The pigmented hollow particles prepared in Example N 5 are placed in the automatic particle loading apparatus. A retinol solution in soybean oil (BASF)

  is placed in the syringe filling tray and the device is switched on.

  The particles thus filled are collected to measure the retinol. The particles are fragmented in an ultrasonic probe in an ice bath. The amount of retinol released is measured by HPLC (high pressure liquid chromatography). We find that the particles

  are loaded up to 0.5% by weight, with a yield of 90% retinol.

EXAMPLE 10

  Loading of PC 12 rat pulmonary carcinoma cells into polysaccharide hollow particles and measurement of cell viability The hollow particles prepared according to example N 1 are washed with bi-distilled water and then sterilized in an autoclave at 120 ° C. pressure of 1.2 bar for 20 min. The particles are then washed under laminar flow host with DMEM cell culture medium (Dulbecco's Modified Eagle's Medium, Sigma) and then placed in the reservoir of the particle loading device. PC12 rat carcinoma cells (Greene L.A. et al., 1976, Proc. Natl. Acad. Sci. U. S.A, 73, 2424-28) are suspended in medium

  of new DMEM culture and placed in the syringe of the automatic loading device.

Then the device is switched on.

  The hollow particles encapsulating the PC 12 cells are recovered and put immediately

  in an oven at 37 ° C. in DMEM cell culture medium.

  To measure the viability of the cells thus encapsulated, the hollow particles are mechanically fragmented and the released cells are taken up in DMEM culture medium. Their viability is characterized by measuring the dehydrogenase activity of the mitochondria (toxicological test XTT, Sigma). The results show that the cells thus encapsulated are alive and active. The repeated test at regular time intervals indicates that the viability and the cellular activity are maintained after eight months of storage of the

  particles loaded with PC 12 cells in the culture medium.

EXAMPLE N011

  Comparison of kinetics of enzymatic degradation of particles as a function of the rate

of crosslinking.

  The degradation rate of the polysaccharide wall of particles P (17) and P (8.5) of Example N 1 is studied. 0.5 g of each type of particle is weighed and then placed in the presence of amyloglucosidase (A 7255, Sigma) at a content of 40 mg in 10 ml of acetate buffer. The suspension obtained is placed in a water bath at 55 C. The glucose released by the hydrolysis by amyloglucosidase of the crosslinked starch is assayed as a function of time. The assay is carried out by the specific DNS (di-nitro-salicylic acid) method

  reducing sugars for both samples and for a range of control glucose.

  The results obtained, expressed as a percentage of released sugar, are as follows: time particles P (8.5) particles P (17)

0 0% 0%

  i5 min 17% 3% 1h 36% 9% 2h 72% 22% 3h 80% 38% 4h 84% 61% 5h 90% 65% 2.4

Claims (21)

  1 - Particle formed of a polymer matrix characterized in that its section is between 0.1 and 5 mm, preferably between 0.2 and 2 mm, and in that it comprises at least one   section cavity greater than 0.01 mm.   2- Particle according to claim 1, characterized in that said polymer matrix is a   polysaccharide matrix, preferably based on modified or unmodified starch.   3- Particle according to one of claims 1 and 2, characterized in that a compound consisting   with at least one of the following compounds: functional compound, ionic compound, compound modifying at least one property of said polymer matrix, combination of these   compounds, is attached chemically or physically to said polymer matrix.   4- Particle according to one of claims 1 to 3, characterized in that said cavity contains   one of the following phases: gaseous, liquid, solid, mixed.   - Particle according to claim 4, characterized in that said cavity contains at least one   a chemical or biological entity of interest.   6- Particle according to claim 4, characterized in that said cavity contains a liquid phase and at least one of the following elements: biologically active cell, fraction biologically active cell.   7- Particle according to one of claims 1 to 6, characterized in that said particle is   covered with a film modifying the surface properties of said polymer matrix.   8- Process for the preparation of a particle according to one of claims 1 and 2, characterized in   what it consists of: dissolving, in a reactor, at least one monomeric compound in the presence of a crosslinking agent to obtain a reaction mixture, blowing a gaseous fluid into the reactor with stirring until a mixture is obtained viscous saturated with micro-bubbles, fractionate the viscous mixture into isolated polymer particles, place the isolated polymer particles in a dispersing medium until complete gelation. 9- Process according to claim 8 for the preparation of a particle according to claim 3, characterized in that it consists in reacting at least one chemical compound with the reaction mixture, said chemical compound being chosen from the following compounds: a compound carrying a chemical function, a compound carrying an ionic charge, a   compound modifying the physico-chemical properties of the polymer matrix.   - Process for the preparation of a particle according to one of claims 4 to 7,   characterized in that it consists of:   fix a particle according to one of claims 1 to 3 on a support,   pierce the wall of said particle, inject a substance of interest into said cavity, said substance consisting of one of the following phases: gaseous, liquid or solid, said phase being pure or incorporating a chemical or biological entity,   recovering said particle whose cavity contains said substance.   11- Application of a particle according to one of claims 4 to 7, to at least one of   following uses: therapeutic, cosmetic, diagnostic, food, phytosanitary,   hygienic, prevention and treatment of degradation of the natural environment.   12- Device for filling with a substance of interest, the cavity of said particle, for implementing the method according to claim 10, characterized in that it comprises: - means (10) for applying a first force on said particle so as to hold it on a first face (11) of a support plate (12) arranged to generate a reaction force to said first force, - means (13) for making a breakthrough in the wall ( 1) of said particle so that it opens into said cavity (2) when said particle is held on said support plate by said first force, and - means (14) for introducing, by said breakthrough, a determined quantity of 'a substance   of interest in said cavity (2).   13- Device according to claim 12, characterized in that the means (10) for applying a first force on said particle so as to maintain it on a first face of the support plate arranged to generate a reaction force to said first force , are   constituted by a through hole (15) produced in said support plate (12), said plate   support being made of a relatively waterproof material, the maximum section of said orifice (15) being less than the minimum section of the particle, and means (16) for creating a pressure difference on either side of said support plate , the strongest pressure   being applied on the side of the first face (11) of the support plate (12).   14- Device according to claim 13, characterized in that it further comprises means 29 for bringing the particle to the orifice 15, the latter means 29 comprising a reservoir (17) comprising at least one opening (18), said reservoir being able to contain said particle so that, between said reservoir and the particle, a relatively large pressure drop can be created, and means (19) for positioning said support plate and said reservoir relative to the other in a first determined location (A) so that said opening (19) is located opposite the first face (11) of the support plate (12) and substantially in the axis of said orifice (15) so that , under the action of the difference of   pressure, the particle comes to position at the entrance of this orifice.   - Device according to one of claims 13 and 14, characterized in that the means (13)   to make a breakthrough in the wall of said particle so that it opens into said cavity when said particle is held on said support plate by said first force are constituted by at least a first needle (21) and means for moving said first needle and said support plate relative to each other, while maintaining the pressure difference, so that said first needle is able to penetrate   in said cavity (2) by crossing the wall (1) of said particle.   16- Device according to claim 15, characterized in that the means for moving said first needle and said support plate relative to each other while maintaining the pressure difference, so that said first needle is capable of penetrate into said cavity by crossing the wall of the particle, and the means (14) for introducing, by said breakthrough, a determined quantity of the substance of interest into said cavity (2), are constituted by an injection device, preferably by a first syringe (22) capable of containing a sufficient quantity of substance of interest, means (23) for connecting said first needle and said first syringe, means for bringing said support plate and said first needle mounted in cooperation with said first syringe at a second determined location (B), means (24) for moving said first syringe (22) relative to said support plate (12) q when it is positioned at said second determined location (B), and means (25) for controlling the injection of the substance by said first needle (21) into said cavity (2) by controlling said first syringe (22),   while maintaining the pressure difference.   17- Device according to one of claims 12 to 16, characterized in that it comprises a   receptacle (27), means for moving said receptacle and said support plate relative to one another at a third determined location (C) while maintaining said pressure difference, and means for, on the one hand cancel said first force after the desired amount of substance of interest has been introduced into said cavity (2), and secondly apply a second force (28) on said particle to detach it from said support plate (12) and   so that it is able to be recovered in said receptacle (27).   18- Device according to one of claims 12 to 17, characterized in that it comprises   means (30) for creating mechanical forces in a direction substantially tangential to said   first face (11) of said support plate (12).   19- Device according to one of claims 12 to 18, characterized in that it comprises   in addition to means (33) for aspirating the atmosphere contained in said cavity (2) substantially simultaneously with the introduction of said substance, depending on the amount of   subsantce introduced into said cavity.   20- Device according to claim 19, characterized in that the means (33) for aspirating the atmosphere contained in the cavity comprise at least a second needle (34) and a second syringe (35), means (36) for binding said second needle (34) and syringe (35), said support plate (12) being located at said second determined location (B), means for moving said second needle linked to the second syringe so that said second needle (34) come through the wall (1) to open into the cavity (2), and   means (37) for controlling the second syringe (35) so that it is aspirating.   21- Device according to any one of claims 12 to 20, characterized in that it   comprises at least three support plates (12-1,2,3), means (40) for moving in space said three support plates so that they can successively assume three first, second and third determined positions ( A, B, C), these three positions being respectively: the first position (A) in which a particle is fixed on the first face (11) of a support plate, the second position (B) in which the cavity ( 2) of the particle is filled with a determined quantity of the substance of interest, and the third position (C) in which the particle with its cavity (2) filled with product, is   recovered in a receptacle (27).   22- Device according to claim 21, characterized in that the means (40) for moving in space the three support plates are constituted by means for performing a simultaneous rotation (R) of said three support plates around a axis of rotation (W) so that they pass respectively and successively through the three said first, second and third positions, and means for translating between two positions (T) of said three support plates along said axis of rotation ( W)   23- Device according to one of claims 21 and 22, and claim 20, characterized in that   that the means for moving the first needle (21) linked to the first syringe (22) and the second needle (34) linked to the second syringe (35) relative to the support plate consist of at least one plate (41 ), means (42) for connecting the two syringes (22,) to the plate (41) so that the two first and second needles (21, 34) are located on two concurrent lines, means (50) for moving the plate along two axes (in X and Y), and means (48, 49) for respectively displacing the two needles (21,   34) on the two concurrent lines.   24- Device according to claim 23, characterized in that it comprises means (43)   for determining the presence and the position of said first and second needles (21, 34).   - Device according to one of claims 22 to 24, characterized in that each of the three   support plates has a plurality of holes (15).   26- Device according to one of claims 13 to 25 and claim 17, characterized in that   that, when it is intended to be used in an environment where an atmospheric pressure and a gravitational force prevail, the means for creating a pressure difference are constituted by means (45) for creating, with the second face (47) of the support plate (12) on which said orifice (15) opens, a sealed enclosure (46) and means (48) for producing the vacuum in said enclosure (46), and that said second force is constituted by the force 1 O of gravitation.