WO2004105734A1

WO2004105734A1 - Method of preparing microcapsules

Info

Publication number: WO2004105734A1
Application number: PCT/CA2004/000803
Authority: WO
Inventors: Patrice Hildgen; Jean-Michel Rabanel; Philippe Mercier
Original assignee: Valorisation Recherche, Societe En Commandite
Priority date: 2003-05-28
Filing date: 2004-05-28
Publication date: 2004-12-09

Abstract

The present invention relates to a method of preparing a permeable and sized microcapsule having a suitable mass transfer capability. The process basically comprises the step of: a) preparing a polymeric degradable particle and optionally loading it with an active ingredient. b) coating said polymeric degradable particle obtained in step a) with a coating mixture by emulsion polymerization, thus forming a coated particle of desired thickness; and c) selectively degrading the coated particle obtained in step b), in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form at least one sized cavity inside said coated particle. The invention also relates to a permeable and sized microcapsule having a suitable mass transfer capability obtained by the aforementioned method, and uses of such a microcapsule.

Description

METHOD OF PREPARING MICROCAPSULES

A) Field of the invention

The present invention relates to a method of preparing permeable and sized microcapsules having suitable mass transfer capabilities. More precisely, the present invention relates to a method of preparing permeable and sized microcapsules having suitable mass transfer capabilities, wherein a polymeric degradable particle is first prepared, then coated with a particular coating mixture, and finally selectively degrading the coated particle obtained in the previous step so as to form a microcapsule containing a cavity. The so obtained microcapsule may optionally be loaded with biologically active ingredients and may be used for cell encapsulation or as drug delivery system.

B) Background of the invention Cell encapsulation to deliver specific therapeutic biomolecules, has received much attention as a transplantation technique. Encapsulation of cells in a semi-permeable device allows for the entry of molecules essential for cell metabolism (nutriments, oxygen, growth factors, etc.) and outward diffusion of cell metabolites, waste products and active molecules. However, cells and larger molecules of the host immune system are kept away from the transplanted cells so as to avoid using highly toxic immuno-suppressant drugs (Morris, P.J., 1 996 "Immunoprotection of therapeutic cell transplants by encapsulation", Trends in Biotechnologies, 14, pp. 1 63 to 1 67). Immuno- isolation also allows for the use of animal cells lines, known as xenotransplantation, and immortalized cell lines, eventually genetically modified, instead of primary human cell lines. The active molecule can be continuously secreted or in response to host stimulus, for example glucose concentration for encapsulated pancreatic islets. Other advantages of encapsulation consist in the possibility to target specific organ or body compartments, thus minimizing systemic dosage and potential side effects. The capsule can be considered as a "niche", a cell friendly microenvironment with the presence of an extra-cellular matrix, which can serve as cell scaffold. The use of such a capsule can also prevent excessive cell growth and eventually allow for the removal of the cells if problems arise during the course of treatment. Different applications of this concept are currently under investigation ("Technology of mammalian cells encapsulation, Advanced Drug Delivery Reviews, 42 (2000), pp. 29 to 64, Uludag et al.), including central nervous system (CNS) applications, which is well described in an scientific article entitled "Cell delivery to the Central Nervous System", Advanced Drug Delivery Reviews, 42, (2000), pp. 81 -102, Shoichet et al.

Different approaches with macroscopic and microscopic devices have been tested. Macroscopic devices (more than 1 ,5 mm) are usually implanted by surgical procedures (idem. Uludag et al. (2000)). Macrocapsules are usually formed with synthetic co-polymers such as: polyacrylonitrile-polyvinyl chloride (PAN-PVC), polyethersuflone (PES), poly-tetra-fluoro-ethylene (PTFE) or polypropylene (PP) for greater stability (Advanced Drug Delivery Reviews, 33, (1 998), pp. 87-1 09, Li, R.H.). These devices are known for their stability, high loading capacity and retrievability, though a problem associated with them consists in that mass transfer is not optimal. Such is due to the poor surface/internal volume ratio and membrane thickness. Fibrous tissue deposition is frequent and the size of the device makes implantation invasive and lowers a patient comfort. Moreover, the constant need for surgical procedures can impose a recurrent risk of infection.

Many micro-encapsulation systems (under the 1 mm range) have been described in earlier works, such as in the scientific journals entitled: Sciences, 146, (1 964), pp. 524-525 (Chang T.G.) and Science, 210, (1 980), pp. 908- 910 (Lim F., et al.). Most of these micro-encapsulation systems rely mainly either on gentle hydrogel cell embedding or synthetic membrane formation by co-extrusion of a cell preparation and capsule material. Natural polyelectrolyte polymers such as alginates can form gel when in contact with electrolytes of opposite charge such as cations, namely Ca²⁺, Ba²⁺ or poly-L-Lysine, and they can form capsules by emulsification or extrusion (see Science, 210, (1 980), pp. 908-91 0 (Lim F., et al.)). In an attempt to better control the preparation parameters, synthetic hydrogel polymers such as polyphosphazene, or blends of polymethacrylates have been proposed as well as the combination of natural and synthetic polymers. Use of agarose, forming gel upon heating, has been described for bead preparation. Early work on nylon capsules (Sciences, "Semi-permeable microcapsules", 146, pp. 524 to 525 (Chang, T.G.)) have lead to the development of a method of encapsulation by synthetic membrane formation by interfacial precipitation of polymethacrylate polymers, which was described in Advanced Drug Delivery Review, 33, (1 998), pp. 87-1 09 (Li, R.H) and Advanced Drug^' Delivery Reviews, 42, (2000), pp. 29-64 (Uludag et al.).

With micro-encapsulation, mass transfer of various products such as molecules secreted by encapsulated cells, nutriments, drugs or products of enzymatic reactions, is optimized (high surface/internal volume ratio); thus increasing cell viability and allowing a faster secretory response to an external signal. But, such a micro-encapsulation technique also has its limitations. These limitations include mechanical fragility and instability of ionic interactions in hydrogels and batch-to-batch variability, which leads to differences in permeability properties. This is particularly the case for natural hydrogels, wherein such materials entail problems of variable biocompatibility of materials, limited cell loading capacity, stress on cells during encapsulation procedures and non homogenous repartition of cell in beads which could eventually lead to host immunological responses. Other limitations are further described in an scientific article entitled "Engineering challenges in cell- encapsulation technology", Trends in Biotechnologies, 14, (1996), pp. 158- 161 (Colton C.K.).

To overcome some of the aforementioned limitation, it is known to the

Applicant that Kappa Biotech, a French company, has recently developed a new concept in the area of micro-encapsulation with the aim to develop applications for CNS delivery of encapsulated cells. The latter is described in an International patent application, which was published under no. WO 02/05943 (Mercier et al.) on January 24^th, 2002. Generally speaking, this International application concerns a method for filling hollow polymeric particles. Briefly, the approach is to encapsulate cells after the synthesis of capsules by automatic microinjection, thus avoiding cellular stress during the entrapment step, and non-homogeneous repartition of cells in the beads. However, to achieve this, the critical step has become to master a reliable method to produce soft hollow particles of the desired size, permeability and is compatible with the injection procedure.

More particularly, International publication no. WO 02/05943 A1 distinguishes itself from the present application in that it describes a process for preparing a gas during amylopectin reticulation by bubbling. The so described process is very different from the one described in the present invention, namely in that it does not suggest or disclose the possibility that active ingredients can be incorporated in the polymeric degradable particle. Moreover, the process defined in the aforementioned international publication does not work. Indeed, the only feature that seems to work in this International publication is automatic cell injection. Few methods have been described to prepare hollow particles in the micrometer and millimeter range and which are compatible with cell viability. Preparation of hollow particles has been described essentially for nano- capsules, for drug and peptide delivery system, for aroma capsules in food preparation, for contrast agents in medical imaging (Journal of Microencapsulation, 18(2), (2001 ), pp. 1 59-1 71 , Bjerknes et al.), and for floating gastro-intestinal drug delivery device (Journal of Microencapsulation, 16(6), (1 999), pp. 71 5-729, Lee et a/.). Classical techniques, such as spray- drying and double solvent emulsion, are not usually compatible with cell viability. Other techniques have been developed, including self-assembling capsules, vesicular polymerization, template approach using melamine formaldehyde particle coated with polyelectrolytes and emulsion/suspension polymerization, but the latter were not designed for cell encapsulation and they offer only limited capacity. Such problems are well described in an article entitled "Polymer nanocapsules", Chemical society Reviews, 29, (2000), pp. 295-303 (Meier. W.) .

To the Applicant's knowledge, covalent gel hollow microcapsules have not much been described previously. The first method investigated by Kappa Biotech involves the introduction of air micro-bubbles in a specially designed reactor during the amylopectin gel crosslinking reaction. The hollow particles obtained with the aforementioned approach have allowed the validation of the concept with dopamine secreting PC1 2 cell lines for CNS delivery. Such is also partially described in International publication WO 02/05943 A1 mentioned hereinabove. For further information, reference can also be made to the Journal of Microencapsulation, 18(3), (2001 ), pp. 323-334 (Mercier et al.) . However, it is worth mentioning that the preceding method was not satisfactory, as product yield remained low. Thus, there is a need for a novel method of preparing permeable and sized microcapsules having suitable mass transfer capabilities, and which may preferably serve as a template for capsule wall formation. Moreover, there is a need for such a method to prepare permeable and sized microcapsules, which can preferably allow for improved cell encapsulation. There is also a need for microcapsules having the aforementioned properties, and in which its physical characteristics such as size, morphology and porosity can be varied by using the method according to the present invention. There is a further need for a method for preparing a microcapsule that overcomes all of the above- mentioned drawbacks.

SUMMARY OF THE INVENTION

A first object of the invention is to satisfy the above needs.

For this purpose, the invention provides a process for preparing a permeable and sized microcapsule having suitable mass transfer capabilities, said method comprising the step of: a) preparing a polymeric degradable particle and optionally loading it with an active ingredient. b) coating said polymeric degradable particle obtained in step a) with a coating mixture by emulsion polymerization, thus forming a coated particle of desired thickness; and c) selectively degrading the coated particle obtained in step b), in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form at least one sized cavity inside said coated particle. An advantage with the above-mentioned method for preparing microcapsules resides in that the size of the cavity can be varied. Indeed, other components such is size, morphology and porosity of the particle may also be changed.

A second object of the invention lies in a permeable and sized microcapsule having suitable mass transfer capabilities as obtained by the method mentioned hereinabove.

A third object of the invention lies in the use of a permeable and sized microcapsule having suitable mass transfer capabilities as prepared by the method as defined hereinabove for cell encapsulation, for the preparation of microreactors and/or microreservoirs, and the preparation of multi- compartmental capsules.

Other objects and advantages of the present invention with the apparent upon reading the following non-restrictive description of several preferred embodiments thereof, made with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 is a schematic illustration of the products obtained during the steps of the method for preparing microcapsules according to the present invention. In this Figure 1 , step (1 ) represents a polymeric degradable particle that has been prepared in this step; step (2) represents the polymeric degradable particle that is obtained after coating in step 2; step (3) represents the so coated polymeric degradable particle after it has been subjected to selective degradation to form a cavity therein; and in step (4), represents the microcapsule loaded with cells and/or active ingredients.

Figure 2 are illustrations of microcapsules obtained by the method according to the present invention: A) Core particles containing amylopectin particles crosslinked with 10% (w/w) trimetaphosphate (TMP), stained with chromium oxide powder, size: 250-355 micron (in 0.05 M NaOH); B) idem particle obtained after coating with an amylopectin gel crosslinked with epichlorohydrin (particles sieved: 425-500 microns); C) core particle subjected to selective degradation, assay: incubation, NaOH 1 N/37°C/48 hours, core particles are stained with methylene blue. Bar represents 0.500 mm.

Figure 3 are illustrations of the PLGA core particles after they have been subjected to the steps of coating and selective degradation according to a preferred embodiment of the invention (Assay PGE0003). A) Coated PLGA particles between 425 and 710 microns; and B) Fraction of particles between 425 to 710 microns after 5 hours hydrolysis (NaOH 1 N/37°C). Arrows indicate hollow particles with completely degraded core. Bar represents 0.500 mm.

Figure 4 are illustrations of particles when they have been subjected to the step of coating according to the present invention and the effect of Span 80™ therein. A) Assay PGE003: 0% Span 80™/Rushton/400 rpm; B) Assay PGE001 : 0.1 % Span 80™/Rushton/200 rpm; and C) Assay PLE027: 0.2% Span 80™/ Rushton/800 rpm. All fractions: 425 to 710 microns. Bar represents 0.500 mm.

Figure 5 are illustrations of particles when subjected to the coating according to a preferred embodiment of the present invention and the effect of polyethylene glycol distearate (PEGDS) and viscosity thereon. A) particle containing PLGA/5% PEGDS. Coating was conducted with a concentrated amylopectin/epichlorohydrin reaction mix; B) idem particle after undergoing the step of hydrolysis with NaOH 1 N/65 hours. Arrows show degraded core with presence of non-degraded PEGDS; C) Coated PLA particles (no PEGDS added). Coating was conducted with a concentrated amylopectin/epichlorohydrin reaction mixture (assay PLE014, fraction: 425 to 710 microns). Bar represents 0.500 mm.

Figure 6 are illustrations of core particles after having been subjected to the step of coating according to a preferred embodiment of the present invention. A) "Template" amylopectin/trimetaphosphate (AP/TMP) gel microparticle (250 to 350 microns) colored by chromium oxide in an amylopectin/NaOH/epichlorohydrin coating mixture; B) "Template" core particles PLA/FITC-Dextrans 70kD (PL034) in amylopectin/NaOH/epichlorohydrin coating mixture. Microscopy (X10); C) Green fluorescence microscopy (X10). Bar represents 0.500 mm.

Figure 7 are illustrations of microcapsules obtained by the method according to the present invention. The illustrations are by a SEM. Control amylo-pectin gel particle: Assay C001 : A) Control AP gel particles: Fraction 425 to 710 microns, light microscopy, A') idem in SEM; B) Coated PLA particle with AP gel (assay PLE01 1 ), after 60 hours NaOH 1 N /37°C hydrolysis: Fraction 425 to 710 microns, light microscopy, B') idem in SEM.

Figure 8 are illustrations of microcapsules obtained by the present invention and depicting the release of fluorescent markers. A) Coated PLA- FITC-Dextrans 4kD particles (assay PLE029); B) Coated PLA-FITC-Dextrans 10kD particles (assay PLE030); C) Coated PLA-FITC-Dextrans 70kD particles (assay PLE027); D) Coated PLA-FITC-Dextrans 70kD particles (assay PLE028). All fractions: incubation NaOH (1 N) for 96 hours and at pH equilibration (7.0). Fractions 425 to 710 microns observed in green fluorescence microscopy.

Figure 9 is a graph demonstrating the diffusion of fluorescent markers. Two batches of particles, PLE020 (dark symbols) and PLE023 (open symbols), were tested after a complete hydrolysis of polymeric degradable particle (preferred embodiment of the present invention). Fluorescence added to the external medium was followed for 6 hours and the results are expressed as a percentage of a control assay with no particles. Squares: FITC-Dextrans 4kD, Triangles: FITC-Dextrans 10kD, Circles: FITC-Dextrans 70kD.

Figure 10 are illustrations of microcapsules obtained by the method according to the present invention and their diffusion of FITC-Dextrans. All assays were carried out on batch PLE020 after complete degradation of the polymeric degradable particle. At t = 0, fluorescent markers were added to the external medium. Particles were observed in green fluorescence microscopy at t = 24 hr. A) FITC Dextrans 4kD; B) FITC-Dextrans 10kD; C) FITC-Dextrans 70kD; and D) Diffusion of FITC-Dextrans 70kD into a defective hollow particle (microcapsule).

Figure 1 1 are illustrations of microcapsules obtained by the method according to the present invention, which are to preferably be used as a microreservoir (Figure 1 1 A) or a microreactor (Figure 1 1 B).

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the following terminology is exclusively for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and appended claims, the expressions "hollow particle" and "microcapsules" or any variant thereof may be used interchangeably in the context of the present description.

Similarly, the expression "core particle" and "template particle" may be used to define the "polymeric degradable particle" as used in the appended claims.

The expression "mass transfer", as defined by the IUPAC Compendium of chemical terminology, relates to a "spontaneous process of transfer of mass across non-homogeneous fields. The driving force can be difference in concentration (i.e. diffusion gradient) or partial pressure of the component.". As per the present invention, the expression "mass transfer" may also relate to the diffusion of substances that could either penetrate inside the microcapsule, for example gas, nutriments, reactive agents, etc., or get out of the microcapsule, for example via secretion.

As aforesaid, the object of the present invention is to provide a method of preparing a permeable and sized microcapsule having suitable mass transfer capabilities. This method comprises the steps of: a) preparing a polymeric degradable particle and optionally loading it with an active ingredient, b) coating said polymeric degradable particle obtained in step a) with a coating mixture by emulsion polymerization, thus forming a coated particle of desired thickness; and c) selectively degrading the coated particle obtained in step b), in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form at least one sized cavity inside said coated particle. A) Preparing the polymeric degradable polymer

In the first step of the method, denoted step a), a polymeric degradable particle is prepared. Such a polymeric degradable particle can be prepared by various techniques known to a person skilled in the art. Indeed, polymers that can be used as choice of polymeric degradable particle according to the present invention include, but is not limited to: polyesters, polyanhydrides, polyamides, polyorthoesters, polyacrylcyanides, polylactide, poly(lactide-co- glycolide), polycaprolactone, polyhydroxybutyrate, their copolymers and mixtures thereof. There, of course are other suitable polymers and combinations thereof, which can be used as choice of polymeric degradable polymer particle.

B) Coating the polymeric degradable particle

In the second step of the method, denoted step b), the polymeric degradable particle, such as PLA and/or PLGA, is to be coated with a coating mixture by emulsion polymerization. By conducting this step, the Applicant can prepare a coated particle of desired thickness.

Indeed, this step comprises coating of the polymeric degradable particle obtained in the preceding section. Typically, a natural polysaccharide such as an amylopectin is dissolved in a NaOH solution under stirring so as to form a solution. Preferably, epichlorohydrin and/or another crosslinking agent known to a person skilled in the art is then added to this solution, thus forming a coating mixture. The polymeric degradable particles, being treated for example by sieving, are then added to the aforementioned coating mixture and is preferably incubated for a few minutes. The viscous mixture is then preferably introduced into paraffin oil under stirring. It is worth mentioning that the solution can further contain a surfactant, though such is not necessarily required. It is worth mentioning that the paraffin oil can by replaced by another type of oil such as silicone oil. However, paraffin oil is preferred since it is non-toxic and inert. It is also worth mentioning that the introduction of the viscous mixture into the paraffin oil can be done by a direct transfer or by injection.

Indeed, suitable types of polysaccharides may include and is not limited to starches, modified starches, alginate, amylopectin, cellulose, amylose, chitosan, xanthan and other modified celluloses. There, of course, exist other types of natural polysaccharides that may be considered.

Preferably, the crosslinking agent used in step b) may include trimetaphosphate, epichlorohydrin and other chemical compounds that are accepted by the FDA standards. Though other commonly used reticulating agents such as: dichlorodiethyl ether; dibasic/tribasic carboxylic acid (both carboxyl etherify OH groups); anhydrides (acetic); divinyl sulfone; diepoxides; cyanuric chloride; di-isocyanates; 1 ,6-hexanedibromide; N,N methylenebisacrylamide; esters of propynoic acid; imidazolium salts of polybasic carboxylic acids; aldehydes such as formaldehyde, acetaldehyde, dialdehyde, glutaraldehyde (toxic); and N,N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC), may be used.

Indeed, it is also worth mentioning that the coated particle obtained in step b), preferably has a thickness of about 75 to 100 microns. However, for other applications such as: microreservoir, microreactors, multicomptimental capsules and the like, the thickness could preferably be from 500 nm to 500 microns. C) Selective Hydrolysis

In the third step of the method, denoted step c), the coated particle obtained in section B) is selectively degraded, in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form a sized cavity inside the coated particle.

It is worth mentioning that reaction wherein one changes pH of a medium and/or hydrolytic degradation is quite well known to a person skilled in the Art. However, it is beneficial to use such a step, in that it allows the Applicant to selectively adjust the size of the cavity in the coated particle, in either basic or acidic conditions, thus allowing the microcapsule to contain foreign matter.

In fact, it is at this stage in the method of preparing a permeable and sized microcapsule having suitable mass transfer capabilities, is that one can modify the nature of the microcapsule. For example, one can:

(1 ) obtain a microcapsule containing a cavity by hydrolysis of the core which contains no additional substances; (2) obtain a microcapsule by hydrolysis of the core which contains an active substance, wherein the active substances finds itself free in the reservoir (i.e. the cavity contained within the microcapsule). In the case that the microcapsule contains more than one cavity, each of these cavities could contain an active substance; and (3) not carry out any hydrolysis on the core(s), thus obtaining a microcapsule containing an active substance which is liberated by diffusion through the matrix of the core and then through the coating. Applications

It is understood that the nature of the microcapsules obtained by the method according to the present invention will depend on the applications considered. For example, a microcapsule with one or more compartments can be used for the insertion of cells into an artificial organ application or in a biotechnological application. Microcapsules can also be used as a microreservoir system, which is able to liberate drugs at a constant drug rate. Preferably, the drug can be a protein, genetic material, a peptide and/or small molecules.

Indeed, suitable drugs may include and are not limited to: plasmids for gene therapy, proteins (i.e. deficient enzymes, G-CSF, erythropectin, growth hormones, FSH and the like), and those known to person skilled in the art to have low biodisponibility or stability problems. Such drugs may include: omeprazole, enanapril and the like.

It is also to be understood that the microcapsules obtained by the method according to the present invention, with or without a cavity formed therein by the step of selective degradation, can also be used as a drug delivery system. It is worth mentioning that in the application of oral drug delivery, the goal of the microcapsule is to increase the biodisponibility of the drug. This goal can be achieved since the coating of the microcapsule acts as a mucoadhesive agent.

It is also worth mentioning that when part of the coating of the microcapsules is pH sensitive, the surface of the microcapsule becomes porous. Moreover, the microcapsules containing one or more cavities; thus being multi-compartmental may also contain a reactive material. Indeed, when the microcapsule erodes, the reactive material can diffuse into a cavity where a reaction can occur and form an active material, such as NO. It is worth mentioning that the active material can also diffuse outside of the microcapsule. In fact, the control of drug release is obtained by the porosity and pore size of the microcapsule. Preferably, the release rate is a near zero order mechanism.

Preferably, the mean size of core particle obtained from the above- mentioned techniques is preferably from 500 nanometers to 500 micrometers. Of course, it is possible to modify the mean size of the obtained particles.

Moreover, it is worth mentioning that the microcapsule obtained by the method according to the present invention can also be used as a micro- reactor.

EXAMPLES

The following examples are illustrative of the wide range of applicability of the present invention and are not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any method and material similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described. Material & Methods

Materials

Polylactide (PLA) was prepared in the laboratory. Briefly, a catalyst such as tetraphenyl tin (Aldrich) was mixed with a precursor 3,6-dimethyl-1 ,4- dioxane-2,5-dione (Aldrich) in a 1 /10 000 proportion (w/w) and introduced in a round bottom flask under a continuous flush of an inert gas such as argon. Preferably, the flask was heated at 180°C for 3 hours. By-products were dissolved in acetone and precipitated in water. The polymer was dried and freeze-dried. The molecular weight was measured by Size Exclusion Chromatography (SEC) with a polystyrene standard (results of analysis: Mw = 57 300, Mn = 25 750). Polylactide-co-glycolide (PLGA) was from Boehringer- Ingelheim (RG 504H, 50:50 lactide/glycolide) with a Mw of 48 000, partially hydrolysed amylopectin (Glucidex 2) was from Roquette (France); paraffin oil, Epichlorohydrin and Span 80™ were from Fluka; fluorescein isothiocynate (FITC) dextrans, trimetaphosphate (TMP), sodium carbonate and polyethylene glycol distearate (PEGDS) were from Sigma, organic solvents were from Laboratoire MAT (Montreal), partially hydrolyzed polyvinyl alcohol (9-10000 PM) was from Aldrich.

EXAMPLE 1 :

Preparation of amylopectin {AP) /trimetaphosphate {TMP} core particle

Preferably, 2g of trimetaphosphate and 20 g of amylopectin (Glucidex 2) were stirred in 36 ml of H₂O for 2 hours. 4 ml of sodium carbonate was added and the preparation was incubated for 90 minutes at 42°C with frequent agitation. The preparation was transferred slowly into 600 ml of paraffin oil under stirring (750 rpm, Caframo mixer) overnight at 25°C. Water was added and after sedimentation, the particles were washed, re-dispersed in 0,05 M NaCI and sieved. Coating of amylo-pectin core particles

Preferably, 5g of amylopectin was dissolved in 6 ml of NaOH (2N). After adding epichlorohydrin (ratio: 1 mole per 1 ,5 mole of sugar monomer), 10g of core particles are mixed and transferred into 600 ml of paraffin oil under stirring (500 rpm, Caframo mixer) for 6 hours. Particles were then recovered by sedimentation in water, sieved (mesh sizes: 150, 355, 425 and 710 microns) and washed to remove salts, traces of oil and epichlorohydrin.

Selective degradation assays Preferably, 1 g of coated particles are introduced into 50 ml of NaOH

(1 N) and incubated at 37°C under gentle orbital agitation.

EXAMPLE 2:

Preparation of PLA and PLGA core particles Preferably, PLA and PLGA core particles were prepared according to the solvent extraction-evaporation technique. Typically, 500mg of PLGA or PLA were dissolved in 3,5 ml of chloroform and injected in 250 ml of 0,3% PVA (w/v) and kept under stirring for 12 hours (Caframo stirrer). Particles were then collected, sieved (sieves: 150 and 350 microns) and washed before coating. Viscosity of the polyester solution in solvent (5-20% w/v) and stirring speed (150 to 350 rpm) were adjusted for desired particle size.

Coating of the PLA and PLGA core particles

In a preferred embodiment, 2 to 5g of amylopectin (Glucidex 2) were dissolved in 2,5 to 7 ml of NaOH (2N) for one hour under stirring. Preferably, epichlorohydrin was added in a ratio of 1 /1 ,5 or 1 /9 (ratio mole of epichlorohydrin per mole of sugar monomer). Sieved core polyester particles were added and incubated under stirring for 1 to 5 minutes. The viscous mixture was then introduced in 400 to 800 ml of paraffin oil under stirring (straight blade or Rushton propeller, at a speed of 250 to 900rpm, Caframo stirrer) in the presence of surfactant Span 80™ (from 0% to 0,5% vol. /vol.) for 6 to 12 hours. The presence of the surfactant is only a preferred embodiment of the invention. Indeed, the preferred introduction of the surfactant can be either done by direct transfer or by injection. In the latter case, the mixture was introduced in a 5 ml syringe and injected by a syringe pusher (Harvard 1 1 ) at a velocity of 45 ml /hr. The needle (18 Gauge) was completely immerged in paraffin oil. Particles were recovered by sedimentation in water, sieved (mesh sizes: 150, 355, 425 and 710 microns) and washed to remove salts, traces of oil and epichlorohydrin. Drained particles fractions were weighed to calculated yield.

Selective hydrolysis

In a preferred embodiment, the coated PLA or PLGA particles can be suspended in NaOH (1 N) solution and incubated at 37°C for 24 to 96 hours. Particles are collected, sieved and washed in Millipore™ water before further use (fraction 425 to 710 microns retained for analysis).

Characterization of microcapsules Optical Microscopy.

All particles, including polyester core particles and hollow particles, were examined with a light microscope (Zeiss Axiovert™ S100, X10, Zeiss,

Germany) or by making use of a binocular (Nikon™, X 0,5 or X2).

Microphotographs were recorded with digital camera using Northern Eclipse software and analyzed for particle size with Optimas (v6.0) image analysis software and examined for morphology by visual inspection. Scanning electron microscopy

Control particles and final hollow particles fractions were examined on a Jeol™ scanning electron microscope.

Fluorescence study Release study

Fluorescein isothiocyanate Dextrans (FITC-Dextrans) were introduced in polyester core particles as an emulsion. 50 μl of a FITC-Dextrans aqueous solution was added to the polyester/chloroform mixture, sonicated for 20 seconds on a 550 Sonic Dismenbranor™ (Fisher). This primary emulsion was then transferred in a 250 ml 0,3% PVA under stirring (see section annotated Preparation of PLA and PLGA core particle). The ratio of polyester/FITC- Dextrans was about 1 mg to 500 mg, except for batch PLE034: 10 mg to 500mg. After coating, microparticles were incubated in NaOH (1 N) solution at 37°C, and examined under microscope, on a Zeiss Axiovert S100™ microscope (Zeiss, Germany), which was equipped with a set of emission/excitation filters (excitation: 495nm, emission 517nm) and a fluorescence lamp. Digital pictures were recorded with a digital camera and use of Northern Eclipse software was also made.

Diffusion.

In a preferred embodiment, the 425 to 710 micron fraction of coated particle batch PLE020 was incubated in NaOH (1 N) solution for 72 hours at 37°C to selectively degrade the core particles. After sieving and repeatedly washing, 500mg of the fraction was re-suspended in 1 ml of H₂0 containing the fluorescent probe (FITC-Dextrans, 4kD, 10kD or 70 kD) and incubated at room temperature. Aliquots of 10μl were collected and analyzed on a spectro- fluorometer. A control assay containing 1 ,5ml of H₂O with the same quantity of fluorescent probe was also incubated in the same conditions. Fluorescence diffusion results were expressed as a percentage of the control at each time point in time for each of the FITC Dextrans tested. Similar experiments were simultaneously conducted and particles were observed under fluorescence on a Zeiss Axiovert S100 microscope (Zeiss, Germany) equipped with a set of emission/excitation filters and a fluorescence lamp. Pictures were recorded with a digital camera and use was made to Northern Eclipse software.

Results & Discussion

Assays with amylopectin core particles Amylopectin core particle preparation and coating.

In yet another preferred embodiment and as seen in both Figure 2, an amylopectin particle crosslinked with a crosslinking agent such as trimetaphosphate (TMP), also referred to as AP/TMP particle, were prepared to be used as "template" or "core" particle, according to the preparation scheme represented in Figure 1 . A TMP crosslinked amylopectin (AP) matrix is very sensitive to basic conditions. To illustrate this point a AP/TMP particle of about 500 microns is completely degraded in NaOH (1 N) in less than 2 hours at room temperature. On the other hand, an epichlorohydrin crosslinked amylopectin matrix is much more resistant to basic conditions. Coating of the core AP/TMP particle by emulsion polymerization with an amylopectin/epichlorohydrin gel gave good results. This result is shown in Figure 2B.

Selective degradation

Nevertheless, as represented in Figure 2C, different degradation assays were done and these assays were found to be unsuccessful or largely incomplete. Core AP/TMP particles were selectively stained by methylene blue. The presence of the dye (methylene blue) after a 48-hour incubation period in a strong basic condition indicated the resistance of the core particle matrix for degradation. If the incubation period were to last longer, for example 4 to 5 days, the whole particle would eventually completely disintegrate. It is thus hypothesized that the preparation of hollow particles was hampered by diffusion and reaction of the crosslinking agent within the polysaccharide core particles at the "coating" stage. This second crosslinking reaction, led to a less selective degradation of the core, i.e. an incomplete degradation and weakened capsule walls. Thus, the material chosen as template particle must not react with the crosslinking agent.

Validation of new approach with polyester core particle Core particle preparation and coating

The premise was that the size of the core particle must correspond to the size of the cavity expected, for example around 300 microns for assays of amylopectin/TMP core particles (see Figure 2). PLA or PLGA particles were prepared according to the solvent emulsion/evaporation technique described in the Journal of Controlled Release, 17, (1 991 ), pp. 1 to 22 (Arshady, R.), to produce large particles of about 350 microns. The coating of polyester core particles was first carried out by using the same protocol developed to coat amylopectin/TMP core particles. The thickness of the coating was then determined to be about 500 nm to 500 microns to compromise between mechanical stability and mass transfer of various compounds such as nutriments, reactive substances, secreted substances, enzymes, drugs, genetic material, etc. Coating was carried out by an emulsion polymerization of a solution of amylopectin and polyester core particles in paraffin oil under stirring. The coating was stabilized by an epichlorohydrin crosslinking reaction described in Starch, 37, (1985), pp. 297-306 (Kartha et al.). Although some coated polyester particles were obtained in the initial experiments, the yield is very low. The results can be seen in Figure 3. As a result, there was formation of big lumps composed of polyester particles embedded in an amylopectin matrix. The cavities thus obtained were larger than the core polyester particles. This phenomenon can be explained by a partial degradation of the polyester template particle when it is in contact with the strongly basic amylopectin gel and/or mechanical action of the particle under stirring and/or swelling of the capsule under washing at the end of the process. Similar results are also obtained with PLA core particles (data not shown).

Selective degradation

Polyester particles are degradable in both acidic and basic conditions. Preliminary experiments on uncoated polyester particles confirm that basic conditions are the fastest. This has been confirmed by a scientific article published in the Journal of Microencapsulation, 3, (1986), pp. 203-212 (Makino et al.). It has been further reported that reactions conducted in basic conditions at 37°C are at least two times faster than those conducted at room temperature. Degradation of PLA particles are 4 to 5 times slower than PLGA (Resomer RG 504H™) particles (data not shown). The coating of the core particle does not affect the extent of the degradation. The end result, as shown in Figure 3, shows hollow particles with residual polyesters after 6 hours for PLGA and 48 hours for PLA.

The preliminary experiments also confirm the feasibility in preparing hollow particles by this method and point out the parameters to be optimized, such parameters including core particle size, selective polyester hydrolysis and core particle coating reaction.

Optimization of particle template coating

Based on early assays, the Applicant was able to identify the main factors affecting the efficiency of the polyester core particle coating. These factors may include: stability of emulsion in paraffin oil, stirring speed, geometry of mixer, viscosity of amylopectin mixture, interaction between the polyester's surface and the amylopectin mixture. It is worth mentioning that PLA, as choice of polymer for the core particle preparation was done in an attempt to reduce the cost of future preparations.

Stability of emulsion

In early experiments, low yield of coated polyester particles seems partially due to the formation of big lumps composed of polyester particles and an amylopectin matrix gel. When introduced in paraffin oil, the emulsion was well dispersed but within minutes, lumps were formed. The droplets, composed of polyester particles surrounded by an amylopectin gel in formation, do not seem stable enough. To overcome this apparent instability problem, a non-ionic surfactant such as Span 80™ (Sorbitan monooleate) at different concentrations can be preferably used in paraffin oil to stabilize the emulsion. Such a feature can be seen in Figure 4. Indeed, it was found that a concentration 0,02% v/v of surfactant was sufficient to prevent lump formation without affecting the particle size and morphology. The effect of surfactant concentration is in relation to the stirring speed and geometry of the mixer as well as oil viscosity. A series of experiment were done to optimize the results, however this data is not provided.

Polyester surface properties

In spite of the elimination of lumps, the polyester particle coating yield was low (% of particle with a polyester core within the 425 to 71 0 micron size fraction) and lower than the yield obtained with an amylopectin gel particle coating, as demonstrated in Figure 2. It can be hypothesized that such a result is due to the hydrophobic surface of polyester particles, which could thus be an obstacle to gel coating. To resolve this problem, polyethylene glycol distearate (PEGDS) can preferably used as a surface modifier. PEGDS is composed of two stearate moieties at each of its extremities and contains a spacer of PEG (Mn around 930). It is documented that when PEGDS is introduced into polyester particles, the hydrophilic PEG form a loop, while being segregated on to the surface, and the stearate moieties stay buried in the more hydrophobic medium of the PLA or PLGA (see the International Journal of Pharmaceutics, 174, (1 998), pp. 101 -109 (Lacasse et al.)). Different concentrations, from about 1 to 10% w/w relative to polyester, can be introduced in polyester particle synthesis. The coating yield was not improved, and more importantly, clusters of "naked" polyester particles were observed. This could be attributed to the attractive Van der Wall interactions between PEG layers (see Advances in Colloid and Interface Sciences, 27, (1987), pp. 189-209 (de Gennes, P.G.)). The surface modifier could also have different properties according to its density. It has further been documented in the International Journal of Pharmaceutics, 174, (1 998), pp. 101 -109 (Lacasse et al.) that a 0,1 % concentration of PEGDS inhibits polyester particle adhesion to macrophage but not 1 %.

Viscosity

Further to the experiments with PEGDS, the viscosity of the coating mix was modified. In an initial experiment, amylopectin of about 40% w/v was dispersed in NaOH (1 N) solution, however some aggregation was still observed. When PEGDS is used at 60% w/v, the amylopectin concentration gave the results shown in Figure 5A. Although capsule walls were thick, i.e. to 200 microns, for optimal mass transfer, the morphology of the particle was as expected. Selective degradation assays (NaOH 1 N/37°C) gave good results, except for the presence of some residual material, probably non-degraded PEGDS, as shown in Figure 5B. Experiments repeated in the same conditions without PEGDS show that the initial viscosity is the main factor to influence the coating yield, not the surface modifier as seen in Figure 5C. However, when the core particle is dispersed in amylopectin, the viscosity of the mixture is the function of not only the water content but also of the extent of the reaction of epichlorohydrin, primarily governed by the incubation duration and the incubation temperature. These parameters are likely to influence not only the coating yield but also the permeability properties the gel capsule walls.

Injection

A highly viscous amylopectin solution is necessary for the success of the polyester particle coating reaction. Moreover, viscosity increases as a function of the epichlorohydrin crosslinking reaction, as it progresses over time. It is, thus, difficult to disperse the emulsion in a reproducible manner. Hence, automatic injection of the mixture was introduced: the amylopectin/core particle mix was injected with a syringe pusher with a 18 gauge needle immerged in paraffin oil. Repeated experiments with the same conditions, such as stirring speed and incubation time, show an average yield of 43% of particles in the range of 425 to 710 microns, a marked increase in comparison to prior transfer methods (data not shown). In these conditions, it has been estimated upon visual inspection that between 25% to 50% of the particles in this size range have a polyester core particle inclusion. These results can be confirmed upon inspection of Figure 5C.

Core particle size

The premise was that the core particle must have the same diameter as the cavity to be formed, i.e. preferably about 1 to 500 microns. However, observations show that particle sizes of about 175 to 200 microns is the ideal size for cell encapsulation applications only, as cavity diameters are around twice the core particle diameter, as seen in Figures 3 and 4. Core particle synthesis was adapted accordingly. The main parameters to be adjusted were the viscosity of the starting polyester/organic solvent solution and the stirring speed of the emulsion. Sizes of the particles were assessed with Optimas^® image analysis software, Area Equivalent Diameters and Ferret diameters measured gave typical results of 190 μm with a standard deviation (SD) of + /- 35 μm after optimization of the parameters such as viscosity, stirring speed and PVA %.

Coating process

In a preferred embodiment, polyester particles are not dried; instead they are just washed and sieved before use. Dried particles are rapidly degraded when , in direct contact with strongly basic amylopectin/epichlorohydrin reaction mix. Observation of PLA particles in the amylopectin mixture before crosslinking occurs, show a thin layer of water around each PLA particle (see Figure 6B), unlike the amylopectin gel particle in the same situation (see Figure 6A). It is further shown that the surface of polyester particles has at least some affinity for water. This phenomenon can also be attributed to residual PVA molecules adsorbed on the particle surface. The water layer acts as a shield between the polyester and the basic solution (NaOH 2N). In the case of polyester particles containing FITC-Dextrans 70kD (batch PL034) and as can be seen when comparing Figures 6B and 6C, fluorescence can be observed in the water layer surrounding the particles. This could be explained by the segregation of hydrophilic molecules at the surface of the particle as already observed with PEG (see the International Journal of Pharmaceutics, 174, (1998), pp. 101 -109 (Lacasse et al. 1998)). Also, one can expect some polyester hydrolysis at the surface of the particle; the latter inducing release of some fluorescence in the water layer. The presence of this water layer and possible surface erosion of core particle could contribute to the size of cavities observed relative to core polyester particle diameter, as seen in Figures 3 and 5. Optimization of selective degradation

In a preferred embodiment of the invention, the so formed polyester core particles are very compact, and such can be attributed to the high viscosity of the initial polyester/chloroform mix. The choice to use PLA instead of PLGA, increases the incubation time necessary to completely degrade the core particle. The latter can take up to 96 hours for PLA. To preserve the structure of the gel capsule walls and to speed up the process, optimization will need to be carried out. Methods such as double emulsion to weaken polyester particle structure and a choice of a PLA with lower molecular weight should be considered, as per the Journal of Controlled Release, 30, (1994), pp. 161 -173 (Park, T.G.).

Hollow particle characterization Scanning Electron Microscopy

The hydrated particles were examined by scanning electron microscopy at room temperature and under vacuum. Unlike frozen samples, the amylopectin gel undergoes dehydration over time and eventually collapses in these conditions. Control particles, made of crosslinked amylopectin (non hollow particles), were first examined (see Figure 7). As seen in Figure 7A', the pictures taken in the initial moments show a smooth surface for native particles. Fractions with hollow particles were then analyzed, before and after polyester core particle hydrolysis. Upon inspection of Figure 7B', one can observe particles at the initial moment that appear like a deflated balloon.

Porosity, FITC-Dextrans Release.

To study the effective porosity of the gel capsule, the Applicant has chosen to use FITC-Dextrans as molecular weight markers. The diffusion of fluorescence from the coated core particle upon basic degradation under basic conditions into the medium was observed in fluorescence microscopy. Six different assays were conducted and their results are represented in Table 1 provided hereinbelow:

TABLE 1 - Some particles prepared for FITC-Dextrans permeability experiments.

As seen in Figure 8, fluorescence microscopy of individually coated core particles show that, the 70kD marker is retained within the capsule (assay PLE027, see Figure 8C), while the 4kD and 10kD markers (assay PLE029 and assay PLE030 respectively, see Figures 8A and 8B) seem to diffuse through the gel capsule wall. As represented in Figure 8D and in assay PLE028, where the coating was done with a lower ratio epichlorohydrin/sugar monomer (i.e. 1 /9 instead of 1 /1 ,5), the 70kD seems to diffuse in the medium; thus emphasizing the role of degree of crosslinking on permeability to FITC- Dextrans. Porosity: FITC-Dextrans diffusion

Penetration of FITC-Dextrans added to the medium containing the hollow particles was followed over time. The diffusion of the fluorescence inside the hollow particles was compared with respect to a control without any particles added in the medium for six hours. It was expected that fluorescence remains high in the medium for 70kD markers (no penetration in the particles), and decreases over time for 10kD and 4kD markers as diffusion equilibrium is reached between the external marker concentration and the marker concentration in accessible regions of the particle. The two different batches (PLE020 and PLE023), prepared under the same conditions were selected to rule out the possibility of batch-to-batch variations. The results obtained with the two batches are seen in Figure 9. In the case of the 70kD marker, the concentration in the external medium remained at a plateau signifying little or no PLEO23 and/or PLE020 diffusion into the particles as expected. 4kD and 10kD markers showed decrease in fluorescence, but slower than expected. Moreover the value of fluorescence is greater than the expected value, and such varies between batches. One explanation could be attributed to insufficient washing of particles after incubation in basic conditions. Indeed, it has been shown that the release of NaOH in the medium can affect the intensity of fluorescence (see Methods in Enzymology, 174, (1989), pp. 131 -145 (Ohkuma, S.)). This could also account for the initial burst of fluorescence seen in the first 30 minutes.

Observation in fluorescence of the individual hollow particles under a fluorescent microscope showed that the 4kD and 10kD markers diffused into the hollow particles within 24 hours. These results are shown in Figures 10A and 10B. However, the 70kD marker does not seem to penetrate into the hollow particle, such can be seen in Figure 10C. However, a defective hollow particle could allow for the penetration of the 70kD marker inside the particle, such can be seen in Figure 10D.

Permeability: porosity & diffusion. Permeability and/or porosity of the capsule to proteins are essential characteristics of the microcapsules. One can expect that the control of the porosity will be an important concern for regulatory agencies to allow clinical trials.

The principles governing capsule gel permeation are similar to those of size-exclusion chromatography. The ability of a protein to cross the capsule wall depends on gel pore size, size and shape of the protein and interactions between the protein and the gel network (ionic or hydrophobic). Molecular weight cut-off determination using FITC-Dextrans has to be cautiously interpreted as gel permeability is not directly dependent on molecular weight (Mw), but rather on physical size and shape of the molecule. Dextran in solution adopts a flexible coil conformation while proteins adopt a more globular and compact conformation. Correlation between molecular weight (Mw) and physical size (Rη: viscosity radius) has been established for dextrans and proteins. These correlations are described in the scientific publication entitled Analytical Biochemistry, 242, (1996), pp. 104-1 1 1 (Brissova et al.)). If dextrans size increases linearly with Mw, proteins on the other hand assemble in structure of similar size, even if Mw vary substantially. As a consequence, if Mw cut-off is estimated here to be between 10kD and 70kD with dextran, it might correspond to a protein cut-off between 30kD and 400kD. However, dextrans are useful because they allow for the determination of permeability without a confounding effect of interactions with the gel network. Amylopectin gel is a neutral polymer network and therefore its interactions with neutral markers such as dextrans are not expected to be significant for permeability determination.

The novel method for preparing microcapsules thus produces particles with a range of permeability characteristics in accordance with immuno- isolation parameters.

Other applications

Indeed, the microcapsules obtained by the method according to the present invention can be used as a micro-reactor. When the microcapsules are used as a micro-reactor, several substances can be loaded in different core particles. For example, one batch is prepared with PLA microspheres containing FeSO4. A second batch can be prepared with PLA to yield PLA microspheres containing sodium nitrite. After the coating, a microcapsule could include particles from the first and second batch. In this case there is no need of degrading the polyester core. A slow diffusion of both reactive in the medium allows the reaction and generates nitrogen monoxide (NO) . The microcapsule thus acts as a micro-reactor thereby slowly generating NO by a constant rate through the hydrogel.

When the microcapsules obtained by the method according to the present invention are to be used as a multi-compartmental drug carrier, several substances can be loaded into the core particles. For example, one batch can be prepared with PLA microspheres containing anti-inflammatory drugs, while a second batch is prepared with PLA to yield PLA microspheres containing an opioid drug. After the step of coating the microcapsule, the microcapsules could include particles obtained from both the first and second batches. The cores could be degraded or not. Alternatively, the microcapsules obtained by the method according to the present invention can also be used as a micro-reservoir drug delivery system. In this connection, one substance can be loaded into the core microsphere, and the particle can then be coated with the hydrogel. The core is then degraded. The drug is free inside the cavity and can be released by diffusion through the gel at a constant rate. An example of such an application can be for example the use of an anticancer drug for brain delivery.

Conclusion

Thus, the novel method of preparing permeable and sized microcapsules as set forth in the present application is successful. Morphology, size and porosity of the particles can be adjusted. This novel method can be extended to other materials for core particle preparation and coating. Indeed, the use of a wide variety of polymeric degradable core materials can be used; and thus allow for selective degradation in acidic conditions, for example by using polyorthoester polymers. The coating can then preferably be done with trimetaphosphate (TMP) as choice of crosslinking agent, which produce links that are sensitive to basic conditions, but less sensitive to acidic conditions. The advantage of TMP as choice of cross-linking agent is considered to be more acceptable in terms of toxicity. Coating can also be done with other natural or synthetic hydrogel and crosslinking methods. Thus, the method according to the present invention could also be useful to create multi- compartmental drug delivery devices, wherein a drug can be trapped in the polyester hydrophobic medium, then released during degradation at physiologic pH while the hydrophilic coating can be involved in hydrophilic drug storage, release regulation and biocompatibility of the devices. While several embodiments of the invention have been described, it will be understood that the present invention is capable of further modifications, and this application is intended to cover any variations, uses or adaptations of the invention, following in general the principles of the invention and including such departures from the present disclosure as to come within knowledge or customary practice in the art to which the invention pertains, and as may be applied to the essential features hereinabove set forth and falling within the scope of the invention.

Claims

1 . A method of preparing a permeable and sized microcapsule having a suitable mass transfer capability, said process comprising the step of: a) preparing a polymeric degradable particle and optionally loading it with an active ingredient, b) coating said polymeric degradable particle obtained in step a) with a coating mixture by emulsion polymerization, thus forming a coated particle of desired thickness; and c) selectively degrading the coated particle obtained in step b), in either basic or acidic conditions, by a pH change and/or hydrolytic degradation so as to form at least one sized cavity inside said coated particle.

2. The method according to claim 1 , characterized in that the polymeric degradable particle is made of at least one compound selected from the group consisting of polyesters, polyanhydrides, polyamides, polyorthoesters, poly- acrylcyanides, polylactide (PLA), poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL) , polyhydroxybutyrate, their copolymers and mixtures thereof.

3. The method according to claim 1 , characterized in that the coating mixture comprises a polysaccharide and a cross-linking agent.

4. The method according to claim 3, characterized in that the polysaccharide is selected from the group consisting of amylopectin, starches, modified starches, amylose, chitosan, xanthan, alginate, modified cellulose and their mixtures.

5. The method according to claim 3 or 4, characterized in that the cross- linking agent is selected from the group consisting of trimetaphosphate (TMP), epichlorohydrin, dichlorodiethyl ether, di- and tri-basic carboxylic acid, anhydrides, divinyl sulfone, diepoxides, cyanuric chloride, di-isocyanates, 1 ,6- hexanedibromide, N,N-methylenebisacrylamide, esters of propinoic acid, imidazolium salts of polybasic carboxylic acids, aldehydes and N,N-(3- dimethylaminopropyl)-N-ethyl carbodiimide.

6. The method according to claim 1 , characterized in that the coated particle obtained in step b) has a thickness of about 500 nanometers to 500 microns.

7. The method according to claim 6, characterized in that the coated particle obtained in step b) has a thickness of about 500 nanometers to 500 microns for an application wherein the microcapsule is to be used as a micro-reservoir, a micro-reactor and/or a multi-compartmental microcapsule.

8. The method according to claim 6, characterized in that the coated particle obtained in step b) has a thickness of about 0.5 to 200 microns for an application wherein the microcapsule is to be used for cell encapsulation.

9. The method according to claim 1 , characterized in that in step b) use is also made of a surfactant to stabilize the emulsion during emulsion polymerization.

1 0. The method according to claim 1 , characterized in that in step b) use is also made of a surface modifier for modifying the polymeric degradable particle surface and thus improve its mechanical properties.

1 1 . The method according to claim 1 , characterized in that the size of the cavity is between 0.5 and 400 microns.

1 2. The method according to claim 1 1 , characterized in that the size of the cavity is between 250 and 350 microns for a microcapsule of 500 microns.

1 3. The method according to claim 1 , characterized in that the active ingredient is selected from the group consisting of hormones, peptides, pain killers, genetic material, drugs, enzymes, and combinations thereof.

14. The method according to claim 1 , characterized in that said microcapsule containing at least one cavity is obtained by hydrolysis and does not contain any active ingredient therein.

1 5. The method according to claim 1 , characterized in that said microcapsule containing at least one cavity is obtained by hydrolysis and contains an active ingredient therein.

1 6. The method according to claim 1 , characterized in that said microcapsule does not undergo hydrolysis and contains an active ingredient, which is liberated by diffusion through the coated portion of said microcapsule.

1 7. A permeable and sized microcapsule having a suitable mass transfer capability as obtained by the method as defined in any one of claims 1 to 1 6.

1 8. Use of a permeable and sized microcapsule having a suitable mass transfer capability as prepared by the method as defined in any one of claims 1 to 1 6 for cell encapsulation, a micro-reactor, a multi-compartmental drug carrier, a micro-reservoir and/or a drug delivery system.