Background of the Invention The encapsulation of materials in polymeric microcapsules having permeable or semipermeable membranes surrounding a liquid core is well known in the art. A wide variety of different approaches,, based on different polymer chemistry, different processes for membrane formation and different encapsulation technologies have been tried.

An important application of these techniques is in the encapsulation of biologically active species. In general, these techniques have the potential for the treatment of diseases requiring enzyme or endocrine replacement as well as in nutrient delivery via the encapsulation of enzymes and bacteria. Encapsulation is currently employed in the food, agriculture and biotechnology and biomedical industries. Examples of these and potential applications are the encapsulation of islets for the treatment of diabetes mellitus, the use of encapsulated bioartificial organs targeted at treating neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and Huntington's chorea, and in the control of chronic pain and the administration of human growth factors.

The immunoisolation of biologically active materials has been the subject of recent reviews, see Willaert & Baron (1996) and Jen et al (1996). The technology of encapsulation (Colton, 1996), the inflammatory response of transplanted tissues (Morris, 1996), the modification of polymer surfaces to enhance biocompatibility (Hubbell, 1994), the use of polyacrylates for encapsulation (Sefton & Stevenson, 1993 ; Stevenson & Sefton, 1993), the use of water-soluble polymers for immunoisolation (Hunkeler, 1998), a review of polymers for bioartificial organs (Hunkeler, 1997) and an overview of tissue engineering (Baldwin & Saltzman, 1996) have also been examined by researchers in this field.

One approach for forming capsules is based on complex formation between oppositely charged polymers. In this method, a solution of a first polymer including a material to be encapsulated is preformed in droplets between about 0.2 and 3.0 mm in diameter and contacted with the second oppositely charged polymer. Typically, droplets of the first polymer and the material to be encapsulated are generated using a capillary or spraying device and contacted with the second polymer, e. g. by falling into a precipitation bath. The reaction between the polymers at the surface of the droplet forms a membrane around a liquid core including the encapsulated material. These polyelectrolyte complexes (PECs) have been studied due to their potential application as microcapsules for medical implants. A variety of approaches, based on various polymer chemistries, processes for membrane formation and encapsulation technologies have been evaluated. However, over the past two decades the overwhelming majority of scientists have

restricted their studies on the alginate/poly-L-lysine polyelectrolyte complex system, where solid alginate/calcium beads are coated with a solution of oppositely charged poly-L-lysine (PLL) and subsequently converted into a permeable capsule by liquefying the ionotropically gelled anionic polysaccharide.

By way of example, US Patent No: 4,582,799 (Jarvis, Jr.) discloses a method of encapsulating cells to promote recovery of low molecular weight non-secreted products from high molecular weight contaminants. In this method the cells encapsulated within a membrane are lysed after the product is produced by the cells, allowing the product to escape from the cells while retaining the higher molecular weight cell debris within the capsule.

The authors disclose the use of a membrane with a molecular weight cut-off of 100 kDa. The capsules are formed from alginate having a Mw between 50-80 kDa and above (sodium alginate solution of 0.6 to 2.4 %w/v) and poly-L-lysine (PLL) having a Mw of about 35 kDa in the presence of calcium ions and optionally including a coating step using a cross-linking polymer such as polyvinylamine (PVAm) to improve the mechanical strength of the capsules.

A further example of alginate/PLL/PVAm capsules is provided in US 4,663,286 (Tsang et al) which uses these polymers to encapsulate viable cells. In these capsules, a membrane is formed from multiple layers of PLL which are then coated with high molar mass PVAm having a Mw of 50-150 kDa. The capsules obtained are permeable for molecules having a molar mass of at least 150-160 kDa.

Other examples of encapsulation systems using alginate/PLL/PVAm are disclosed in Grdina et al (1984) Biotech 84 USA, A235-246; and Wheatley et al (1991) J.

Appl. Polym. Sci. 43,2123-2135.

Wang et al. (1992) Biotechnology and Bioengineering 40, 1115-1118 disclose beads based on an alginate/CaCl2/PVAm system, which are then coated with sodium alginate.

Brissova et al (1998) J. Biomed. Mater. Res. 39,61-70 disclose the encapsulation of islets by the complexation of sodium alginate and cellulose sulphate with poly (methylene-co-guanadine) (PMCG) in the presence of calcium and sodium ions, and that these capsules have improved mechanical strength as compared to alginate/PLL capsules. In some embodiments, the capsules were coated with PVAm.

The system discussed in this paper represents the closest prior art of which the inventors are aware.

Our earlier application, WO00/01373 discloses, among others, an encapsulation system employing as a positively charged polymer oligosaccharides of Mw less than 10 kDa (e. g. chitosan) and alginate.

However, it remains a problem in the art in finding methods for controlling the balance between the permeability of the capsules and their mechanical strength. There is also a problem in the art in finding reactants and reactions conditions which are compatible with biological materials, in particular as many of the biological materials that might be usefully encapsulated,

for example mammalian cells, require neutral or slightly alkaline pH to remain biologically viable.

Summary of the Invention Broadly, the present invention relates to novel methods of producing microcapsules based on two oppositely charged polymers, in particular to optimise the permeability and mechanical stability of microcapsules.

The capsules are formed by introducing liquid droplets from an aqueous solution of a charged (usually anionic) polyelectrolyte (or a blend) into an aqueous solution of a counter-charged (usually cationic) polyelectrolyte (or a blend). The membrane is formed during the reaction time of the opposite-charged polymers. The capsules are optionally subject to an additional coating step.

The microcapsules are well suited for cell encapsulation, applications for biomedicine and transplantation, among other uses.

Accordingly, in one aspect, the present invention provides a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane, the method comprising: forming a droplet from-an inner polymer solution comprising at least one positively or negatively charged polymer and the core material; contacting the droplet with an outer polymer solution comprising at least one polymer having an opposite charge to the polymer or polymers forming the droplet so that the polymers react to form a membrane around the droplet; and

optionally coating the droplet by contacting it with a coating polymer solution ; wherein at least one of the polymer solutions comprises polyvinylamine (PVAm) or a copolymer of polyvinylamine, said PVAm or copolymer having a molar mass of less than 20 kDa.

Previously, all encapsulating systems employing PVAm have used molar masses of 20 kDa and above. It is surprisingly shown in the present work that the use of lower molar mass PVAm can lead to increased mechanical stability of the resultant capsules.

Processes previously used for obtaining substantially pure PVAm or its salts tend to be very elaborate, since PVAm cannot be obtained from the hypothetical vinyl- monomer formally recognized as the repeating unit. There are various different ways to synthesise PVAm of low molar mass, such as less than 20 kDa, but the conditions are usually drastic, using strong mineral acids or bases at relatively high temperatures, and often under pressure. These reaction conditions are not easy to carry out, especially on polymeric materials, and since the reactions are attended by side reactions it is difficult to obtain a pure polymeric product. The present invention, however ; in this and following aspects, preferably employs PVAm prepared by radical polymerisation of N-vinylformamide followed by hydrolysis (preferably stoichiometric hydrolysis, preferably stoichiometric base hydrolysis) to PVAm, preferably followed by precipitation as a salt (preferably PVAm-HCl) and desaltation, e. g. by dialysis. This method is

suitable for producing low molecular weight PVAm of suitable purity.

Preferably the molar mass of the PVAm or copolymer is less than 19,18, or less than 17 kDa. Generally, the molar mass will be at least about 10 kDa. Especially preferred molar masses are about 16 kDa and about 17.5 kDa."About"in this context preferably means +2 kDa, more preferably 1 kDa, most preferably 0. 5 kDa.

References to the molar mass of the PVAm or copolymer refer to the average molar mass in a population of molecules.

Preferably the PVAm or copolymer thereof is included the outer polymer solution and/or the coating polymer solution. Where more than one of the solutions includes PVAm or a copolymer thereof, the molar mass restriction need only apply to the polymer in one of the solutions (preferably the outer polymer solution), but may apply to more than one solution.

A preferred copolymer of PVAm is poly- (vinyl alcohol)-co- vinylamine (PVA/PVAm).

While the prior art has employed various substances as encapsulating polymers, it was not realised until now that copolymers of PVAm were suitable for use in encapsulation methods.

Accordingly, in a further aspect, the present invention provides a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane, the method comprising:

forming a droplet from an inner polymer solution comprising at least one positively or negatively charged polymer and the core material; contacting the droplet with an outer polymer solution comprising at least one polymer having an opposite charge to the polymer or polymers forming the droplet so that the polymers react to form a membrane around the droplet; and optionally coating the droplet by contacting it with a coating polymer solution; wherein at least one of the polymer solutions comprises a copolymer of polyvinylamine.

A preferred copolymer is poly- (vinyl alcohol)-co- vinylamine (PVA/PVAm), which, we have found, can be used in place of PVAm in encapsulation systems, both as an outer polymer and as a coating polymer. However, it is also proposed that other copolymers of PVAm may also be used according to this aspect.

The copolymer is preferably included in the outer polymer solution and/or, when used, the coating polymer solution.

In a further aspect, the present invention provides a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane, the method comprising: forming a droplet from an inner polymer solution comprising at least one positively or negatively charged polymer and the core material; contacting the droplet with an outer polymer solution comprising at least one polymer having an opposite charge to the polymer or polymers forming the

droplet so that the polymers react to form a membrane around the droplet; and optionally coating the droplet by contacting it with a coating polymer solution; wherein the outer polymer solution and/or the coating polymer solution comprises at least two polymers.

This method is distinct from certain prior art methods which sequentially contact the droplet with different outer polymer solutions, in that the different polymers are present in the same solution.

Preferably, the at least two polymers include PVAm (or a copolymer thereof) and another polymer. A particularly preferred such other polymer is PCMG (polymethyl-co- guanidine). The PVAm or copolymer thereof may have molar mass less than 20 kDa.

The at least two polymers can be in any suitable proportion, according to the desired properties of the resultant capsule. Preferred ratios (by weight) are between 1: 10 and 10: 1. For PVAm (or PVAm copolymer) and PCMG, especially preferred ratios are between 5: 1 and 1: 5, more preferably between 3: 1 and 1: 3, more preferably between 3: 1 and about 1: 1, more preferably about 7: 3 and still more preferably about 1: 1.

It has been found that a mixture of polymers can impart surprising beneficial properties on the resultant capsule. For example, the use of a mixture of PCMG and PVAm in the outer polymer solution leads to capsules having comparable or only slightly lower mechanical

stability and higher permeability, compared to the use of PCMG alone in the outer polymer solution.

Preferably, at least the outer polymer solution comprises at least two polymers. In such embodiments, the coating step may be omitted. A preferred contact time between the droplet and such an outer polymer solution is about 3 minutes.

In a further aspect, the present invention provides a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane, the method comprising: forming a droplet from an inner polymer solution comprising at least one positively or negatively charged polymer and the core material ; contacting the droplet with an outer polymer solution comprising at least one polymer having an opposite charge to the polymer or polymers forming the droplet so that the polymers react to form a membrane around the droplet; and optionally coating the droplet by contacting it with a coating polymer solution; wherein the droplet is subjected to a gelation step prior to contacting it with the outer polymer solution and wherein the outer polymer solution comprises PMCG and/or a copolymer of PVAm.

The use of a two-step method allows the production of small capsules having a diameter of down to 300 pm.

Where capsule diameter is small (e. g. below 1 mm, 0.8 mm, 0.6 mm or 0.5 mm), PEG (polyethylene glycol) may be added to the gelation solution to prevent floating of the

gelled capsules on the gelation solution. Low molar weight PEG is preferred, e. g. 1 kDa PEG.

The gelation step preferably comprises contacting the droplet with a solution of a multivalent cation, preferably a solution of Ca2+ ions, more preferably a CaCl2 solution. While CaCl2 concentrations of about 1 wt % are generally used to bring about gelation of the droplet (whether in a separate step or simultaneously with membrane formation), the concentration of CaCl2 (or equivalent free ion concentration) may be raised above 1 wt % in the manufacture of small capsules, preferably to at least 1.2 %, more preferably at least 1.3 or 1.4 most preferably to at least 1.5 %.

In a further aspect, the present invention provides a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane, the method comprising: forming a droplet from an inner polymer solution comprising at least one positively or negatively charged polymer and the core material; contacting the droplet with an outer polymer solution comprising at least one polymer having an opposite charge to the polymer or polymers forming the droplet so that the polymers react to form a membrane around the droplet; and optionally coating the droplet by contacting it with a coating polymer solution; wherein the droplet is subject to a gelation step simultaneously with contacting the droplet with the outer polymer solution and wherein the outer polymer solution comprises polyvinylamine or a copolymer thereof.

Again, a preferred copolymer of PVAm is PVA/PVAm.

Known encapsulation methods using PVAm as the outer polymer all comprise a separate gelation step.

The outer polymer solution preferably includes a polyvalent cation, more preferably polyvalent Ca2+ ions, more preferably a solution of CaCl2. Preferred concentrations are as indicated previously.

The various aspects of the invention, singly or in combination, provide the following advantages: the ability to control the permeability of the capsules (and hence their suitability for different uses) in the range of <10 to >110 kDa; the ability to produce capsules having wide range of mechanical stabilities (and, again, hence their suitability for different uses), from <40 to >200 capsule ; and the ability to simultaneously control the permeability and stability of capsules ranging in size from 0.4 to 3 mm diameter, by controlling the parameters which define the membrane formation.

In the present invention, the inner polymer solution preferably comprises alginate (alg) (a polyanion). It . may be used in the absence of other polyanions, preferably at a concentration of over 1.0 and less than 1.8%, more preferably 1.2 0.2 (more preferably 0.15 or 0.1) wt %. In the absence of other polyanions, the alg is preferably in solution in NaCl, typically NaCl at about 0.9 wt %.

Alternatively, alg may be used in combination with cellulose sulphate (CeS), preferably at a weight ratio of between 1: 2 and 2: 1 alg: CeS, more preferably at a weight ratio of about 1: 1.

Typically, the polyanion (e. g. alg and/or CeS) may have a molar weight in the range 100-1000 kDa, more preferably in the range 100-500 kDa.

However, other polyanions such as carrageenan or carboxymethylcellulose may be used in place of or in addition to alg and/or CeS.

Except where otherwise indicated, preferred outer polymer solutions comprise PMCG, optionally in combination with PVAm or a copolymer thereof (preferably PVA/PVAm). The droplet is preferably contacted with such outer polymer solutions for about 3 minutes. In certain embodiments, however, PVA or a copolymer thereof is used without PMCG.

Preferred coating polymer solutions, where used, comprise PVAm or a copolymer thereof (a preferred copolymer being PVA/PVAm). Preferred durations for contact between the droplet and the coating polymer solution are less than 3 minutes, more preferably less than 2 minutes, most preferably about 1 minutes ("about"meaning 30 seconds, more preferably 15 seconds).

Generally, the reaction time between the droplet and the outer polymer solution (and optionally a coating polymer solution) will be chosen to achieve the desired capsule characteristics. In particular, the present invention

provides a method comprising selecting a desired permeability and/or mechanical stability for a capsule, preparing a capsule from an inner polyanion solution (preferably alg, optionally with CeS) and an outer polycation solution (preferably PMCG) and contacting the droplet with a coating polymer solution (preferably of PVAm or a copolymer thereof) for a sufficient time to achieve the desired permeability and/or mechanical stability. A surprising relationship between these properties and reaction time has been discovered, as indicated elsewhere herein.

However, in one embodiment particularly preferred for mechanical stability, the inner polymer solution comprises alginate in the absence of CeS, the outer polymer solution comprises PVAm or a copolymer thereof (preferably PVA/PVAm) and no coating solution is used.

Copolymers of PVAm may have different proportions of PVAm, preferably at least 5 wt%, more preferably at least 50%, more preferably at least 75%, more preferably at least 85%, and preferably up to 99%, more preferably up to 98%, 95% or 90%. A preferred PVA/PVAm copolymer is obtained using 10% vinyl acetate and 90% N- vinylformamide.

In some embodiments, the polymer solutions may include low molecular weight monovalent metal salts (e. g. Na+ or K+), or agents to promote isotonicity (e. g. mannitol) or buffer solutions. In the encapsulation of biological materials (cells, bacteria etc), these and other additives can be included to help to maintain the osmotic

pressure between the cells and polymer solutions in equilibrium.

For example, the solutions preferably contain physiological concentration of NaCl (in addition to other species as appropriate), i. e. around 0.9 wt% NaCl.

The method, particularly when used to encapsulate biologically active materials, e. g. mammalian cells, may employ physiological pH conditions with a pH between 6.5 and 8.5. This, like the use of physiological NaCl concentration, may promote maintenance of biological activity. A preferred physiological pH range is about pH 6.6 to 7.5 ("about"meaning 0. 05), more preferably about 6.8 to 7.4. However, pH in excess of 7.5, >7.6, >7.7, >7.8, or even 8.0 may also be employed. Previous methods have not achieved encapsulation using such high pH values. pH values greater than 7.5 may in particular be preferred for encapsulation methods employing PVAm or a copolymer thereof when reduced permeability is desirable.

In contrast to PMCG, the stoichiometry of the complexation reaction of PVAm or its copolymers with cellulose sulphate and/or alginate is dependent on the degree of protonation of PVAm. PVAm cannot be fully protonated at physiological pH (Sumaru et al (1996) J.

Phys. Chem. 100,9000); the maximum degree of protonation was 82% and the lower the pH, the higher the degree of protonation. As pH increases e. g. above physiological ones, the degree of protonation of PVAm (or its copolymers) is reduced, resulting in smaller microcapsule pores, and lower permeability.

Conveniently, the capsules can be produced by dropping one of the polymer solutions into a bath of the other.

Preferably, the concentration of the polymers is in the

range 0.1 to 5.0 wt%, more preferably in the range 0.5 to 2.0-wt%. Particularly preferred concentrations for PVAm or copolymers thereof are at least 1.0 wt%. The droplets can be formed by passing one of the polymers through a capillary, by extrusion with gas or a liquid, using a spinning disk or by electrostatic generation.

Preferably, the droplets are formed from the polyanion (e. g. alg, CeS), with the polycation (e. g. PVAm, PVA/PVAm and/or PCMG) being included in the outer polymer solution.

At least one step of the method may be carried out at a temperature of more than 25°C, more preferably at least 30°C, most preferably about 37°C. The density of the solution is reduced with increased temperature, so the droplets have less of a tendency to float on the surface of the solution (which can lead to uneven capsule formation). This is particularly preferred when producing small capsules (e. g. as defined elsewhere herein). Preferably this applies at least to the gelation step (which may be separate from or combined with the step of contacting the droplet with the outer polymer solution).

Preferably, the capsules produced using the method have a diameter between 50 pm and 5.0 mm. The diameter of the capsules can be controlled by varying the flow rate of the inner polymer solution or by controlling physical parameters such as the applied voltage, if electrostatic droplet generation is used, or the fluid stripping velocity, if the capsules are made by air/fluid stripping.

The thickness of the membrane around the capsules is typically from 2 to 15% of the capsule diameter, and more preferably from 5 to 10%. Preferably, the membrane is permeable or semi-permeable. The encapsulation method can also allow the molecular weight cut off of the membrane to be controlled between about 1 to about 300 kDa, particularly between about 10 and >110 kDa, by selecting the appropriate parameters, including polymers, polymer concentrations, salt concentrations, pH, temperature and reaction time.

Conveniently, the core material can be encapsulated by mixing it with the polymer solution which is formed into droplets. The method is particularly useful for encapsulating biological or biologically active materials, including cells, bacteria, enzymes, seeds, antibodies, drugs, cytokines or hormones, as it can be carried out biologically compatible pH, preferably at a pH greater than about 6.5, preferably at a pH between about 6.6 and 7.5, more preferably at a pH between about 6.8 and 7.4. However, pH in excess of 7.5, >7.6, >7.7, >7.8, or even 8.0 may also be employed, particularly in steps involving PVAm or a copolymer thereof. Previous methods have not achieved encapsulation using such high pH values.

In a further aspect, the present invention provides a method which comprises following the encapsulation of a core material according to one or more of the above aspects, the further step of formulating the capsules in a composition or other product. The capsules may be formulated with pharmaceutically acceptable ingredients, e. g. pharmaceutically acceptable carriers, adjuvants,

excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, and/or surfactants (e. g., wetting agents).

Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990 ; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

In a further aspect, the present invention provides a polymeric capsule comprising a semipermeable membrane encapsulating a core material produced or as producible according to one or more of the above aspects.

In a further aspect, the present invention provides a polymeric capsule comprising a semipermeable membrane encapsulating a core material produced or producible according to one or more of the above aspects, for use in a method of medical treatment.

As used herein, weight % figures, and weight ratios are expressed as grams per 100 ml of solution, i. e. 1% represents 1 g in 100 ml of solution. The term"about"in relation to weight % or weight ratio values implies 20%, more preferably 15%, most preferably 10% of the figure given, unless otherwise indicated.

Embodiments of the present invention will now be described by way of example and not limitation.

Materials and Methods Synthesis of Polyvinylamine (PVAm) To obtain well-defined, defect-free, PVAm we used poly (N-vinylformamide) as the precursor. The synthesis of PVAm involved 2 steps: (1) radical polymerization of N- vinylformamide to poly (N-vinylformamide); and (2) stoichiometric base hydrolysis of poly (N-vinylformamide) to PVAm and then precipitated as a PVAmHC1 salt. The hydrolysis was carried out under basic conditions in order to achieve complete conversion.

For radical polymerization, 4 g of N-vinylformamide was dissolved in methanol and 0.38 g of initiator (AIBN) was used, with 2-mercaptobutanol as a chain transfer agent for regulation of molar mass. The polymerization was carried out at 60°C for 24 h. For stoichiometric base hydrolysis, the poly (N-vinylformamide) was dissolved in NaOH 1.7 M and heated at 80°C for 6 h.

The polymer solution was then acidified by concentrated HC1 solution. Precipitated PVAm. HCl was washed with methanol and dried in vacuum. The product was desalted by membrane dialysis with Milli-Q (Millipore, Bedford, Warrington, PA) grade water. The products of each step were confirmed by IR measurements.

Synthesis of Poly- (vinyl alcohol)-co-vinylamine (PVA/PVAm) The synthesis of PVA/PVAm also required two steps: (1) radical polymerization of N-vinylformamide and vinylacetate to poly- (N-vinylformamide)-co-acetate ; (2) stoichiometric base hydrolysis of poly- (N-

vinylformamide)-co-acetate to PVA/PVAm. The procedure was as set out for PVAm, using different molar ratios between the two monomers, N-vinylformamide and vinylacetate, respectively.

Capsule Production The standard production method and its variations are listed in Table 1. The following materials were used: alginate (alg)-Keltone HV (Kelco Chemical Company, UK); cellulose sulphate (CeS) (Batch A006986201, Acros Organics, Geel, Belgium) ; polyvinylamine (PVAm) (synthesised by Gabriela Grigorescu) ; poly- (vinyl alchohol)-co-vinvylamine (PVA/PVAm) (synthesised by Gabriela Grigorescu) ; poly-methylene-co-guanidine (PMCG) (Scientific Polymer Product, Ontario, NY).

Table 1. Production Method for Quaternary Capsules Solution Example 1 Variations Polyanion 0.6% Keltone HV, 0.6% 1.2% Keltone HV in CeS in PBS 0.9% NaCl Polycation PMCG 1.2% in 0.9% 1-4% PVAm, 10-50 NaCl, pH 7.4 kDa in 0.9% Nazi ; 1-4% PVA/PVAm in 0.9% NaCl Washing Bath 0.9% NaCl- Coating 1% PVAm in 0.9% 0. 5%-1% PVAm, 16-20 NaCl, pH 7.4,16 kDa kDa in 0.9% Nazi ; 1-4% PVA/PVAm in 0.9% NaCl

Production Example 1 Variations step Atomization With air-stripping- Gelation 10 min. 1-10 min. Washing 1 min- Membrane 3 min 10s-10 min Formation Washing 1-3 min Coating 3 min lOs-30 min The mechanical stability of microcapsules was determined with the Texture Analyzer (TA-2xi, Stable Micro Systems, Godalming, UK). The experiment was done in series of twenty. Results are expressed as mean standard deviation.

The determination of the molar mass cut-off was realised with a size exclusion chromatograph, equipped with Shodex protein KW-G and KW-804 columns, and an refractive index detector, using polydisperse dextran mixture of known molecular size.

Example 1 PMCG/Alg/CeS/CaCl2 (the"quaternary"system used in our laboratory) microcapsules have high permeability (110-200 kDa). In order to keep the permeability in the range accessible only to oxygen, nutrients and secretory products of encapsulated cells, but not for antibodies

(the ideal permeability should be around 110 kDa), we coated quaternary capsules with PVAm.

Capsules of typical diameters of 1.5 mm were produced.

The inner polymer solution, 0.6% Alg and 0.6% CeS in 0.9% NaCl, was dropped with an air-stripping system into the gelation bath consisting of 1% CaCl2. After a washing step in 0.9% NaCl the beads were transferred into the outer polymer solution, 1.2% PMCG in 0.9% NaCl. The capsules were coated by transferring them into a 1% PVAm solution. The capsules were washed three times in 0.9% NaCl.

Table 2. Properties of the capsules of Example 1 Reaction pHPVAm molarPermeability Mechanical Time mass (kDa) (kDa) Stability (min) (g/capsule) 1 7. 4 16 <110 12531 3 7. 4 16 <40 8226 1 7.4 17.5 <110 135.85~38 1 7. 4 32 <110 167. 99. 78 Surprisingly, longer reaction time led to increased permeability and reduced strength. This is in contrast to systems employing PMCG alone or PVAm alone, in which mechnical stability is directly proportional to reaction time of the outer polymer (i. e. PMCG or PVAm) with the inner polymer. It is thought that when the quaternary polymer is coated, there is an interaction between the PMCG and PVAm (i. e. the outer and coating polymers), the interaction being dependent on reaction time.

Example 2 Capsules were produced as in Example 1, but they were coated for 1 min with 0.5% PVAm solution. Capsules of typical diameters of 1.8 mm were produced.

Table 3. Properties of the capsules of Example 2 Reaction pH PVAm molar Permeability Mechanical Time mass (kDa) (kDa) Stability (min) (g/capsule) 1 7.4 17.5 >110 98.4~18. 6 1 7. 4 32 >110 92. 412 1 7. 4 45 >110 93. 8528. 5 1 7. 4 60 >110 56. 49. 3 Surprisingly, reduced molar mass of PVAm led to increased mechanical stability. In comparison with Example 1, incubation for the same length of time in a weaker PVAm solution led to a less mechanically stable, more permeable capsule.

Example 3 Capsules were produced as in Example 1 but the diameter of the capsules were decreased to 400 pm and the reaction times were adjusted to 10 sec. In order to prevent capsule floating in the gelation bath the calcium concentration was increased to 1. 5% and 1% polyethyleneglycol (Mw 1000 Da) was added and the temperature was increased from 20-25 to 37°C.

Table 4. Properties of the capsules of Example 3 Reaction pH PVAm molar Permeability Mechanical Time mass (kDa) (kDa) Stability (s) (g/capsule) 10 7. 4 16 <110 82. 5 The resultant capsules were permeable and of low strength.

Example 4 PMCG/PVAm/Alg/CeS/CaCla microcapsules were prepared as in Example 1 but the outer polymer was a mixture of PMCG and PVAm and no additional coating step was performed. The mixture had different ratios between PMCG and PVAm, the total concentration of both polycations in 0.9% NaCl being 1.2%.

Table 5. Properties of the capsules of Example 4 Ratio Reaction pH PVAm molar Perm-Mechanical PMCG: Time mass (kDa) eability Stability PVAm (min) (kDa) (g/capsule) 7: 3 3 7. 4 16 <110 9737 1: 1 3 7. 4 16 <110 7625 The resultant capsules were both strong and permeable.

Example 5 PVAm/alg/CaClz microcapsules were prepared with a simultaneous gelation and membrane formation step. 1.2% alginate was dropped into 1% PVAm and 1% CaCl2.

Table 6. Properties of the capsules of Example 5 Reaction pH PVAm molar Permeability Mechanical Time mass (kDa) (kDa) Stability (min) (grams/capsule) 10 7. 1 16 <10 209103 The resultant capsules were very strong with low permeability.

Example 6 PVA/PVAm/alg/CaCl2 microcapsules: copolymers of vinyl alcohol and vinylamine, with different percentages of vinyl alcohol represent another class of polycations which can be used to produce microcapsules. This copolymer can be applied as substitute of PVAm in Examples 1-5. The following example is for a copolymer obtained using 10% vinyl acetate and 90% N-vinylformamide as monomers.

Table 7: Properties of the capsules of Example 6 Reaction pH PVA/PVAm Permeability Mechanical Time molar mass (kDa) Stability (min) (kDa) (grams/capsule) 5 7. 4 20 <110 12627. 5 As compared with the capsules of Example 5, the permeability is increased and the mechanical stability decreased. The mechanical stability is still, however, greater than that obtained using the quaternary system.

Applications The method described herein can be applied to the encapsulation of a wide variety of materials. The method is particularly relevant to the encapsulation of biological or biologically active materials. The encapsulation can be used to physically protect the encapsulated material, e. g. from mechanical or environmental, or where the capsules are for implantation into a human or animal, to help to prevent or reduce an immune rejection to the material. Particularly preferred applications include: (a) Encapsulation of living animals and plant cells, for example cells for implantation in a patient, e. g. to replace cells lost through disease, cells which are transformed to express a therapeutically useful polypeptide, artificial cells, and bioartificial organs.

(b) Encapsulation of bacteria/nutrients, particularly for use in the food industry, as all components utilized in the method can be of food grade.

(c) Encapsulation of vitamins, drugs, polypeptides and enzymes, particularly for use in the cosmetics and pharmaceutical industries References The references cited herein are all expressly incorporated by reference.

Willaert & Baron, Rev. Chem. Eng., 12: 5,1996.

Jen et al, Biotechnol. Bioeng., 50: 357,1996.

Colton, Trends Biotechnol., 14: 158,1996.

Morris, Trends Biotechnol., 14: 163,1996.

Hubbell, Trends Poly. Sci., 2: 20,1994.

Sefton & Stevenson, Adv. Polym. Sci., 107: 143,1993.

Baldwin & Saltzman, in Fundamentals of Animal Cell