WO2008134807A1 - Method for manufacturing a porous polymer matrix - Google Patents

Abstract

Disclosed herein are methods for manufacturing a porous polymer matrix, optionally containing a bioactive material. The methods comprise the steps of providing a solution comprising a polymer dissolved in a solvent, and causing the polymer to precipitate from the solvent to form a polymer matrix.

Description

METHOD POR MANUFACTURING A POROUS POLYMER MATRIX

FIELD OF THE INVENTION

The invention relates to a method for manufacturing a porous polymer matrix. The porous polymer matrices of the invention may be used in the medical field for various purposes including the controlled release of bioactive molecules or as scaffold systems to support cell growth and development .

BACKGROUND ART

Microporous polymer matrices can be used to manufacture implantable, insertable or topical matrix-type or depot devices for controlled release of bioactive molecules such as contraceptive steroids, vaccines and microbicides.

They may also be used for manufacturing three dimensional (3-D) scaffold systems to support cell growth and development in tissue engineering.

The release of hydrσphilic species from matrix-type or depot devices can be influenced by ^vpore-type' diffusion mechanisms. The ability to prepare such devices with control over the pore structure, connectivity and tortuosity would therefore be useful for fine tuning release kinetics- Control over the pore structure, connectivity and tortuosity would also be advantageous in the preparation of ^• microporous polymer matrices for use as scaffold systems to support cell growth in order to optimise cell-scaffold interaction.

Various techniques have been employed to prepare polymer matrices with controlled pore size, shape and connectivity. Such techniques include solvent- casting/particle leaching, thermally induced phase separation, gel casting, solid free form fabrication such as selective laser sintering, and super critical fluid processing. Micro/macroporous poly (L-lactide) (PLA) implants have also been produced by direct machining of 50θμm channels in a block of microporous, gel cast material .

A precipitation casting technique for producing microporous, polycaprolactone (PCL) matrices is described in WQ 01/38428. The process described in that document involves the gradual crystallization of the polymer due to solvent extraction across a PCL membrane formed in-situ at a solution/non- solvent interface.

The process described in WO 01/38428 has been used to form polymer matrices for controlled delivery of a low molecular weight hydrophobic drug (progesterone) or a hydrophilic antibiotic (gentamicin sulphate) with retained bioactivity in vitro. In the former case, steroid loadings up to 32% w/w were achieved and release rates of 1,5-7 μg/mg matrix/day for 11 days were obtained by variation of steroid content and matrix density.

It would be advantageous to provide alternate methods by which to manufacture a porous polymer matrix.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a method for manufacturing a porous polymer matrix (preferably a microporouB polymer matrix) , the method comprising the steps of;

(a) providing a solution comprising a polymer dissolved in a solvent, and

(b) causing the polymer to rapidly precipitate from the solvent to form a polymer matrix.

Typically, the method further comprises the further step (c) of removing the solvent from the polymer matrix. This is particularly important if the polymer matrix is to be used in the medical field. In some embodiments, the solvent is removed from the polymer matrix by drying the matrix, typically in air under ambient conditions. ϊn some embodiments, the step of removing the solvent from the polymer matrix comprises immersing the polymer matrix in a non-solvent in which the polymer is insoluble, followed by drying the polymer matrix.

In some embodiments, the solution further comprises a suspension of a particulate, and wherein the particulate is dispersed throughout the polymer matrix.

In some embodiments, the solution further comprises a bioactive molecule dissolved m the solvent with the polymer, whereby the bioactive molecule is dispersed throughout the polymer matrix. In such embodiments, the bioactive molecule becomes incorporated into the polymer matrix when the polymer precipitates from the solvent.

In a second aspect,, the present invention provides a . method for manufacturing a porous polymer matrix (preferably a micrσporous polymer matrix) , the method comprising the steps of : (a) providing a solution comprising a polymer dissolved in a solvent, the solution further comprising a particulate suspended in the solution; and

(b) causing the polymer to precipitate from the solvent to form a polymer matrix, wherein the particulate is dispersed throughout the polymer matrix.

Typically, the method further comprises the further step (c) of removing the solvent from the polymer matrix.

In some embodiments, the solvent is removed from the polymer matrix by drying the matrix, typically in air under ambient conditions. . In some embodiments, the step of removing the solvent from the polymer matrix comprises immersing the polymer matrix in a non- solvent in which the polymer is insoluble, followed by drying the polymer matrix.

In some embodiments, the solution further comprises a bioactive molecule dissolved in the solvent with the polymer, whereby the bioactive molecule is dispersed throughout the polymer matrix. In such embodiments, the bioactive tnolecule becomes incorporated into the polymer matrix when the polymer precipitates from the solvent .

The inventors have found that when a polymer matrix in which a particulate and/or bioactive molecule is dispersed throughout the polymer matrix is formed by the methods of the present invention, relatively high loadings of the particulate and/or bioactive molecule in the polymer matrix can be achieved. Typically, the particulate and/or bioactive molecule is evenly or substantially evenly distributed throughout the polymer matrix.

In some embodiments, the particulate is soluble in the non-solvent. In such embodiments, immersing the polymer matrix in the non-solvent can result in the particulate dissolving in the non-solvent and being removed from the polymer matrix, leaving behind a substantially "empty" matrix with pores of a similar size to the particulate. Thus, as the polymer matrix has an inherent porosity typically of about 5-lOμm (the primary pore structure) , incorporation of particulates of a specific shape and size range in the solution allows the formation of a matrix with a defined surface and internal pore architecture (the secondary pore structure) .

In some embodiments, the particulate is particles of a natural polymer such as a protein (e.g. albumin, collagen, gelatin) , an enzyme (such as lyεozyme) , alginate, chitosan, a polysaccharide (e.g. inulin, search, dextran) , a cellulose derivative (e.g. methylcellulose) , a sugar (e.g. lactose), an oligosaccharide ester derivative, a synthetic polymer such as polyethylene glycol (PEG) , polyethylene oxide (PEO), a copolymer of poly (ethylene oxide) -poly (propylene oxide) (e.g. Poloxamer, Pluronic, Tetronic copolymers) , polyvinylpyrrolidone (PVP) or polyvinylalcohol, or a water soluble inorganic material (a salt) . These particulates are soluble in water and other polar solvents, and thus water or other polar solvents can be used to remove such particulates from the polymer matrix. For example, water or another polar solvent could be used as the non-solvent to remove such particulates from the polymer matrix together wich the solvent. Alternatively, water or another polar solvent can be used to remove such particulates from the polymer matrix as a separate step to the removal of the solvent from the polymer matrix.

In some embodiments, the particulate is a bioactive particulate. In such embodiments, it will typically be desirable to maintain the bioactive particulate in the polymer matrix and Che bioactive particulate will either not be soluble (or only slightly soluble) in the non- solvent, or the polymer matrix will not be immersed in a non- solvent .

In some embodiments, the bioactive molecule or the bioactive particulate is a corticosteroid hormone, an antiviral, a microbiocide, a vaccine, a peptide/ polypeptide growth factor, an antibiotic, an anti -cancer drug, melatonine, an anticoagulant (e.g. low molecular weight heparin), an anti- inflammatory, an antifungal, or a miotic. The polymer is typically caused to precipitate in a mould such that the polymer matrix has a. predetermined three dimensional shape. The polymer matrix can therefore be formed having an appropriate shape for use as a topical matrix- type or depot device for controlled release of a particulate and/or bioactive molecule, or having an appropriate shape to provide a 3D scaffold system to support cell growth and development in tissue engineering. Advantageously, the methods of the present invention can be used to produce a thick section (i.e. 3D) polymer matrix in which a particulate and/or bioactive material is substantially evenly distributed throughout the entire matrix.

Typically, the porous polymer matrix is a microporous polymer matrix. As used herein the term "microporous polymer matrix" will be understood to mean a polymer matrix having micron-sized pores - i.e. pores of less than about lOOOμm diameter. Typically, the porous polymer matrix has an average pore size of less than about 500μm diameter .

Materials having micron-sized pores can advantageously be used in numerous applications in the medical field including as biodegradable polymer matrices, depot-type devices and tissue engineering. Advantageously, biodegradable polymers having a porous structure have improved fluid ingress, which increases the surface area of the material and results in a higher rate of resorption of the polymer. Furthermore, the release of hydrophilic drugs from depot-type devices in which drug particles are dispersed in a porous polymeric matrix is influenced by ''pore-type' diffusion mechanisms during which drug εolubilisation is followed by diffusion through the network of fluid- filled pores and channels. In. addition, the surface chemistry and microstructure of tissue engineering scaffolds have been shown to influence cell distribution and morphology and, importantly, cell proliferation and differentiation In particular, the pore structure controls the key processes of nutrient supply to cells, metabolite dispersal, cell signalling and the type and extent of tissue ingrowth.

Typically, the polymer is a semi -crystalline polymer, and precipitation occurs when the polymer crystallises out of the solvent .

In some embodiments, a blend of polymers can be dissolved in the solvent in order to provide a polymer matrix comprising a blend of polymers.

Typically, the polymer is polycaprolactone [PCL] , poly (L- lactide) fPLA] or high L-lactide-containing polydactide co-glycolide) [PLG] copolymers which tend to crystallise from solution, polyglycolide [PGAl or oligosaccharide ester derivatives, or a combination of two or more of these polymers.

The solvent is typically an organic solvent. Depending on the polymer (s) used, a suitable solvent for use in the methods of the present invention may be acetone, ethyl acetate, xylene, toluene, dimethyl sulfoxide (DMSO) or dimethyl fσrmamide (DMF) . Suitable non-solvents include water and alcohols (e.g. methanol and ethanoZ) .

Typically, the polymer is caused to precipitate by cooling the solution, for example, to a temperature of less than -5°C, more typically less than -25°C. The inventors have found that by cooling polymer solutions to such low temperatures, the polymer rapidly precipitates from the polymer solution. Typically, the polymer precipitates from the solution to form a polymer matrix in less than 10 minutes, more typically in about 2 to 5 minutes. Because of the rapid precipitation of the polymer, any particulates suspended in the polymer solution (or any bioactive molecules dissolved in the solvent) are dispersed throughout the precipitated polymer matrix. As ' will be apparent to a person skilled in the art, typically not all of the particulate (or bioactive molecule) present in the solution will be incorporated into the resultant polymer matrix. However, at least some of the particulate (or bioactive molecule) present in the solution is dispersed throughout the polymer matrix. Typically, the particulate (or bioactive molecule) is evenly or substantially evenly distributed throughout the polymer matrix.

The methods of the present invention can advantageously be used to provide a microporous polymer matrix with a relatively high loading of particulate (e.g. about 44% w/w of particulate protein) .

In contrast to the methods of the present invention, the process for preparing a polymer matrix described in

WO 01/38428 may take hours or days to form the polymer matrix. As a result of this slow formation of the polymer matrix, at least some sedimentation of any particulates in the polymer solution is likely to occur during the formation of the polymer matrix.

In a third aspect, the present invention provides a three dimensional porous polymer matrix (preferably a microporous polymer matrix) manufactured using the method of the first or second aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be described with reference to the following drawings, in which:

Figure 1 shows graphs depicting the weight distribution of lactose (upper graph) and gelatin (lower graph) particles of various size ranges in the upper, middle and lower portions of tubular shaped microporous PCL matrices containing lactose or gelatin prepared as described in Example 1 and Example 2 ;

Figure 2 shows a graph depicting the cumulative quantity of lactose released over time from the microporous PCL matrices containing lactose particles of various size ranges prepared as described in Example 1 and Example 2,-

Figure 2 shows a graph depicting the cumulative quantity of gelatin released over time from the tniσroporous PCL matrices containing gelatin particles of various size ranges prepared as described in Example l and Example 2 ;

Figure 4 shows scanning electron micrographs of (A) a PCL matrix, (B) a PCL matrix containing lactose particles (90- 125μm) , and (C) and (D) the PCL matrix of (B) after release of the lactose particles ,-

Figure 5 shows scanning electron micrographs of (A) a PCL matrix containing gelatin particles (125-250μm) , (B) the PCL matrix of (A) after release of the gelatin particles, (C) Lactose powder (90-l25μm) , and (D) gelatin powder (125-250μm) ;

Figure 6 shows internal microtomographs of (A) the polymeric phase of a PCL matrix containing lactose particles (90-125 μm) after release of the lactose particles, and (B) the pore structure of the PCL matrix after release of the lactose particles;

Figure 7 shows internal microtomographs of (A) the polymeric phase of a PCL matrix containing gelatin particles (125-250 μm) after release of the gelatin particles, and (B) the pore structure of the PCL matrix after release of the gelatin particles;

Figure 8 shows rcicroporous PCL matrices produced using a method in' accordance with the present invention and cast in the form of tubes and cylinders;

Figure 9 shows a vaginal insert produced using a method in accordance with the present invention for applications in vaccine and microbiocide delivery,- and

Figure 10 shows graphs depicting the biological activity of lysozyme following release from microporous PCL matrices containing various loadings of lysozyme and produced using a method in accordance with the present invention over the course of 8 hours (upper graph) and 21 days (lower graph) .

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION The present invention relates to methods for manufacturing porous polymer matrices, preferably microporous polymer matrices.

In the methods of the present invention, a polymer is provided dissolved in a solvent and is subsequently caused to precipitate from the solvent to form a polymer matrix.

Any polymer that is soluble in the solvent and can be caused to precipitate from the solvent to form a polymer matrix may be used. Preferred polymers are semi- crystalline polymers such as polycaprolactone [PCLl , poly (L-lactide) [PLA] or high L-lactide-eontaining poly(lactide co-glycolide) [PLG] copolymers which tend to crystallise from solution, polyglycolide [PGA] , or oligosaccharide ester derivatives.

PCL and/or PLA are especially useful polymers because they can be slowly degraded under physiological conditions (e.g. once implanted in a human or animal body) .

In some embodiments, two or more polymers may be used to impart the advantageous properties of each of the polymers to the resultant polymer matrix. For example, one or more different polymers (synthetic or natural) may be used with PCL to form a polymer blend, thereby modulating the physico-chemical properties, drug release or cell interaction of the resultant polymer matrix. The degradation characteristics of the polymer matrix could be controlled, for example, by blending PCL with poly(α- hydroxy acids) such as PLA and PLG since their degradation rates can be varied readily from several weeks to over a year by cσpolymerization, control of molecular weight, and crystallinity . Combinations of semi-crystalline and amorphous polymers may also be useful, provided the polymers co-precipitate from the solvent.

Useful, non-water soluble polymers apart from PLA and PLG include copolymers produced from lactide and non-lactide monomers such as lactones (e.g. ε-caprolactone) or ethylene glycol, PMMA, PU, copolymers containing a thermoplastic elastomer or hydrogel-forming copolymers such as poly (hydroxyethyl methacrylate) . Such polymers are used or have been investigated for use as implant materials and have advantageous properties such as proven biocompatibility (e.g. PMMA has been used in joint prosthesis and poly(hydroxyethyl methacrylate) has been used in contact lenses) and flexibility (e.g. PU, thermoplastic elastomers) .

PCL may also be used with water soluble polymers including polyethylene glycol (PEG) , polyethylene oxide (PEO) , copolymers of poly (ethylene oxide) -poly (propylene oxide) (e.g. Poloxamer, Pluxonic, Tetronic copolymers) or polyvinylpyrrolidone (PVP) to prepare polymer matrices having a water-soluble phase. Such polymer matrices may be useful for controlling drug release from the matrix.

Other materials may be added to the polymer solution to vary certain properties of the precipitated polymer matrix such as degradation rate, density, thermal, mechanical, morphological and chemical characteristics.

The polymer is provided dissolved in a solvent . The solution of the polymer dissolved in the solvent may be prepared by dissolving the polymer in the solvent, with some heating if necessary. In embodiments where the solution further comprises a bioactive molecule dissolved in the solvent, the bioactive molecule can be added to the solvent at the same time as the polymer or before or after the polymer has been dissolved in the solvent. If Che bioactive molecule is heat sensitive, the bioactive molecule is typically added to the solvent after the polymer has been dissolved.

Similarly, in embodiments where the solution further comprises a particulate suspended in the solution, the particulate can be added to the solvent at the same time as the polymer or before or after the polymer has been dissolved. The particulate may be suspended in the solution using techniques known in the art. The particulate can be dispersed throughout the solution immediately before precipitation of the polymer by vigorously stirring the solution.

Depending on the polymer (s) used, suitable solvents for use in the methods described above include acetone, ethyl acetate, xylene, toluene, dimethyl sulfoxide (DMSO) and dimethyl formaraide (DMF) . If necessary, combinations of miscible solvents may be used. The suitability of a particular solvent for use in the methods may be readily determined by a person skilled in the art. It may be necessary for the concentration of the polymer in the solvent to be above a certain level. Optimisation of the polymer concentration may be readily carried out.

For example, it would be apparent to those skilled in the art that a certain polymer concentration and molecular weight may be required because sufficient chain entanglements are necessary in the polymer solution and subsequently in the precipitated polymer matrix to provide mechanical cohesion of the matrix.

For example, in embodiments where PCL is used to form the polymer matrix, the concentration of the polymer dissolved in the solution may be in the range of l to 50% w/v, preferably, 10 to 20% w/v.

In the method of the first aspect, the polymer is caused to rapidly precipitate from the solvent to form a polymer matrix. In the method of the second aspect, the polymer is caused to precipitate from the solvent to form a polymer matrix in a manner such that the particulate is dispersed throughout the polymer matrix. Typically, the polymer is caused to precipitate from the solvent to form the polymer matrix by rapidly cooling the solution (e.g. from an ambient temperature) to a temperature below 0⁰C, preferably below -25°c. For example, the solution may be cooled by immersing it in dry ice (-78.5⁰C). Measurements by the inventors have shown that immersing a mould containing a polymer solution in dry ice reduces the temperature of the solution to ca. -25°C after 2 minutes and to σa. -50⁰C after 5 minutes.

In some embodiments, the polymer is caused to precipitate from the solvent to form a polymer matrix by cooling the solution to a temperature below about 0⁰C in less than 5 minutes, preferably less than 2 minutes. In some embodiments, the polymer is caused to precipitate from the solvent to form a polymer matrix by cooling the solution to a temperature below about -5°C in less than 5 minutes, preferably less than 2 minutes.

In some embodiments, the polymer is caused to precipitate from the solvent to form a polymer matrix by cooling the solution to a temperature below about -25°C in leas than 5 minutes, preferably less than 2 minutes.

In some embodiments, the polymer is caused to precipitate from the solvent to form a polymer matrix by cooling the solution to a temperature below about -50⁰C in less than 10 minutes, preferably less than 5 minutes, more preferably less than 2 minutes -

Typically, the solution is cooled to a temperature above the freezing point of the solvent . other methods to induce or assist precipitation could also be used, such as by adding a "seed crystal" to the solution or by adding a precipitant to the solution.

When the polymer (or polymer blend) precipitates from the solvent, a polymer matrix is formed. Such polymer matrices will have an intrinsic porosity, depending on the polymer. For example, if PCL is used, the resultant polymer matrix will have a microporosity of about 5-10μm.

The invention provides a number of routes by which a bioactive material may be incorporated into the polymer matrix. For example, in embodiments where a bioactive molecule is also dissolved in the solvent with the polymer, the bioactive molecule becomes incorporated into the polymer matrix and is typically substantially evenly distributed throughout the matrix. The bioactive molecule may, for example, become entrapped when the polymer precipitates (molecular dispersion) , or the dissolved bioactive molecule may precipitate separately on exposure to the non- solvent and be entrapped as particulates within the polymer matrix.

The bioactive molecule (e.g. a hydrophobic steroid) may, for example, be released from the polymer matrix by diffusing through the polymer phase (partition-type mechanism) and fluid-filled pores (diffusion-type mechanism). For example, hydrophilic molecules (e.g. gentamicin sulphate antibiotics) diffuse through fluid- filled pores. Thus the porosity and connectivity throughout the polymer matrix affects the rate of release of such molecules from the polymer matrix.

Similarly, in embodiments where a particulate (which may^¬ be a bioactive particulate that is insoluble in the solvent) is suspended in. the polymer solution, when the polymer is caused to precipitate, the particulate becomes incorporated into the polymer matrix and is typically substantially evenly distributed throughout the matrix.

The methods of the invention are particularly useful for incorporating a particulate in the polymer matrix at high loading and with good distribution. High loadings and good distribution of the particulate can be achieved using the present invention as rapid precipitation of the polymer minimises sedimentation o£ the particulate during the precipitation of the polymer. Particulate protein loading of about 44% w/w can be achieved using the methods of the present invention.

Examples of useful particulates include particles of natural materials such as gelatin and lactose. Others include particles comprising polysaccharides (inulin, starch, dextran, cellulose and derivatives) , sugar spheres, particles comprising bioactives such as antibiotics, spray dried therapeutic polypeptides such as growth factors, proteins such aε vaccine antigens and decalcified freeze dried bone (DPDB) .

Other particulates include bioceramics such as hydroxyapatite and tricalcium phosphate_/ which have been ^■widely used for production of bone substitutes, carbon, calcium carbonate, bioactive ceramics 'Bioglass' and magnetic particles.

For example, inclusion of particulates of bioceramics (e.g. hydroxyapatite or tricalcium phosphate) in the polymer matrix would be expected to increase the hardness and density of the matrix, promote adhesion of bone cells to the matrix and osteoconduction. inclusion of a water soluble particulate of gelatin or polyethylene glycol (PEG) would be expected to increase the hydrophilicity of the matrix and provide pathways for release of incorporated bioactives.

In some embodiments, the particulate is a synthetic polymer. Examples of particulate synthetic polymers include PMMA powders such aε those used in bone cements, polyesters, polyamides, biodegradable polymers (PLA, PGA, PLG) , polyphosphazines, polyorthoesters, polyanhydrides and oligosaccharide ester derivatives.

ϊn some embodiments, particles of the polyesters PLA, PGA, PLG can be suspended as particulates in the polymer (e.g. PCL) solution prior to precipitation (e.g. by rapid cooling) to form the polymer matrix. in preferred embodiments, the selected polyester particles will not dissolve in the non-solvent (e.g. methanol), and would therefore be retained in the matrix initially. Biodegradable polymer particles (e.g. PLA, PLGA, polyorthoester) would resorb from the matrix in vivo over time. Non-degradable polymer particles (e.g. PMMA) would remain in the matrix, but could be useful for bone cell attachment .

Other particulates include 'chopped fibre' particulates such as alumina, carbon and synthetic polymers such as polyester 'Dacron' , polyamides, polyglycolic acid (PGA) and polydioxanone (PDS) . Discontinuous, electrσspun, natural and synthetic fibres could also be usefully incorporated in the matrix.

In some embodiments, the size of the particulate will be in the range of from about 10μm to about 500/xm, for example, in the range of from about 45μm to about 250/xτn-

In some embodiments, after formation of the polymer matrix, the polymer matrix is removed from the remaining solvent .

Typically, the methods of the present invention comprise the step of removing at least some of the residual solvent from the polymer matrix. This is particularly important where the polymer matrix is to be used in the medical field as many organic solvents, such as acetone, are toxic and can only be present in devices for medical use in less than parts per million. In some embodiments, the solvent is removed by immersing the precipitated polymer matrix in a non-solvent in which the polymer is insoluble, followed by drying the polymer matrix.

The non-solvent may be any solvent in which the polymer is substantially insoluble but which is miεcible with the solvent. A preferred non-solvent is methanol- Alternative non-solvents may be other alcohols or water or mixtures of non-solvents.

When the polymer matrix is formed in a mould, the polymer matrix may be immersed in the non- solvent before or after the polymer matrix is removed from the mould.

In certain embodiments in which the particulate (e.g. lactose) is soluble in the non-solvent (e.g. water), immersing the polymer matrix in the non-solvent will extract substantially all of the particulate from within the matrix, which provides a substantially "empty" matrix useful for tissue engineering, for example.

However, in other embodiments, the particulate is not soluble (or is only slightly soluble) in the non-solvent and the non-solvent is used merely to remove the solvent from the polymer matrix. In a subsequent step, if a substantially "empty" matrix is desired, substantially all of the particulate from within the matrix can be extracted by immersing the matrix in a solvent in which the particulate is soluble (but in which the polymer matrix is insoluble) . The inventors have found that if the particles are extracted at the same time as the solvent, matrix shrinkage and distortion may occur.

In embodiments of the methods of the present invention in which particulates are removed from the formed polymer matrix, the resultant polymer matrix is a substantially "empty" matrix. Such a polymer matrix can be thought of as having a primary pore structure and a secondary pore structure. The primary pore structure is provided by the precipitated polymer matrix having an inherent porosity (e.g. an inherent porosity of about 5-10μm) . The secondary pore structure is provided by the pores left behind when the particulate was extracted from the polymer matrix, which are a similar size to the size of the particulate. Thus, incorporation of particulates of a specific shape and size range in the solution allows the formation of a matrix with a defined surface and internal pore architecture (the secondary pore structure) . By controlling the size and loading of the particulate, it is possible co control the connectivity of the pores throughout the matrix. For example, high loadings of particulates result in more, larger scale connections between the secondary pores .

Control of pore size and connectivity can/ for example, be useful in the manufacture of polymer matrices for cell scaffolds. Cell -scaffold binding can block pores of inadequate size and geometry. Ingrowth of bone rather than fibrous tissue has been found to predominate in porous polymethylmethacrylate (PMMA) implanted in bone when the pore size was around 450μm, while the presence of macropores (150-300 μm) highly interconnected by micropores (<50 μm) has been found to be conducive to ingrowth of fibrocartilaginous tissue in polyurethane meniεcal implants.

Micro-CT studies conducted by the inventors have shown that, whilst the pores of the secondary structure formed in 26% gelatin loaded matrices are not all connected by large (>15μm) channels, which are important for cell ingrowth, they are sufficiently connected (eg by the 5- lOμm pore structure) to permit extraction of the majority of the particulate phase. However, higher gelatin-loaded materials (44%) showed more large scale connections between the ^x secondary' pores.

Whilst substantially all of the particulate will be removed from the matrix following' immersion in a solvent in which the particulate is soluble, the presence of small amounts of particulate may be advantageous. For example, the persistence of gelatin in the interior and surface of a polymer matrix may advantageously display cell adhesion sequences for binding with iπtegrin cell surface receptors.

In a preferred embodiment of the present invention, a method is provided for the manufacture of a thick-section, microporous polymer matrix (or a microporous polymer matrix incorporating a high loading of evenly distributed particulate) . The method comprises the following steps: (a) introducing a solution of a polymer in a solvent (or a suspension of a particulate in a polymer solution) into a mould;

(b) immersing the mould in dry ice (-78.5⁰C) for 2 to 5 minutes to precipitate and harden the polymer matrix;

(c) removing the hardened matrix from the mould and immersing the matrix in a non-solvent (e.g. for 24 h) to extract the solvent; and

(d) drying the matrix (e.g. in air under ambient conditions) to remove residual solvent/non- solvent .

In such, embodiments, acetone is the preferred solvent.

In such embodiments, PCL is the preferred polymer, but^' other synthetic polyesters such as PLA or high L-lactide content PLG copolymers which will dissolve in acetone and tend to Crystallise from solution can also be employed.

The cooling time in step (b) is preferably minimised because prolonged cooling times can result in non-uniform shrinkage of the formed polymer matrix.

In an alternative to the step (c) described above, the solvent may be removed by adding non-solvent on top of the matrix which is retained in the mould. In other embodiments, the solvent is removed by drying the matrix without use of a nσn-solvent.

This method may be used to provide block-form (i.e. 3D) polymer matrices which may be machined to the required form for implantation e.g. bone graft substitutes. For example, the solution in step (a) may be poured into a mould which replicates the shape of Che tissue to be repaired e.g. craniofacial bone segments. Further, inserts may be moulded in to the polymer matrix to provide attachment points to host tissue e.g. suture sewing sites.

A structural component, such as a reinforcing metal spiral for tubular structures or a Nitinol shaped memory effect device, may be coated with a microporous polymer matrix by placing it in the mould in step a) .

Composite materials may be produced by impregnation of fibre preforms with PCL solution at step (a) followed by hardening. Use of water-soluble fibres enables development of an internal pore structure following dissolution. For example, fibres, mats and textiles can be made from a water soluble polymer such as polyvinylalcohol (PVA) . These could be added to the PCL. solution, which is subsequently converted into the polymer matrix. The fibres can then be solubilised and extracted to form a polymer matrix with an internal pore structure replicating the original fibre pattern.

The microporous polymer matrices of the invention are . widely applicable in soft and hard tissue repair. They may be used as a binder for bioceramics and as a delivery system for growth factors. Macropores of controlled size and shape may be formed on the surface and in the interior of the polymer matrix by extraction of water-soluble particulates (e.g. lactose or gelatin) to enhance tissue ingrowth.

The microporous polymer matriceβ are also useful for fabricating controlled release rate delivery systems for pharmaceuticals such as contraceptive hormones, microbiocides, vaccines and therapeutic peptide/ polypeptide growth factors (e.g. bone morphogenetic protein (BMP) and DNA) . In the case of therapeutic peptides/polypeptides, controlled local delivery can overcome problems of short half lives and rapid absorption in vivo which can limit the efficacy of injected, soluble preparations. Growth factors may be incorporated in microporous polymer matrices by simple admixing of spray dried particulates with the polymer solution before precipitation of the polymer matrix.

The microporous polymer matrices produced in accordance with the invention may find application as implantable, depot-type, delivery devices for anti-cancer drugs such as leutinising hormone releasing hormone (LHRH) used to treat prostate cancer and for drugs εuch as Carnustine (BCNU) used in brain tumour therapy. Production of 'matrix-type' transdermal delivery systems for melatonine, low molecular weight heparin and proteins is envisaged. Controlled release, vaginal inserts for vaccine antigens (such as the human papilloma virus (HPV) vaccine used for treating cervical cancer) or microbiocides for controlling the transmission of HIV infeccion devices may also be readily manufactured using the invention. Examples of microbiocides include the nucleotide reverse transcriptase inhibitor (NRTI) analogue - tenofovir or the nonnuσleoεide reverse-transcriptase inhibitor (NNRTI) - TMC-120.

Routes of drug administration for polymer matrix devices formulated using the methods of the present invention include ocular, sub-cutaneous, intramuscular, intra brain implantation, vaginal and transdermal.

The present invention may be used to produce microporous PCL matrices and biocompoεite matrices in the form of tubes for use in soft tissue engineering. The surface pore structure formed in gelatin- loaded PCL matrices following gelatin extraction is anticipated to encourage cell colonization and integration with host tissue. The persistence of gelatin in the interior and surface of the matrices may^' advantageously display cell adhesion sequences for binding with integrin cell surface receptors, In addition, the potential for incorporating various growth factors (e.g. VEGF), anticoagulants (e.g. heparin) and anti-bacterials (e.g. gentamicin) in PCL matrices would overcome some of the limitations of conventional blood vessel substitutes based on Dacron and ePTFE.

EXAMPLES

Preferred embodiments of the present invention will be described by way of example only, with reference to the following examples.

PCL (Mw 115_/000 Da, Capa 650) was obtained from Solvay Interox, Warrington, UK. Lactose particles and gelatin particles (bloom 125, bovine skin) in three size ranges (45-90, 90-125, 125-250 μm) were obtained by sieving the as-received powder (Sigma Chemicals) .

Example 1 Preparation of lactose-loaded PCL matrices

PCL (1.7 g) was dissolved in 10 ml acetone with gentle heating at approximately 50⁰C to produce a 17% w/v solution. In three separate preparations, lactose particles (which are insoluble in acetone) of each defined size range (i.e. 45-90, 90-125 and 125-250 μm) were dispersed in the PCL solution using a glass rod to give a final loading of 40% w/w. Each lactose suspension was transferred into a mould comprising a 5 ml polypropylene (PP) syringe body with a centrally located 1 ml PP syringe body. This produced a tubular casting (5.5 mm internal diameter, 10.3 mm external diameter x 30 mm) of interest for production of substitute blood vessels. The mould was then suspended in dry ice for 2 minutes. Following crystallization and hardening of the PCL phase, the polymer matrix was removed from the mould and immersed in methanol (50 ml) for 24 h to extract the acetone. The castings were removed from the methanol bath and any acetone/methanol remaining in the matrices was allowed to evaporate in air under ambient conditions for 1-2 days. If required_/ the lactose (which is insoluble in methanol) particles may subsequently, be extracted from the PCL matrix by immersing the loaded matrix into water or saline .

Example 2 Preparation of gelatin-loaded PCL matrices Gelatin loaded PCL matrices in tubular form were produced as described above in Example 1 for lactose-loaded materials. In two separate preparations, gelatin particles (in the si2e ranges of 45-90 μm and 90-125 μm) were added to 17% w/v PCL solutions to give a protein concentration of 40% w/w. in a separate preparation, gelatin particles of size range 125-250μm were added to 17% w/v PCL solution to give a 30% w/w loading. If required, the gelatin particles may subsequently be extracted from the PCL matrix by immersing the loaded matrix into water or saline.

The dried PCL castings present a. soft textured, porous and highly flexible character, free of large scale cracks and voids in the surface and interior.

The influence of cooling time in dry ice on the shrinkage behaviour of unloaded matrices (i.e. a PCL matrix which is prepared from PCL solutions without dispersed particulates) is shown in Table 1. As can be seen, increasing the cooling time in dry ice results in increasing shrinkage and distortion of the material on drying . Table 1 - Influence of cooling time in dry ice on shrinkage of unloaded-PCL matrices

5 min in dry Tubes Shrinkag Cylinders Shrinkag ice e (%) e (%)

Diameter on 11 .13 ± 11.1 ± 0. 11 de-mould 0.56 mm mm

Diameter after 10 .86 + 2 4 10.9 + 0. 05 1.8 methanol 0.06 mm mm treatment

Diameter on 10 .06 ± 9 6 9.85± 0. 22 11.3 drying 0.11 mm

10 min in dry Tubes Shrinkage Cylinders Shrinkage ice ⁽%) (%)

Diameter on 11 .13 ± 11.10 t de-mould 0. 05 mm 0.12 mm

Diameter 10 .76 ± 3.3 10.36 ± 6.7 after 0. 11 mm o.ll mm methanol treatment.

Diameter on 10 .01 + 10. 1 9. 93 + 10 .5 drying 0.12 mm 0.06 mm

Example 3 Determination of the lactose and gelatin content and distribution of PCL microporous matrices

The distribution of lactose and gelatin content in the matrices prepared as described in Examples 1 and 2 was measured to assess the influence of the casting method on loading uniformity and release rate.

Three individual samples from the top 5mm, mid-region, and base of each of the lactose-loaded PCL tubes (i.e. PCL matrices loaded with lactose having particle sizes 45-90, 90-125 and 125-250 μm) were each dissolved an 2ml DCM. The polymer was precipitated by addition of distilled water (8 ml) and the sample tubes were shaken overnight on a Vibrax VXR system to evaporate the DCM. The lactose concentration in distilled water was determined by HPLC (Waters) with refractive index detection and compared with a calibration curve constructed using a series dilution of lactose in distilled water (20-1000 /xg/ml) . The lactose loading of the matrices was subsequently calculated as % w/w.

Three individual samples from the top (5mm) , middle and base of each of the gelatin-loaded PCL matrices (i.e. PCL matrices loaded with gelatin having particle sizes 45-90 μm and 90-125 and 125-250Mm) were each dissolved in 2 ml DCM. PBS (8ml) was added to precipitate the PCL and the sample tubes were shaken overnight as described above to evaporate the solvent. Samples of gelatin in PBS solution were analysed in triplicate to determine the protein content using the BCA assay. Aliquots (10 μl) were transferred to a glass tube and 2 ml BCA working reagent were added. A stock solution of gelatin in PBS was used to produce calibration samples (Q-SOO μg/ml) . All test and calibration samples were incubated at 37⁰C for 30 min before measuring the LJV abεorbance using a Hitachi U2000 spectrophotometer at a wavelength of 562 nun. The protein extracted in PBS was subsequently compared with a calibration curve to calculate the gelatin loading of PCL matrices as % w/w.

Results

The loading of the matrices is shown in Figure 1. A high proportion of the particle content of the starting PCL solution was measured in the finished casting and a good particle distribution was obtained throughout the material indicating that sedimentation effects are limited. The lactose particle distribution is improved relative to gelatin., particularly in samples produced using the largest particles, indicating that particle density and shape may influence the resultant polymer matrix (e.g. because the sedimentation rate of a particle in a fluid depends on particle density and particle diameter and shape, which could be different for gelatin and lactose) .

Example 4 In vitro release of lactose and gelatin from PCL microporous matrices

Samples of lactose-loaded PCL matrices prepared as described in Example 1 were accurately weighed and immersed in 10 ml distilled water at 37 'C for 15 days. The release medium was replaced completely by fresh distilled water at 1 day intervals and the concentration of lactose in the samples was determined by HPLC (Waters) . The amount of lactose released was calculated using a calibration curve as described above and expressed as cumulative release (%) versus time (Figure 2) .

Accurately weighed samples of PCL matrices loaded with gelatin powder in various particle size ranges prepared as described in Example 2 were immersed in 10 ml PBS (pH 7.4). The sample tubes were retained at 37'C for 21 days and the release medium was replaced completely by fresh PBS at 1 day intervals. The amount of gelatin released was analysed after 24 h and then every 2 days up to 21 days using the BCA total protein assay and expressed as cumulative release (%) versus time (Figure 3) .

Results

As can be seen in Figure 2, approximately 90% of the original lactose content of the PCL matrices was extracted into distilled water over 9 days. A major "burst" release phase was observed during the first 24h, followed by a plateau region after 3-4 days. The similarity in release pattern of differently sized lactose particles indicates that the particle size ranges investigated have little influence on release kinetics beyond the initial burst phase .

As can be seen in Figure 6, micro-CT analysis revealed large numbers of internal pores following lactose extraction. This rapid and highly efficient release behaviour is attributed to lactose particles protruding through the matrix surface and contact and interconnections between particles which promote fluid penetration and solubilisation of deeply embedded particles. As can be seen in Figure 3, PCL matrices containing gelatin particles (of size range 45-90, 90-125 and 125-250 μm respectively) displayed gradual and sustained release of the protein phase in PBS at 37 "C. Approximately 40% of the gelatin content was released in 24 h for 45-90 μm particles, and the remaining protein was slowly and almost completely extracted over 11 days. Almost 90% of the gelatin load was released over 21 days, for particles in the higher size ranges. Gelatin-loaded PCL matrices prepared using the largest particles (125-250μrn) exhibited the lowest amount of protein release during the first day, and a more uniform, sustained profile for the duration of the release period. This behaviour may be explained by the lower specific surface area of the larger particles which decreases the dissolution rate.

As will be appreciated from the above experiments, the methods- of the invention- may be used to produce polymer matrices that exhibit highly efficient and sustained release of protein, which may be useful for controlled delivery of macrotnolecules such as vaccines and growth factors such as b-FGF and active protein C.

Example 5 Thermal characteristics

The thermal characteristics (peak melting point (τ_m) and percentage crystallinity) of PCL matrices (prepared using a similar process to that described in Examples 1 and 2, but without adding lactose or gelatin to the PCL solution) , lactose- loaded PCL matrices and gelatin- loaded PCL matrices (prepared as described in Examples 1 and 2) were determined using a TA Instruments DSC 2920. Specimens were accurately weighed (minimum 5 mg) , sealed in aluminium pans and programme -heated from O'C Co 90 'C at a rate of 10'C/min. Sample percentage crystallinity was estimated from the reported heat of fusion of 139. S J/g for fully crystalline PCL (28) .

Results

Incorporation of small lactose or gelatin particles in PCL matrices tended to result in a lower Tm compared with unloaded PCL matrices (Table 2) , suggesting the presence of smaller crystallites and/or crystal imperfections. The increased crystallinity of PCL matrices resulting from inclusion of 45-90 and 125-250μm lactose particles indicates an improved nucleating ability of the extraneous solid. However, the reduced PCL crystallinity in both lactose and gelatin-loaded matrices containing intermediate size particles is more noticeable and suggests that the morphology and surface characteristics of the 9o-l25μm powder fraction impedes nucleation and growth of PCL crystals.

Table 2 - Thermal properties and porosity of PCL tubular castings

Formulation of PCL PCL Lactose-loaded PCL matrix Gelatin. loaded PCL matrix matrix

Particle size , 45-90 SO-125 135 -350 4S-90 90-125 125-250 ranges (μm)

Tm ( "C) SS.2 €4.9 64.0 66-1 6S.7 «5.2 67.S

CrysCal1inity (%) 71.5 i 74. S * «5.9 ± 74.9 _t 71. a + S2 β ± 72.0 s

0.7 1.4 5.7 3.7 S-O 2.1 3.5

Porosity (%) 75.S ₁ 74. S± 76.2+ 78.8 ♦ 13.9 i 73.8 ± 80.8 i

2.1 12.5 11.3 8.3 2.4 10.5 12.2

Example 6 The morphology of a PCL matrix, and lactose-loaded and gelatin- loaded PCL matrices (prepared as described in Examples 1 and 2) both before and after in vitro release of the lactose/gelatin a.3 described in Example 4 were examined using a Philips x30 scanning electron microscope (SEM) . Specimens were mounted on aluminium sample stubs and sputter-coated with platinum prior to examination in the SEM at a voltage of 15 kV. The results are shown in Figure3 4 and 5.

Results

Unloaded, microporous PCL matrices display irregular pore shapes and sizes in the 5-10 μm range (Figure 4A) .

Lactose-loaded PCL matrices exhibit a rough and porous surface morphology due to an abundance of lactose particles embedded in and projecting from the surface (Figure 4B) . A distinct surface porosity developed after lactose release, with irregular pore shape and size (Figure 4C, 4D) .

Gelatin-loaded PCL matrices present a fairly uniform dispersion of particles close to the matrix surface (Figure 5A) . Protein extraction results in deep pores and surface depressions formed by dissolution of embedded particles (Figure 5B) . The pore size range 70-125μm corresponds to the original particle size distribution, but tnicroporosity is also visible in the range of 5-10 μm resulting from the PCL phase (Figure 5B) . Particles of lactose in the size range of 90-125μm and gelatin in the size range of 125-250μm are shown in Figures 5C and 5D, respectively.

As will be appreciated from the above experiments, the method of the invention permits control of the surface porosity of the polymer matrix by adjusting the size and shape of water-soluble particles incorporated in the starting polymer solution. This facility may be useful for enhancing cell interaction and tissue integration with the material .

Example 7

The porosity of PCL matrices (prepared as described in Examples 1 and 2} after in-vltro release of lactose and gelatin respectively as described in Example 4 was measured by Archimedes' principle using ethanol as the displacement liquid.

The porosity of PCL matrices, measured using ethanol displacement after lactose or gelatin release, was found to be similar to unloaded PCL matrices.

Example 8

3-D images of the polymeric phase and internal pore structure of PCL matrices (prepared as described in Examples 1 and 2) after in vitro release of the lactose/gelatin as described in Example 4 were acquired using X-ray micro computed tomography (Micro-CT) . Longitudinal samples (approximately 2x2x10 mm in height) were cut from microporous PCL tubes and analysed using a SkyscanlO72 desktop X-ray CT scanner at 15 μm voxel resolution (5Ox magnification) , X-ray tube current of 173 μA and voltage of 30 kV. Specimens were mounted vertically on a plastic support and rotated through 360 degrees around the long axis (z-a_xis) of the sample. Three-dimensional reconstruction of the internal pore morphology was carried out using the output format of serial bitmap images and analysed by VG Studio Max 1.2 software .

Results Micro-CT analysis of the internal 3-D structure of PCL matrices, following extraction of gelatin or lactose, identified the solid phase (Figures 6A, and 7A respectively) and macropore component (Figures 6B₁ and 7B respectively) . The pore shape and size corresponds closely with the particles used for matrix production (Figure 5C and 5D respectively) . The formation of the internal pore structure by extraction of lactose or gelatin particles indicates the presence of a well developed network of connections between pores.

Example 9 ^■

Biocomposite matrices in the form of tubes for use in soft tissue engineering and produced using an embodiment of the methods of the invention are shown in Figure 8.

A vaginal insert produced using an embodiment of the methods of the invention for applications in vaccine and microbiocide delivery is shown in Figure 9.

Example 10 Preparation of lysozyme-loaded PCL matrices

Lysozyme (MW 14.3kDa) particles (lθO-200/.m) were suspended in a 17% w/v PCL solution in acetone to produce 10% and 20% w/w suspensions. Lysozyme- loaded PCL matrices were subsequently produced in a similar manner to that described in Examples 1 and 2 by rapid cooling of the suspensions in dry ice followed by solvent extraction in methanol .

Microporous PCL matrices containing lysozyme particles were soft and flexible and free of large scale cracks and voids in the surface and interior. The lysozyme loading in the final matrix was around 3.1 % w/w and 10.5% w/w for matrices produced from 10% and 20% w/w particle suspensions, respectively. This reduction in matrix loading compared with the starting suspension may be explained by the solubility of lysozyme in methanol which is used during matrix formulation to extract acetone. Increasing the initial enzyme loading of the PCL solution from 10% to 20% w/w improved the loading efficiency in the finished matrices from 30% to around 50%.

In vitro release of Lyεozyme from microporouε PCL matrices

The amount of lyεozyme released from PCL matrices in PBS solution at 37 ⁰C was measured using the BCA total protein assay, and was observed to be similar to that for lactose and gelatin as described in Example 4. A burst release phase of 30 and 45% was observed for enzyme-loading of 3.1% and 10.5% respectively during the first day, followed by gradual release up to day 7 when the release profile plateaued. The initial enzyme loading affects the initial burst release. There was no marked difference in the release pattern of the two formulations; around 80% of the initial enzyme content was delivered from the 10.5% w/w lysozytne-loaded PCL matrix within 12 days compared with 60% from the 3.1% w/w lysozyme-loaded PCL matrix. This behaviour indicates a more highly developed network of interconnections between lysozyme particles in the more highly loaded matrices. The remaining enzyme content may be efficiently encapsulated by polymer therefore taking longer to release or not being released -

Biological activity of lyεσzyme following release from POL matrices

Most of the biochemical assays for lysozyme are based on its lytic activity since lysozyme can hydroly2e the 1-4 glycosidic linkage between alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid residues on the cell wall of micro-organisms . The activity of lysozyme released from PCL matrices was determined by measuring the turbidity degree of lysis induced by the enzyme on Micrococcus lysodeikticus cells.

As will be appreciated, an important aspect of protein or enzyme delivery is the stability of the protein in the delivery device during the release period. The lysozyme- loaded PCL matrices described above were retained in PBS release medium. The medium was collected every two days and stored at -20⁰C prior to analysis. As can be seen in Figure 10, the activity of lysozyme released into PBS over the first 8 hours (upper graph) was recorded as 99.8 ± 0.6% compared to native lysozyme, and was similar for 3.4% and 10.5% enzyme loaded PCL matrices. The activity of released lysozyme decreased gradually over 11 days to around 80% of the activity of fresh lysozyme solution (lower graph) . This behaviour may result from denaturation of the released enzyme over the 2 days period in PBS caused by unfolding of the secondary structure at 37⁰C. The enzyme may also be denatured in the matrix at 37⁵C and released in degraded form.

The results indicate that the hydrophobic nature of PCL (which is normally responsible for poor compatibility with proteins, resulting in protein adsorption onto the polymer surface, denaturation and aggregation) may not be a major problem since 60-80% of the enzyme load was released in PBS (lower graph) . However, increasing enzyme degradation may occur in the matrix and/or release medium over extended time periods.

This investigation reveals the potential of microporous PCL matrices to control the release of bioactive factors with retained activity for use in drug delivery and tissue engineering .

In the claims which follow and in the preceding description, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments .

Claims

CLAIMS :

1. A method for manufacturing a porous polymer matrix, the method comprising the steps of: (a) providing a solution comprising a polymer dissolved in a solvent, and

(b) causing the polymer to rapidly precipitate from the solvent to form a polymer matrix.

2. The method of claim 1, further comprising the step (c) of removing the solvent from the polymer matrix.

3. The method of claim 2, wherein the solvent is removed from the polymer matrix by immersing the polymer matrix in a non- solvent in which the polymer is insoluble, followed by drying the polymer matrix.

4. The method of any one of claims 1 to 3, wherein the solution further comprises a suspension of a particulate, and wherein the particulate is dispersed throughout the polymer matrix.

5. A method for manufacturing a porous polymer matrix, the method comprising the steps of:

(a) providing a solution comprising a polymer dissolved in a solvent, the solution further comprising a particulate suspended in the solution,- and (fc>) causing the polymer to precipitate from the solvent to form a polymer matrix, wherein the particulate is dispersed throughout the polymer matrix.

6. The method of claim 5, further comprising the step (c) of removing the solvent from the polymer matrix.

7. The method of claim 6, wherein the solvent is removed from the polymer matrix by immersing the polymer matrix in a non-solvent in which the polymer is insoluble, followed by drying the polymer matrix.

8. The method of claim 7, wherein the particulate is soluble in the non-solvent.

9. The method of claim 8, wherein the particulate is selected from the group consisting of particles of one or more of proteins, enzymes, alginate_/ chitosan, polysaccharides, cellulose derivatives, sugars, oligosaccharide ester derivatives, polyethylene glycol (PEG) , polyethylene oxide (PEO) , copolymers of poly (ethylene oxide) -poly (propylene oxide), polyvinylpyrrolidone (PVP) , polyvinylalcohol and water soluble inorganic materials (salts) .

10. The method of any one of claims 4 to 9, wherein the particulate is a bioactive particulate.

11. The method of claim 10, wherein the bioactive particulate is selected from the group consisting of particles of one or more of corticosteroid hormones, antivirals, microbiocides, vaccines, peptide/polypeptide growth factors, enaymes, antibiotics, anti-cancer drugs, melatonine, anticoagulants, anti-inflammatories, antifungals and miotics.

12. The method of any one of claims 1 to 11, wherein the solution further comprises a bioactive molecule dissolved in the solvent, whereby the bioactive molecule is dispersed throughout the polymer matrix.

13. The method of any one of claims I to 12, wherein the polymer is caused to precipitate in a mould whereby the polymer matrix has a predetermined three dimensional shape.

14. The method of any one of claims 1 to 13, wherein the porous polymer matrix is a microporous polymer matrix.

15. The method of any one of claims 1 to 14, wherein the polymer is a semi -crystalline polymer.

16. The method of any one of claims l to 15, wherein a blend of polymers is dissolved in the solvent.

17. The method of any one of claims 1 to 16_/ wherein the polymer is selected from one or more of the group consisting of polycaprolactone, poly (L-lactide) , high L-lactide-containing poly(lactide co-glycolide) copolymers which tend to crystallise from solution, polyglycolide (PGA) and oligosaccharide ester derivatives .

18. The method of any one of claims 1 to 17, wherein the solvent is acetone .

19- The method of any one of claims 1 to 18, wherein the polymer is caused to precipitate by cooling the solution.

20. The method of claim 19, wherein the polymer is caused to precipitate by cooling the solution to a temperature of less than -25°c.

21. The method of claim 20, wherein the polymer is cooled in dry ice for 2 to 5 minutes.

22. A three dimensional porous polymer matrix manufactured using the method of any one of claims 1 to 21.

23. The porous polymer matrix of claim 22, which is tubular, cylindrical or ring shaped.

24. The porous polymer matrix of claim 22 or claim 23, wherein a bioactive material is dispersed throughout the matrix..