WO2008041001A1

WO2008041001A1 - Porous particles

Info

Publication number: WO2008041001A1
Application number: PCT/GB2007/003796
Authority: WO
Inventors: Kevin Shakesheff; Lloyd Hamilton; Anita Patel; Cheryl Hunter
Original assignee: Regentec Ltd
Current assignee: Regentec Ltd
Priority date: 2006-10-07
Filing date: 2007-10-05
Publication date: 2008-04-10
Anticipated expiration: 2009-04-07
Also published as: GB0619869D0

Abstract

A method for producing porous particles comprising the steps of (i) mixing a first polymer solution with a second polymer solution; (ii) adding a surfactant to the first and second polymer solution to create an emulsion; and (iii) adding the emulsion to a hardening bath to produce a suspension of particles.

Description

POROUS PARTICLES

This invention relates to porous particles, and in particular to methods of making porous particles and to the use of porous particles.

According to a first aspect, the invention provides a method for producing particles comprising the steps of:

• mixing a first polymer solution with a second polymer solution; • adding a surfactant to the first and second polymer solution to create an emulsion; and

• adding the emulsion to a hardening bath to produce a suspension of particles.

Preferably the particles produced are porous.

As the particles produced by the method of the invention are relatively small they may be referred to as microparticles, the terms particle(s) and microparticle(s) are used interchangeably herein. Preferably the particles have a diameter of about 5000μm or less.

Preferably the first polymer solution is produced by dissolving a first polymer in a first solvent. Preferably the resulting solution is an oil phase. The first polymer may be dissolved in the first solvent by stirring/shaking for about 3 hours. The first polymer solution may be aerated. The first polymer solution may be aerated by vortexing. Aerating the solution may incorporate air bubbles into the solution which aid in particle formation.

Preferably the second polymer solution is produced by dissolving a second polymer in a second solvent. Preferably the resulting solution forms an oil phase. The second polymer may be dissolved in the second solvent by stirring/shaking for about 3 hours. The second polymer solution may be aerated. The second polymer solution may be aerated by vortexing.

In addition to the first and second polymers, one or more further polymers may be included. Preferably additional polymers are added or mixed with the first and second polymers before the surfactant is added.

A third polymer may be included with the second polymer in the second polymer solution. The second polymer and third polymer may be mixed by vortexing.

The first and second polymers may be the same or different polymers. Preferably, the first and second polymers are different polymers.

The first and second solvents may be the same or different solvents.

Any additional polymers may be the same or different to the first and/or second polymer.

After mixing the first and second polymer mixture, the resulting solution may be aerated. The polymer mixture may be aerated by vortexing. The polymer mixture may be aerated before the surfactant is added. Preferably, the mixed polymer solutions form an oil phase. The surfactant may be mixed with the polymer mixture by vortexing. Again, aeration of the mixture may incorporate air bubbles, which may assist in particle formation.

Aeration at any stage may be achieved by vortexing the solutions and/or mixtures. The vortexing may be undertaken at from about 400 to about 3000rpm. Preferably the first and second polymer solutions alone or mixed are aerated by vortexing at from about 2000 to about 3000rpm. Preferably when the first and second polymers are mixed with a surfactant they are aerated by vortexing at from about 400 to about lOOOrpm, more preferably from about 600 to about 800rpm. Preferably the oil and water phases are mixed by vortexing to form an emulsion. Preferably the oil and water phases are vortexed for at least 10 seconds.

Preferably the particles produced by the method of the invention are recovered by evaporating substantially all the solvent from the particles once they have formed. The solvent may be removed by evaporation.

Preferably the first and second polymer solutions are mixed before the surfactant is added. Alternatively, the surfactant may be added to the first and/or second polymer solution before the polymer solutions are mixed.

Preferably the first and second polymer solutions are mixed to form an oil phase.

Preferably the surfactant is in a water phase.

Preferably the polymer solutions when mixed with the surfactant form an emulsion, the emulsion preferably contains between about 25% and about 75% by volume of oil phase, and about 25% to about 75% by volume of water phase. The emulsion may contain about 50% oil phase and about 50% water phase, for example, about 50% polymer solution (oil phase) and about 50% surfactant (water phase) . Preferably the emulsion comprises from about 45% to about 55% oil phase and from about 55% to about 45% water phase. Preferably the surfactant stabilises the emulsion produced when the oil and water phases mix.

The particles may be formed using a single emulsion technique.

The emulsion once formed is added to a hardening bath to produce particles. Preferably the particles are formed as a suspension in the hardening bath. Preferably the emulsion is added drop-wise to the hardening bath. Alternatively the emulsion could be introduced into the hardening bath by pouring, spraying, showering, squirting or any other suitable method. Preferably, the emulsion is allowed to stand prior to being added to the hardening bath. Preferably, the emulsion is allowed to stand for about 20 seconds before being added to the hardening bath.

Once the particles have formed in the hardening bath, and are preferably in suspension in the hardening bath, any remaining solvent may be evaporated. Preferably the solvent is evaporated by slowly stirring the suspension of particles. Preferably the solvent is evaporated by slowly stirring for about 8 hours to about 24 hours, preferably about 10 to about 16 hours.

Preferably the hardening bath is a solution comprising an excess of a water phase. Preferably the volume of the hardening bath is at least about 5 times the volume of the emulsion. Preferably the volume of the hardening bath is at least about 6, 7, 8, 9, 10 or more time the volume of the emulsion. The volume of hardening bath needed is determined by a number of factors, including the volume of solvent in the emulsion added to the hardening bath, the chemical composition of the hardening bath and the rate of solvent evaporation. The hardening bath preferably has sufficient volume and/or surface area to expedite solvent removal and to allow particle hardening. Preferably the hardening bath comprises a surfactant. The surfactant may be the same as added to the polymer solution to form an emulsion. The hardening bath may be a solution of PVA (poly vinyl acetate) in water. Preferably the hardening bath comprises a solution of about 0.1 to about 15% PVA in water, preferably about 0.1% to about 10% PVA in water. In one embodiment the hardening bath comprises about 0.3% PVA in water.

Once recovered from the hardening bath the particles may be washed. Preferably the particles are washed by resuspending them in a wash solution, this may be done once or repeated multiple times, preferably three times. Preferably, once the particles have been sufficiently washed the particles are allowed to settle, the excess solution/liquid may then be decanted/removed leaving a particle slurry.

The particles may be dried. Preferably the particles are dried by vacuum drying and/or air drying.

Preferably the particles are formed without the use of porogens or leaching methods.

Particles produced by the method of the invention may range in diameter from about lμm to about 500O₁Um, preferably from about lμm to about 3000μm, or from about lμm to about 1500μm, preferably from about lOμm to about lOOOμm, preferably from about lOμm to about 300μm. Preferably the particles have a diameter of less than 300μm diameter.

The terms "porous particle" and "porous microparticle" are intended to refer to a particle with pores on the surface. Preferably a porous particle according to the invention has either a hollow centre with pores on the surface, or more preferably the particles comprise a number of interconnected surface pores, the pores being connected by a network of channels, voids or passageways within the particle.

The particles may have a porosity of from about 50% to about 98%, or from about 80% to about 98%. Preferably the microparticles have a porosity of about 95%. Porosity is defined as the amount of the pore or void space relative to the total volume occupied by the particle.

The particles may have a density of less than about 1.5, more preferably the density is about 1.

Preferably the particles sink in water. Preferably particles sink due to a combination of the density and the surface properties of the particles. The particles may have a hydrophilic surface which helps them to sink. Particles which sink have the advantage that they are easier to use in cell culture, and that they are easier to include in a formulation for delivery to a human or animal recipient.

The pore size may be from about lμm to about 500μm. Preferably the pore size is large enough to accommodate prokaryote and/or eukaryote cells, such as mammalian cells. Pores of about 20μm to about 50μm will accommodate most cells. The pore size may be uniform or non-uniform throughout the particle. Preferably the pore sizes are non-uniform throughout the particle. The size of the pores may be controllable during the production of the microparticles.

The microparticles may comprise one, two, three, four or more polymers. In one embodiment, only one of the two or more polymers used in the method to produce the particles may actually form the particles. Preferably the first and/or second polymers, and/or any additional polymers, used in the method of the invention are biocompatible and/or bioabsorbable, and/or biodegradable.

The first polymer and/or second polymer and/or any other polymer may be selected from the group comprising poly(D,L-lactide-co- glycolide) (PLGA) , poly lactic acid (PLA) , poly D, L-lactic acid (PDLA) , polyethylene glycol (PEG), polyethyleneimine (PEI), poly (α- hydroxyacids) , polylactic or polyglcolic acids, poly-lactide poly-glycolide copolymers, poly-lactide poly-glycolide polyethylene glycol copolymers, polyesters, poly (ε-caprolactone) , poly (3-hydroxy-butyrate) , poly (s- caprioc acid), poly (p-dioxanone), poly (propylene fumarate) , poly (ortho esters) , polyol/diketene acetals addition polymers, polyanhydrides, poly (sebacic anhydride) (PSA) , poly carboxybiscarboxyphenoxyphosphazene) (PCPP) , poly [bis (p-carboxyphenoxy) methane] (PCPM) , copolymers of SA, CPP and CPM (as described in Tamat and Langer in Journal of Biomaterials Science Polymer Edition, 3, 315-353, 1992 and Domb in Chapter 8 of the Handbook of Biodegradable Polymers, Editors Domb AJ and Wiseman RM, Harwood Academic publishers) , poly (amino acids) , poly (pseudo amino acids) , polyphosphazenes, derivatives of poly [(dichloro) phosphazene] , poly [(organo) phosphazenes) , polyphosphates, polyethylene glycol polypropylene block co-polymers for example that sold under the trade mark Pluronics™, natural or synthetic polymers such as silk, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides (including pectins), alginates, collagen, peptides, polypeptides or proteins, copolymers prepared from the monomers of any of these polymers, random blends of these polymers, any suitable polymer or mixtures or combinations thereof.

Preferably, the polymers used are selected from the group comprising poly (D, L-lactide-co-glycolide) (PLGA) , poly D, L-lactic acid (PDLA), poly lactic acid (PLA) , polyethylene glycol (PEG) and polyethyleneimine (PEI) ,

If PEG is used it may have a MW of from about 100 to about 5000 daltons. Preferably the PEG used is selected from the group comprising

PEGlOO, PEG200, PEG400 and PEGlOOO, preferably the PEG used has a

MW from 100 to 1000 daltons. Preferably the PEG used is liquid.

Preferably if PEG400 is used in the first and/or second polymer solution it is used at a concentration of from about 200mg/ml to about lg/ml, more preferably, from about 300mg/ml to about 900mg/ml or form about

360mg/ml to 720mg/ml.

Preferably if the first polymer is PDLA, PLGA or PLA, the second polymer is PEG.

Preferably if the first polymer is PLGA, PDLA or PLA, the second polymer is a strong cationic polymer such as PEI, and a third polymer, such as PEG, is also used.

The first solvent and/or second solvent may be a volatile organic solvent. The first and second solvent may be the same or different. The first solvent and/or second solvent may be selected from any of the group comprising dichloromethane, methylene chloride, dimethylsulfoxide (DMSO) , N , N, dimethylformamide, 1 ,2-dichloroethane, dichloroacetic acid, acetone, ethylacetate, tetrahydrofuran, n-methyl-pyrrolidone, chloroform, methanol and combinations thereof.

The surfactant used in any step of the method of the invention may be selected from the group comprising polymeric surfactants such as polyvinyl acetate (PVA) and polyoxyethylene-based surfactants, sucrose esters and any other suitable surfactant or combinations thereof. Preferably the surfactant is polyvinyl acetate.

The wash solution may be selected from the group comprising water, distilled water, phosphate buffered saline solution and Hank's balanced salt solution and any other suitable wash solution or combinations thereof. Preferably the wash solution is distilled water.

The particles made by the method of the invention may be treated with a degrading agent to remove an outer surface of the particles and/or to increase the size of pores and/or to increase the number of pores and/or to alter the surface chemistry of the particle. Preferably the particles are treated with a degrading agent for no more than about 24 hours, preferably for no more than about 12 hours. The length of time particles are treated with a degrading agent will be determined on the basis of various factors including temperature, concentration of the degrading agent, quantity of the microparticles being degraded etc.

The degrading agent may alter the surface chemistry of the particles. The surface chemistry may be altered to increase the hydrophilicity of the particle.

A particle produced according to the method of the invention may be treated with a degrading agent to increase the porosity of the particle.

By treating the particles with a degrading agent the size of the pores may be increased.

The degrading agent may be any agent capable of degrading one or more of the polymers which comprise the particle. Preferably the degrading agent is an alkali solution. Alternatively the degrading agent could be an acid solution. Preferably the degrading agent is a solution of NaOH. The NaOH solution may comprise NaOH in ethanol. Preferably, if NaOH is used, it is used at a concentration of about 0.1 to about 0.5N. More preferably about 0.25N. Preferably if NaOH is used it is used in ethanol at a concentration of 96%, for example, the degrading agent used may comprise 30% 0.25M NaOH and 70% 96% ethanol. Preferably the degrading agent acts to increase the size of the pores by eroding the polymer. Preferably the degrading agent chemically alters the surface of the particle, this may have the effect of increasing the hydrophilicity of the particle which increases cell adhesion. An increase in hydrophilicity may also assist in producing porous particles which sink.

The method may include a step of exposing the particles to alcohol, either alone or as part of another solution, for example, in the degrading agent. The alcohol may be ethanol. The alcohol may sterilise the particles. The alcohol may alter the surface chemistry of the particles and increase hydrophilicity. The alcohol may degrade the polymer which forms the particle and may increase porosity. The alcohol may be used as a degrading agent.

Preferably the method does not include a step of homogenising the particles or the solutions used to produce the particles.

The particles may be used as a drug and/or growth factor delivery system; and/or a cell delivery vehicle; and/or a tissue scaffold. The particles may be used as a delivery system for pharmaceutics or therapeutic proteins.

A scaffold made from the particles may be used in vivo or in vitro. A scaffold made from the porous particles may be used in in vitro assays. The particles may provide a scaffold support for tissue formation. The particles may be used alone, re-seeded with cells and/or co-administered with cells.

By using porous particles in a scaffold the overall porosity of the scaffold may be improved, which in turn may improve the mass transport of nutrients/waste products through the scaffold both in vitro culture and in vivo. In vivo, this may improve vascularisation of the scaffold so that the tissue can continue to grow. Another advantage of porous particles is that the surface area available for cell attachment may be increased. Having an increased surface area may allow more cells to be delivered with less scaffold material, this will decrease the quantity of degradation products in vivo. If the pores are large enough cells may grow inside the particles and therefore be protected, to a certain extent, from external mechanical forces. In particular, cells may be protected from shear forces associated with minimally invasive methods used for scaffold delivery.

The particles may be used in a method of cellular therapy. Cellular therapy involves using cells to repair tissues that have been damaged, for example by disease, to generate new tissues with desired functional activities.

Other substances such as growth factors and/or adhesion molecules may be incorporated into the particles during and/or post production. Other substances incorporated into the particles during and/or post production may be selected from any of the group comprising amino acids, peptides, proteins, sugars, antibodies, nucleic acid, antibiotics, antimycotics, growth factors, steroids, synthetic material, adhesion molecules, and other suitable constituents, or combinations thereof. The particles may be adapted to be injectable. Preferably the particles are injectable through a syringe. The particles may be adapted to be injected in vivo .

Preferably the particles are adapted to be cross-linked upon injection to form a scaffold. Preferably solidification into a scaffold takes from about 20 seconds to about 30 minutes, preferably about 2 minutes to about 20 minutes. Preferably the solidification takes less than about 20 minutes, more preferably less than about 15 minutes. Preferably the scaffold is suitable for efficient tissue repair in vitro and/or in vivo.

The particles may be arranged to be cross-linked using a linker molecule to form a matrix or tissue scaffold when in use. The cross linking used may be as described in PCT/GB2004/001419.

The benefit of cross-linking the microparticles is that upon injection the material quickly solidifies creating a scaffold possessing a three- dimensional architecture and an environment suitable for efficient tissue repair.

The current invention describes a simple process for the generation of particles which possess an intraporous network. This invention allows for the efficient delivery of cellular therapies whilst also providing a ready- made support on which cells can proliferate. The structure of the particles would allow for the protection of cells during delivery by minimally invasive methods. The porous nature of the formed particles provide for a high density of potential cell binding per particle whilst not only allowing cells to culture on the surface of the particles but to also migrate within the particles. According to a further aspect, the invention provides a method of producing a porous particle comprising:

• mixing a first polymer solution of PDLA, PLGA and/or PLA with a second polymer solution of PEG; • adding a surfactant to the polymer mixture to create an emulsion;

The emulsion formed may be a water in oil emulsion.

Preferably the amount of the mixed first and second polymer solutions (oil phase) added to the surfactant (water phase) to produce an emulsion is about equal by volume. Preferably about 45% to about 55% by volume of the emulsion is oil phase and about 45% to about 55% by volume is water phase.

Preferably the particles produced by this method are treated with a degrading agent, such as NaOH, to increase the size of the pores in the particle.

Preferably the first and second polymers are dissolved in DCM to form first and second polymer solutions respectively. Preferably the mixed polymer solution is aerated before the surfactant is added. The polymer solution and surfactant may be aerated after they are mixed. Preferably the surfactant used is PVA.

According to another aspect, the invention provides a method of producing a porous particle comprising: • mixing a first polymer solution with a second cationic polymer solution; • adding a surfactant to the polymer mixture to create an emulsion;

• adding the emulsion to a hardening bath to produce a suspension of particles; and • treating the particle with a degrading agent.

Preferably the first polymer solution comprises PLGA, PLA or PLGA. Preferably the second polymer is PEL Preferably a third polymer, such as PEG, is added to the first and second polymer solutions. The third polymer may be included in the second polymer solution with the cationic polymer.

Preferably the degrading agent used is NaOH. Preferably the degrading agent causes pores to be exposed on the surface of the particles.

Preferably the first and second polymers, and any optional further polymers, are dissolved in DCM. The polymer solutions may be aerated before they are mixed. The mixed polymer solution may be aerated before the surfactant is added. The polymer and surfactant emulsion may also be aerated. Preferably the surfactant used is PVA.

The skilled man will appreciate that the preferred aspects of the first method of the invention can be applied to all methods of the invention.

Furthermore, the skilled man will appreciate that the preferred features of the porous particles produced by the methods of the invention can be applied to all aspects of the invention.

According to another aspect, the invention provides porous particles made according to any method of the invention. According to another aspect, the invention provides a porous particle comprising a network of interconnected pores.

According to a further aspect, the invention provides porous particles for use in the production of a tissue scaffold. Preferably the particles are made according to the method of the invention, or have characteristics of porous particles made according to the method of the invention.

According to another aspect, the invention provides the use of porous particles in the manufacture of a medicament for use in the production of a tissue scaffold.

Preferably the particles are made according one or more of the methods of the invention or have characteristics of particles made according to one or more of the methods of the invention.

According to another aspect, the invention provides a scaffold comprising porous particles according to the invention. Preferably, at least some of the porous particles in the scaffold are crosslinked to each other or to other particles.

The scaffold may be seeded with cells and/or may contain one or more therapeutic agents.

In another aspect, the invention provides a process for producing a scaffold with cells comprising the steps of a) seeding cells which are suspended in medium onto microparticles made according to the invention to make a cell and microparticle suspension; and b) incubating the cell and microparticle suspension in order to form a scaffold seeded with cells.

The cells and microparticles may be incubated in vivo, ex vivo or in vitro.

The cells may be mammalian of human or other animal origin. The cells may be stem cells. Preferably the cells are human embryonic stem cells or fibroblast cells. Other suitable cells may be used. The cells may be 3T3 fibroblast cells and/or human embryonic stems cells (line Hl-WiCeIl, USA) . The cells may be osteoblasts, osteocytes, chondroblasts or chondrocystes. The cells may originate from the patient who will use or be treated with the scaffold. The cells may originate from a donor.

Prior to seeding the cells onto the microparticles, the cells may be cultured by incubating in appropriate conditions. Cultured cells may be treated with trypsin to create a single cell suspension for seeding onto the microparticles. Alternatively, cells may be isolated from a source, such as a patient or a donor and seeded directly onto the particles.

The particles and/or the cell suspension may be treated with one or more antimicrobial agents in solution. The one or more antimicrobial agents may be one or more antibiotic and/or antimycotic agents.

The cell and microparticle suspension may be incubated at about 20°C to about 42⁰C, preferably about 37°C. The cell and microparticle suspension may be incubated in an atmosphere of about 3% CO₂ to about 10% CO₂, preferably about 5% CO₂.

The cell and microparticle suspension may be incubated for between about 4 hours to several months.

The cells and/or microparticles may be cryogenically stored.

The scaffold may be checked for successful cell adherence and/or cell viability by treating a sample with a suitable stain, such as Live/Dead stain™, or any other viability or cell type specific stains. The stained cells may be observed and/or quantified under magnification.

According to another aspect, the invention provides a cell culture surface comprising porous particles according to any aspect of the invention. The culture surface may be particularly useful for cells which are sensitive to trypsin and do not grow well in suspension. Cells may be cultured on the porous particles and then administered, say for cellular therapy, along with the porous particles.

According to another aspect the invention provides a medical implant comprising porous particles.

The advantages of the porous microparticles described in accordance with the invention include a) interconnected porosity which will allow the migration of cells throughout a scaffold made from the particles and may also aid nutrient flow to cultured cells; b) the ability to encapsulate cells within the microparticles; c) lower risk of acidic degradation or product build up as less particles are used and the pores allow the degradation products to be quickly dissipated. Acidic build-up and associated drop in pH is a problem for monolithic orthopaedic PLA type screws etc where there is no porosity. This can result in inflammation and implant rejection. By using porous particles the acidic by-products may be dissipated more quickly thus reducing acid build up and pH drop; and d) compatibility with secondary applications such as injectable systems

Embodiments and/or aspects of the invention will now be described in more detail by way of example only, with reference to the accompanying figures.

Figure 1 is a scanning electron micrograph (SEM) of poly(D, L- lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention;

Figure 2 is an SEM, of higher resolution than Figure 1 , of poly (D,

L-lactic acid) /poly (ethylene glycol) porous microparticles made according to a first embodiment of the invention;

Figure 3 is an SEM, of lower resolution than Figure 1 , of poly(D, L-lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention;

Figure 4 is an SEM of poly(D, L-lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention, wherein the polymer mix was aerated for 10 seconds before being added to the surfactant;

Figure 5 is an SEM of poly(D, L-lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention, wherein the polymer mix was aerated for 2 minutes before being added to the surfactant; Figure 6 is an SEM of poly(D, L-lactic acid)/poly (ethylene glycol) porous microparticles made according to a first embodiment of the invention, wherein the polymer mix was aerated for 5 minutes before being added to the surfactant;

Figure 7 is an SEM of poly(D, L-lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention treated for 10 minutes with NaOH;

Figure 8 is an SEM of poly(D, L-lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention treated for 30 minutes with NaOH;

Figure 9 is an SEM of neonatal fibroblasts seeded on NaOH treated poly(lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention. The arrows indicate the position of the fibroblasts;

Figure 10 is a light microscopy image of neonatal fibroblasts seeded on NaOH treated poly(D, L-lactic acid)/poly(ethylene glycol) porous microparticles made according to a first embodiment of the invention stained with toluidine blue;

Figures HA, B and C are SEMs at different resolutions of poly (lactic-co-glycide) acid/poly (ethylene glycol)/poly(ethyleneimine) porous microparticles made according to a second embodiment of the invention;

Figure 12 is an SEM of poly(lactic-co-glycide)acid/poly(ethylene glycol) /poly (ethyleneimine) porous microparticles made according to a second embodiment of the invention, with the porous surface exposed after NaOH treatment;

Figure 13 is an SEM of poly(lactic-co-glycide)acid/poly(ethylene gly col) /poly (ethyleneimine) porous microparticles made according to a second embodiment of the invention with the porous surface exposed after NaOH treatment;

Figure 14 is an SEM of poly(lactic-co-glycide)acid/poly(ethylene glycol) /poly (ethyleneimine) porous microparticles made according to a second embodiment of the invention after treatment with NaOH;

Figure 15 is an SEM of poly (lactic-co-glycide)acid/poly (ethylene glycol)/poly(ethyleneimine) porous microparticles made according to a second embodiment of the invention after treatment with NaOH;

Figure 16 is an SEM of poly(lactic-co-glycide)acid/poly(ethylene glycol) /poly (ethyleneimine) porous microparticles made according to a second embodiment of the invention after treatment with NaOH for 2 minutes;

Figure 17 is an SEM of human embryonic cells seeded on NaOH treated poly(lactic-co-glycide)acid/poly(ethylene glycol)/poly(ethyleneimine) porous microparticles made according to a second embodiment of the invention. The polymer was seen to auto fluoresce under the UV light, the bright, light grey areas indicated live cells. The pattern of staining indicated that there were no dead cells; Figure 18 shows SEMs of poly(lactic-co-glycide)acid/poly(ethylene glycol) porous microparticles made according to the first embodiment of the invention treated with NaOH, in which the PDLA and PEG solutions are mixed by vortexeing for either 0 seconds, 10 seconds, 20 seconds or 30 seconds;

Figure 19 shows SEMs of poly(lactic-co-glycide)acid/poly(ethylene glycol) porous microparticles made according to the first embodiment of the invention treated with NaOH, wherein the ratio of oil phase:water phase is either 40:60, 60:40, 15:85 or 85: 15, wherein the oil phase comprises PDLA and PEG in DCM, and the water phase comprises the surfactant PVA in water; and

Figure 20 shows SEMs of poly(lactic-co-glycide)acid/poly(ethylene glycol) porous microparticles made according to the first embodiment of the invention treated with NaOH, in which the the concentration of PEG used in the second polymer solution is varied. The concentrations used are 180, 360, 460, 660, 720 and 1440mg/ml.

Example 1 - Production of Porous Microparticles from PDLA and PEG

2.4 g of poly D, L-lactic acid (PDLA) was dissolved in 10 ml of dichloromethane (DCM) . This is performed in 2 x 50 ml tubes each containing 1.2 g of PDLA and 5 ml of DCM. 720 mg polyethylene glycol

(PEG400) was dissolved in 2 ml DCM. 1 ml of the dissolved PEG was added to each PDLA 50 ml tube and the two oil phases (the PDLA in

DCM and PEG in DCM) were then mixed together using a Vortex Genie 2 vortex mixer for 2 minutes at speed 10 (which is equivalent to approximately 2700rpm) . Both 50 ml tubes were vortexed simultaneously. 6 ml of a 0.3% PVA solution (poly vinyl acetate in water, manufactured no more than 48 hours previously) was added to each 50ml to be of PDLA and PEG in DCM. The resulting mixture was vortexed for 20 seconds at speed 3 forming an emulsion (speed 3 is equivalent to between about 600 and about 800rpm) . The vial was held using two fingers holding the lid of the vial, placing the vial on the pad of the vortexer without applying any pressure. This emulsion was then added drop wise through a 6 mm diameter pipette tip into 800 ml of a gently stirred solution of 0.3% PVA (in water) . The microparticles mixture was left to stir overnight (no more than 18 hours) . The solvent was allowed to evaporate off and the microparticles were filtered and rinsed with 1 litre of distilled water.

Figures 1 , 2 and 3 are SEMs of microparticles made using this method.

Figures 4, 5 and 6 are SEMs of microparticles made using this method, the particles differ only in the length of time the PDLA in DCM and PEG in DCM were mixed before being added to the surfactant.

Example 2 - Sodium Hydroxide Treatment

In order to optimise cell attachment and increase pore number, porous microparticles made according to the method of Example 1 were sodium hydroxide (NaOH) treated as follows.

3 ml of 0.25 M sodium hydroxide was added to 7 ml of 96% ethanol . Approximately 100 mg of porous microparticles were placed in a 15 ml tube and the NaOH/ethanol solution was added. The tube was then placed on a platform rocker at 30 rev/min for 30 minutes. The tube was then centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded and the pellets rinsed with 0.01M PBS. Figures 7 and 8 are SEMs of particles treated in this way.

Figure 18 shows SEMs of microparticles made using the method of Example 1 and 2, in which the length of time the DLA and PEG in DCM are mixed with the PVA solution before being added dropwise to a PVA solution was varied. In Figures 7 and 8 the PDLA/PEG are mixed with PVA for 20 seconds. Figure 18 shows the results when the PDLA/PEG and PVA are mixed by vortexing for 0 seconds, 10 seconds, 20 seconds or 30 seconds. The results presented show that when the PDLA/PEG (oil phase) is not vortexed to mix it with the PVA (water phase) to form an emulsion large non-porous particles are made. Whereas when the PDLA/PEG and PVA are mixed by vortexing for 10 seconds, 20 seconds or 30 seconds porous particles are produced.

The SEMs in Figure 19 shows the effect of varying the ratio of oil phase to water phase in the method of Example 1 and 2. The oil phase is the PDLA and PEG in DCM, and water phase is the PVA in water. In the method of Example 1 and 2 a 50:50 ratio of oihwater phase is used. The SEMS in Figure 19 illustrate particles produced using a ratio of 40:60, 60:40, 15:85 and 85: 15 oil phase:water phase. The results demonstrate that when the oil phase is decreased and the water phase is increased the porosity of the particles decreases, and when the level of oil phase is decreased to 15% the particles are completely non-porous. When the oil phase is increased, for example to 60:40 and 85: 15 oihwater, large particles are produced. The particles are typically too large for the preferred applications of the invention. Furthermore, the larger particles have poor porosity.

Figure 20 shows the effect of varying the concentration of PEG400 in the second polymer solution in the method of Example 1 and 2. Concentrations of 180, 360, 460, 660, 720 and 1440mg/ml of PEG400 in DCM were used to produced microparticles. The resulting microparticles were compared. The results indicate that when the PEG concentration is decreased to less than 360mg/ml there is a marked decrease in particle porosity. By way of contrast, increasing the concentration of PEG to 720mg/ml and above results in large particles with reduced porosity.

Example 3 - Seeding of Cells on Porous Microparticles

Figure 9 is an SEM of porous microparticles made according to the method of Example 1 and 2 onto which 3T3 fibroblasts are seeded. The cells were seeded using the following method.

A cell suspension of 1.5 x 10⁶ 3T3 fibroblast cells in 200 μl of growth medium was made. This was added to approximately 10 mg of PLA/PEG microparticles (100-200 μm diameter, made according to the method of Example 1) in a 1.5 ml microcentrifuge tube. The microparticles had been NaOH treated in 10 ml of 30% 0.25 M NaOH in 96% ethanol for 10 minutes and serum coated (FCS Gold) by serum absorption over 24 hrs (at RT) . After the cell suspension was added the cell/microparticle suspension was triturated to mix. 800 μl culture medium for the fibroblasts - DMEM containing L-glutamine and 10% FCS, was added and the microcentrifuge tube was centrifuged at 1500 rpm for 5 minutes at room temperature. After centrifuging the microcentrifuge tube was left to stand in an incubator for 24 hrs.

Figure 10 shows another example of microparticles seeded with cells. The microparticles used were produced according to the method of Examples 1 and 2. 3T3 fibroblasts were seeded on to the microparticles using the following method. A cell suspension of 5 x 10⁵ cells in 200 μl of medium was made. This was added to approximately 10 mg of PDLA/PEG microparticles (100-200 μm diameter) in a 1.5 ml microcentrifuge tube. The microparticles had been NaOH treated in 10 ml of 30% 0.25 M NaOH in 96% ethanol for 10 minutes and serum coated (FCS Gold) by serum absorption over 24 hrs (at 37°C) . After the cell suspension was added, the cell/microparticle suspension was triturated to mix. 4 mis of medium was added to the cells and microparticles in the bijou. The bijou was then left static for 24 hours at 37°C with 5%CO₂. The microparticles were then stained with toluidine blue to visualize the location of the cells within the microparticles.

Example 4 - Production of Porous Microparticles from PLGA, PEG and PEI

1.2 g of poly (lactide-co-glycolide) acid (PLGA) was dissolved in 5 ml of dichloromethane (DCM) on a plate shaker for 3 hours. Once dissolved, the mixture was aerated using a Genie 2 vortex mixer at speed 10 for 40 seconds. 100 mg poly (ethyleneimine) (PEI) and 1.08 g of polyethylene glycol (PEG) 400 was mixed on a vortex and dissolved in 3 ml DCM . 1.2 ml of the PEI/PEG/DCM mixture was added to the PLGA/DCM mixture and vortexed at speed 10 for 2 minutes to aerate. Immediately after the vortex, 7.5 ml of 0.3% wt/vol of polyvinyl acetate (PVA) in water solution was added (poured down the side of the container) followed by a further vortex for 1 minute at speed 2. The resulting mixture was then allowed to stand for approximately 20 seconds before adding the solution drop wise via pipette to a 600 ml solution of 0.3% wt/vol PVA (in water) . The resulting mixture was stirred slowly overnight. The container was covered to retard the evaporation rate. The particles were allowed to settle and the top 4/5th of the solution was decanted. 700 ml of distilled water was added to the particle slurry and the particles were allowed to settle, and the top 4/5th of the solution was decanted. The particles were then filtered and washed three times with distilled water before vacuum drying. Figures HA, B and C depict the resulting particles.

In order to optimise cell attachment and expose the surface pores the microparticles were sodium hydroxide treated to remove the outer coat.

Sodium hydroxide treatment was effected by immersing vacuum dried microparticles in a solution containing 30% NaOH (0.25N) and 70% ethanol for 2 minutes. This removed the outer shell from the particles, exposing the porous network (as depicted in Figures 12 to 16) . The particles have 2-5 μm diameter pores.

Example 5 - Seeding of Cells on the Microparticles

Human embryonic stem cells (line Hl - WiCeIl, USA) were cultured in a differentiation media (including dexamethasone and ascorbate) to direct the cells down an osteogenic lineage. After 8 weeks of culture on a mouse embryonic fibroblast feeder layer (13 days old P2) the cells were cultured for 1 day in a differentiation pellet of 100,000 cells. The cells were then treated with trypsin to create a single cell suspension.

Approximately 100 mg of porous microparticles made according to the method of Example 4 were placed in a 15 ml tube and treated with ethanol for 5 minutes. The particles were washed with PBS (phosphate buffered saline) and then soaked in antibiotic/antimycotic solution for 48 hours followed by a further PBS wash. 50,000 cells Hl cells were seeded onto the particles by adding the cells and particles to 5 mis of differentiation medium and incubating at 37°C in an atmosphere of 5% CO₂ for 4 days.

The particles were then treated with Live/Dead stain (from Molecular Probes UK) . As shown in Figure 17 no detrimental effects on cell viability were observed.

Claims

1. A method for producing porous particles comprising the steps of:

• mixing a first polymer solution with a second polymer solution;

• adding a surfactant to the first and second polymer solution to create an emulsion; and

2. The method according to claim 1 wherein the first and second polymers are the same.

3. The method according to claim 1 wherein the first and second polymers are different.

4. The method according to any preceding claim wherein the first polymer and/or the second polymer and/or any other polymer is selected from the group comprising poly (D, L-lactide-co-glycolide) (PLGA) , poly lactic acid (PLA) , poly D, L-lactic acid (PDLA) , polyethylene glycol (PEG) , polyethyleneimine (PEI) , poly (α-hydroxyacids) , polylactic or polyglcolic acids, poly-lactide poly-glycolide copolymers, poly-lactide poly-glycolide polyethylene glycol copolymers, polyesters, poly (ε- caprolactone) , poly (3-hydroxy-butyrate) , poly (s-caprioc acid) , poly (p- dioxanone), poly (propylene fumarate), poly (ortho esters), polyol/diketene acetals addition polymers, polyanhydrides, poly (sebacic anhydride) (PSA) , poly carboxybiscarboxyphenoxyphosphazene) (PCPP) , poly [bis (p-carboxyphenoxy) methane] (PCPM), copolymers of SA, CPP and CPM, poly (amino acids), poly (pseudo amino acids), polyphosphazenes, derivatives of poly [(dichloro) phosphazene] , poly [(organo) phosphazenes) , polyphosphates, polyethylene glycol polypropylene block co-polymers for example that sold under the trade mark Pluronics™, natural or synthetic polymers such as silk, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides (including pectins) , alginates, collagen, peptides, polypeptides or proteins, copolymers prepared from the monomers of any of these polymers, random blends of these polymers, and mixtures or combinations thereof.

5. The method according any of claims 1 to 3 wherein the polymers used are selected from the group comprising poly(D,L-lactide-co- glycolide),(PLGA) , poly D, L-lactic acid (PDLA) , poly lactic acid (PLA) , polyethylene glycol (PEG) and polyethyleneimine (PEI) ,

6. The method according to any of claims 1 to 3 wherein if the first polymer is PDLA, PLGA or PLA, the second polymer is PEG.

7. The method according to any of claims 1 to 3 wherein if the first polymer is PLGA, PDLA or PLA, the second polymer is a strong cationic polymer such as PEI, and a third polymer, such as PEG, is also used.

8. A method of producing a porous particle comprising:

• mixing a first polymer solution of PDLA, PLGA and/or PLA with a second polymer solution of PEG;

• adding a surfactant to the polymer mixture to create an emulsion; • adding the emulsion to a hardening bath to produce a suspension of particles.

9. A method of producing a porous particle comprising:

• mixing a first polymer solution with a second cationic polymer solution; • adding a surfactant to the polymer mixture to create an emulsion;

10. The method according to any preceding claim wherein the first polymer solution and/or the second polymer solution is in an oil phase.

11. The method according to any preceding claim wherein the first polymer solution and/or the second polymer solution is aerated.

12. The method according to any preceding claim wherein after mixing the first and second polymer mixture, the resulting solution is aerated.

13. The method according to any preceding claim wherein the polymer mixture comprising the first and second polymer solutions is aerated after the surfactant is added.

14. The method according to claim 11 , 12, or 13 wherein aeration is achieved by vortexing the solutions and/or mixtures.

15. The method of claim 14 wherein vortexing is undertaken at from about 400 to about 3000rpm.

16. The method according to any preceding claim wherein the surfactant is in a water phase.

17. The method according to any preceding claim wherein the particles produced are recovered by evaporating substantially all the solvent from the particles.

18. The method according to any preceding claim wherein the polymer solutions when mixed with the surfactant form an emulsion, and wherein the emulsion preferably contains between about 25% and about 75% by volume of oil phase, and about 25% to about 75% by volume of water phase.

19. The method according to claim 18 wherein the polymer solutions when mixed with the surfactant form an emulsion, and wherein the emulsion preferably contains between about 45% and about 55% by volume of oil phase, and about 45% to about 55% by volume of water phase.

20. The method according to any preceding claim wherein the emulsion is added to the hardening bath by one of the following methods drop- wise, pouring, spraying, showering or squirting .

21. The method according to any preceding claim wherein the hardening bath comprises a surfactant.

22. The method according to any preceding claim wherein the porous particles are formed without the use of porogens or leaching methods.

23. The method according to any preceding claim wherein the particles produced range in diameter from about lμm to about 500O_jUm.

24. The method according to any preceding claim wherein the particles have a porosity of from about 50% to about 98%.

25. The method according to any preceding claim wherein the particles have a density of less than about 1.5.

26. The method according to any preceding claim wherein the particles sink in water.

27. The method according to any preceding claim wherein all polymers in the porous particles are biocompatible and/or bioabsorbable, and/or biodegradable.

28. The method according to any preceding claim wherein the first solvent and/or second solvent may be a volatile organic solvent.

29. The method according to any preceding claim wherein the surfactant used in any step of the method is selected from the group comprising polymeric surfactants such as polyvinyl acetate (PVA) and polyoxyethylene-based surfactants, sucrose esters and any other suitable surfactant or combinations thereof.

30. The method according to any preceding claim wherein the particles produced are further treated with a degrading agent to remove an outer surface of the particles and/or to increase the size of pores and/or to increase the number of pores and/or to alter the surface chemistry of the particle.

31. The method according to any preceding claim wherein particles are be adapted to be injectable.

32. The method according to any claim 31 wherein the particles are adapted to be cross-linked upon injection to form a scaffold.

33. The method according to any preceding claim wherein the emulsion formed is a water in oil emulsion.

34. Porous particles made according to the method of any of claims 1 to 33.

35. Use of porous particles according to claim 34 in the production of a tissue scaffold.

36. Use of porous particles according to claim 34 in the manufacture of a medicament for use in the production of a tissue scaffold.

37. A scaffold comprising porous particles according to claim 34.

38. A scaffold according to claim 37 seeded with cells and/or containing one or more therapeutic agents.

39. A process for producing a scaffold with cells comprising the steps of a) seeding cells which are suspended in medium onto porous particles according to claim 34 to make a cell and particle suspension; and b) incubating the cell and particle suspension in order to form a scaffold seeded with cells.

40. A cell culture surface comprising porous particles according to claim 34.

41. A medical implant comprising porous particles according to claim 34.