WO2008108736A1

WO2008108736A1 - Particles for delivery of bioactive factors

Info

Publication number: WO2008108736A1
Application number: PCT/SG2007/000065
Authority: WO
Inventors: Jackie Y Ying; Andrew C A Wan; Benjamin Tai
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2007-03-06
Filing date: 2007-03-06
Publication date: 2008-09-12
Anticipated expiration: 2009-09-06

Abstract

Provided herein are cell-responsive biodegradable microparticles, comprising a hydrophilic polymer crosslinked with a crosslinking group which is degradable by a releasing species produced by a cell and a binding species coupled to the polymer for binding and delivering a bioactive factor, such as a growth factor, to a cell. Also provided are methods of synthesis of cell-responsive biodegradable microparticles.

Description

Particles for delivery of bioactive factors

Technical Field

The present invention relates to biodegradable cell-responsive polymer microparticles to s deliver bioactive factors, such as growth factors. The present invention also relates to methods of synthesis of biodegradable cell-responsive polymer microparticles.

Background

The success of tissue engineering and regeneration relies on the proper provision of io bioactive factors to the cells that would potentially constitute the new tissue. These factors may be in the form of cytokines, growth factors or cell adhesion ligands. In situ, the factors may be soluble, tethered to the extracellular matrix (ECM) or localized on the surface membrane of neighbouring cells.

Recent efforts have been devoted towards methods of providing the controlled presentations of bioactive factors for tissue engineering. The dominant general strategy which is used involves the sustained release of growth factors from microspheres or nanoparticles composed of biodegradable polymers such as poly-lactic-co-glycolic acid (PLGA), which may be in turn incorporated within a scaffold matrix. These polymers spontaneously degrade under physiological conditions, resulting in the constant release of encapsulated or bound factors. In0 the case where microspheres are embedded in a matrix, the release profile would mirror the release of growth factor from microspheres alone but with a slower rate, as the growth factor would have to diffuse through the matrix.

While most slow or sustained release delivery systems operate on the basis of a presumed optimal release profile which is then engineered into the system, a recent alternative uses cell-s responsive monolithic materials to deliver growth factors in a manner which more closely mimics the delivery of growth factors in vivo. In cell-responsive materials, growth factors are immobilized on monolithic polyethylene glycol (PEG)-based hydrogels which are cross-linked with specific oligopeptide or polypeptide sequences, which are susceptible to degradation by matrix metalloproteinases (MMPs) produced by cells, thus delivering the factors in response to0 exposure to cell-derived proteinases. Cell-responsive delivery is advantageous, as many growth factors function within a narrow therapeutic range, possibly producing adverse effects when delivered in excess and yet being ineffective when provided at too low a concentration.

Cell-responsive delivery systems may be difficult to use where the bioactive factor is covalently coupled to the degradable hydrogel matrix, due to the sensitivity of growth factorS activity to the structural modifications required to bind the factor to the matrix. In addition, monolithic materials may not be useful in certain applications, such as where the material is provided to the circulation.

Accordingly, it is desirable to be able to provide the cell-responsive delivery of bioactive factors in a form which substantially addresses these shortcomings.

Summary of the Invention

Accordingly, in one aspect there is provided a microparticle for delivery of a bioactive factor, the microparticle comprising a hydrophilic polymer crosslinked with a crosslinking group, the crosslinking group being degradable by a releasing species produced by a cell, and a binding species coupled to the hydrophilic polymer by a coupling group, the binding species being capable of binding and releasing a bioactive factor, and whereby exposure of the microparticle to the releasing species causes release of the binding species.

The hydrophilic polymer may be a polyether. In one embodiment the hydrophilic polymer is branched, such as a branched polyethylene glycol, The crosslinking group may be an oligopeptide or a polypeptide, or an oligosaccharide or polysaccharide.

Where the crosslinking group is an oligopeptide or polypeptide, it may be from about 8 to about

30 amino acids in length.

In certain embodiments the releasing species is an endopeptidase. In certain embodiments, the coupling group is a cysteine residue.

In a particular embodiment, the binding species is heparin or a heparin-like molecule.

In certain embodiments, the microparticle has a diameter of between about 200 and 400nm.

In any of the above embodiments the microparticles may comprise a bioactive factor bound to the binding species. In another aspect there is provided a process for making microparticles for delivery of a bioactive factor, said process comprising: a) preparing a precursor microparticle comprising a hydrophilic polymer crosslinked with a crosslinking group, said crosslinking group being degradable by a releasing species produced in a cell, and a binding species coupled to the hydrophilic polymer by a coupling group, said binding species being capable of binding the bioactive factor; and b) binding the bioactive factor to the precursor microparticle to produce the microparticles.

In one embodiment, step a) comprises: al) providing a first solution comprising the hydrophilic polymer in a first solvent, said hydrophilic polymer having terminal groups capable of reacting with a crosslinker., said crosslinker comprising the crosslinking group; a2) providing a second solution comprising the binding species bound to the coupling 5 species in a second solvent; a3) providing a third solution comprising the crosslinker in a third solvent; a4) combining the third solution with a fourth solvent that is immiscible with the first, second and third solvents to form a dispersion; a5) combining the first solution and the second solution, thereby coupling the hydrophilic io polymer to the binding species to form a mixture; a6) combining the mixture and the dispersion to form a reaction mix; and a7) agitating the reaction mix sufficiently to crosslink the hydrophilic polymer and form the precursor microparticles in the fourth solvent. In one embodiment, the process of additionally comprises I₅ a') separating the precursor particles from the fourth solvent; and a") washing the precursor particles. In one embodiment, step b) comprises: bl) combining a suspension of the precursor particles in an aqueous solvent with a solution of the bioactive factor in a solvent that is miscible with the aqueous solvent to form a0 binding mixture; and b2) allowing the binding mixture to react for sufficient time for the bioactive factor to bind to the precursor particles.

In another aspect there is provided a process for making microparticles for delivery of a bioactive factor, said process comprising: S i) providing a precursor solution comprising a precursor species, said precursor species comprising a hydrophilic polymer, a binding species coupled to the hydrophilic polymer by a coupling group, and the bioactive factor, said bioactive factor being bound to the binding species; and ii) reacting the precursor species with a crosslinker to form the microparticles, said0 crosslinker comprising a crosslinking group which is degradable by a releasing species produced in a cell. In one embodiment, step i) comprises: il) providing a first solution comprising the hydrophilic polymer in a first solvent, said hydrophilic polymer having terminal groups capable of reacting with a crosslinker, said 5 crosslinker comprising the crosslinking group; 12) providing a second solution comprising the coupling species bound to the binding species in a second solvent;

13) combining the second solution with a solution of the bioactive factor in an aqueous solvent to form a first mixture comprising the bioactive factor bound to the binding species, said aqueous solvent being miscible with the second solvent; and

14) combining the first mixture and the first solution, thereby coupling the binding species to the hydrophilic polymer by means of the coupling species to form the precursor species.

In one embodiment, step ii) comprises: iil) combining the precursor solution with a solution of the crosslinker and agitating the resulting mixture sufficiently to crosslink the precursor species and form the microparticles.

Brief Description of the Figures

Figure 1 is a diagrammatic illustration of the synthesis of cell-responsive particles using Method 1. Binding of growth factor (e.g. FGF-2) in the last step occurs by specific structural and charge interactions with heparin.

Figure 2 is a diagrammatic illustration of the synthesis of cell-responsive particles using Method 2. Binding of growth factor (e.g. FGF-2) occurs by specific structural and charge interactions with heparin in the first step of the reaction. Figure 3 is a diagrammatic representation of the interaction between the components of the cell responsive particles. Proteolytic cleavage of the MMP-degradable crosslinker releases heparin with its bound growth factor from the particle.

Figure 4 illustrates light micrographs of cell-responsive particles prepared by (a) Method 1 and (b) Method 2. The scale bar in (a) is 20 micrometers, and in (b) 200 micrometers. Figure 5 provides a graph which illustrates the concentration of free FGF-2 in solution, as determined by ELISA, after filtration to remove the FGF-2-binding particles. This figure demonstrates that cysteine-heparin is necessary for the binding of FGF-2 to the particle.

Figure 6 provides graphs of relative cell numbers (as measured by alamarBlue®) of 3T3 cells cultured with (■) and without (♦) particles loaded with (a) 1 ng/ml, (b) 10 ng/ml, and (c) 100 ng/ml of FGF-2.

Figure 7 provides a graph of relative cell numbers (as measured by alamarBlue®) of hMSCs cultured in a cell pellet (~1 x 10⁵ cells) without particles (•), with particles crosslinked with MMP-degradable peptide with (♦) and without (■) 10 ng/ml of FGF-2, and with particles crosslinked with scrambled peptide (to demonstrate that that particle degradation is sequence specific) loaded with 10 ng/ml of FGF-2 ( ▲ ). Abbreviations

ELISA Enzyme-linked Immunosorbent Assay

FGF-2 Fibroblast Growth Factor-2 hMSCs human Mesenchymal Stem Cells

MMP Matrix Metalloproteinase

PEG-VS poly-(ethylene glycol)-vinyl sulfone

SDF- 1 Stromal-derived Factor- 1

TEOA triethanolamine

Detailed Description

In the context of this specification, the term "comprising" will be understood to imply the inclusion of a stated step or element or integer or group of stems or elements or integers, but not the exclusion of any other step or element or integer or group thereof. Thus, in the context of this specification, comprising means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

Throughout this specification, reference to "a" or "one" element does not exclude the plural, unless context determines otherwise. For instance, reference to "a bioactive factor" should not be read as excluding the possibility of multiple bioactive factors.

The inventors have developed cell-responsive biodegradable microparticles for delivering a bioactive factor.

The term "microparticle" encompasses discrete particles or loose aggregates of particles, wherein the maximum diameter of the individual particles is from 0.1 μm to 100 μm, and more preferably is between 0.2 μm and 50 μm, and is intended to exclude monolithic materials. The microparticles provided herein are able to be produced in a range of sizes and morphologies. In one embodiment, the microparticles are substantially discrete particles which may be produced in a range of sizes from about 200 nm to about 400 nm as measured by a ZetaPALS particle analyser (Brookhaven Instruments Corporation, New York, NY), by variation of the conditions of particle synthesis, as described below. Particles of this embodiment may be produced by Method 1 described herein. For particles of a particular size, the range of particle sizes may vary by approximately ±20 nm.

In another embodiment, the particles may be produced in loose aggregates ranging in size from 40 to 1100 μm, with the individual particles within the aggregates ranging in size from 1 to 30 μm. Particles of this embodiment may be produced by Method 2 described herein. Variation of the sizes of the microparticles of both of these embodiments may be achieved through manipulation of the conditions of particle synthesis. Conditions of synthesis which may influence particle size include the concentration of any one or more of the polymer, the crosslinking group and the binding species which are comprised in the particle, the concentration of surfactant used in the synthesis of the particles, and the degree of agitation during the synthesis of the particles. The ratio of surfactant to polymer, the ratio of polymer to crosslinking species and other ratios of the components used in the synthesis may also affect the particle size, and the polydispersity of the particles. The particle size may also be affected by the nature of the surfactant, the charge on the surfactant and other related factors. The particles may be monodispersed or may have a narrow or broad polydispersity. An increase in the agitation during particle formation commonly leads to a decrease in the particle size. Similarly, an increase in the ratio of surfactant to polymer, or to polymer plus crosslinking group, may also lead to a decrease in the particle size.

In one embodiment, the ratio of molar ratio of functionalized groups on the polymer to coupling group in the synthesis of the microparticles is between about 3 to 1 and 3.5 to 1, for example 3.3 to 1.

In one embodiment, the molar ratio of functionalized groups on the polymer to corresponding functionalized groups on the crosslinker which bind to the functionalized groups on the polymer is between about 1 to 3 and 1 to 4, for example approximately 1 to 3.56. Microparticles of defined size ranges may be produced from a mixture of microparticles of different sizes by the use of size exclusion membranes, meshes or filters. Nuclear track etched membranes, for example, are readily available in pore sizes ranging from 100 nm to 12 μm and greater, and may be used to selectively retain particles of a size greater than the selected pore diameter. The cell-responsive biodegradable microparticles described herein may be made from materials which have previously only been described in monolithic form. The present particles comprise a hydrophilic polymer which is crosslinked with a crosslinking group, and a binding species which is capable of binding the bioactive factor.

The hydrophilic polymer may comprise small organic gel forming molecules, such as peptides or peptide amphiphiles, which may be assembled into supramolecular structures after crosslinking with a crosslinking group. In one embodiment the hydrophilic polymer is a polyether, such as a polyethylene glycol (PEG). It may be an ethylene oxide propylene oxide copolymer, or a copolymer of ethylene oxide with some other monomer, e.g. with some other polyether monomer. The hydrophilic polymer may have a functionality greater than 2, e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more than 10. The functional groups may be at chain ends. They may be located along the chain. They may be located both at chain ends and along the chain. The polymer may be a branched polymer, and may have 1, 2, 3, 4, 5 or more than 5 branches. It may be a hyperbranched polymer. It may be a starburst dendrimer. The functional groups should be capable of reacting with the crosslinlcing group in order to crosslink the polymer. The functional group may be derived from for example hydroxyl, amino, thiol etc. These may be activated towards reaction with the crosslinker by conversion to, for example, a vinyl sulfone or other activated olefmic group. Suitable features for the polymer include (but are not limited to) that it is substantially biocompatible, non-toxic, resistant to protein adsorption, and substantially inert under the biological conditions to which it will be exposed in use. These properties may apply to the polymer before and/or after crosslinking. The polymer may be such that, following crosslinking, it is flexible. It may be capable of forming a hydrogel following crosslinking. The distance between crosslinks may be sufficiently large to provide the desired flexibility and/or water swell. This will vary depending on the nature of the crosslinker and of the polymer, but may be at least about 10 atoms, or at least about 15, 20, 25, 30, 35, 40, 45 or 50 atoms. The distance between crosslinks may be sufficiently small that the polymer when crosslinked is insoluble in water. Thus the distance between crosslinks may be between about 5 and about 100 atoms, or between about 10 and 100, 20 and 100, 50 and 100, 5 and 50, 5 and 20 or 20 and 50 atoms. The molecular weight of the polymer (prior to crosslinking) will depend on the nature of the polymer and on the desired properties of the particles. It may be between about 500 and about 100000, or between about 1000 and 100000, 5000 and 100000, 10000 and 100000, 50000 and 100000, 500 and 50000, 500 and 10000, 500 and 5000, 500 and 1000, 1000 and 100000, 1000 and 10000 or 5000 and 50000, ,e.g. about 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 60-000, 70000, 80000, 90000 or 100000, or may be more than 100000. Individual branches of the polymer may have between about 2 and about 100 monomer units, or between about 2 and 50, 2 and 20, 2 and 10, 5 and 100, 10 and 100, 50 and 100, 5 and 50 or 5 and 20, e.g. about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 monomer units. It will be understood that the distance between crosslinks in the polymer when crosslinked reflects the distance between functional groups capable of crosslinking in the uncrosslinked polymer. The skilled worker will be able to determine by routine experiment the optimum polymer to be used in order to achieve the desired properties.

The synthesis of cell-responsive monolithic PEG-based polymers is described in Lutolf and Hubbell (2003) Biomacromolecules 4: 713-722, the entire contents of which are incorporated herein by reference. The present invention provides methods to produce such PEG-based polymers in microparticle form. Where PEG-based polymers are used, the PEG may be linear or multiarmed, and in one embodiment at least three armed. It will be understood that although specific polymers are described herein, a person of skill in the art would readily recognise that other hydrophilic polymers having the properties described above could be substituted for the PEG polymers without materially altering the invention, and may be incorporated into the microparticles of the invention as described herein for PEG.

The polymer will possess functionalisation which allows interaction with the crosslinker to form a supramolecular structure during particle synthesis. In some embodiments the crosslinker will comprise thiol groups, which may for example be present in cysteine groups in the crosslinker (particularly if the crosslinker is a peptide). In this case the polymer should contain groups which can react with the thiol groups. Suitable groups include olefϊnic groups which are activated towards thiol addition or other Michael addition. They may be electron deficient olefϊnic groups, e.g. vinyl sulfones, acrylates, methacrylates, maleate half esters, maleimido groups etc. These may be made for example by reacting the polymer containing an activatable group such as hydroxyl, thiol, amine etc. with an activating reagent, e.g. vinyl sulfonyl chloride, acryloyl chloride, methacryloyl chloride, maleic anhydride etc.

The crosslinking of the polymer with a crosslinking group allows for the formation of supramolecular structures. The crosslinking group also provides a site which may be degraded by a releasing species produced by a cell, such as a matrix metalloproteinase, thereby allowing the release of fragments of the particle comprising the bioactive factor. The crosslinking group will have at least two sites which are independently able to crosslink with a functionality present on polymer molecules, to allow the crosslinking of at least two polymer molecules together. The releasing species may be produced by a mammalian cell, or may be produced by a bacterium, fungus or yeast. The mammalian cell may be a cancer cell.

In a particular embodiment, the crosslinking group is an oligopeptide or polypeptide which comprises an amino acid sequence which is cleaved by an enzyme which is secreted by a cell or which is present on the plasma membrane of a cell. Amino acid sequences which provide a cleavage site for the releasing species Plasmin, Stromelysin, Elastase, Collagenase, or the plasminogen activators t-PA and u-PA, and which are suitable for inclusion in a crosslinker are disclosed in US patent serial No. 6, 894,022 (Hubbell et al), the entire contents of which are incorporated by reference. Typically the releasing species will be a cell-associated extracellular endopeptidase, such as a member of the matrix metalloproteinase (MMP) family (IUBMB Enzyme Nomenclature Family EC 3.4.24). These metalloendopeptidases are typically able to cleave amino acid sequences found in proteoglycan, fibronectin, and collagen types III, IV, V, IX, and are released by cells which are associated with extracellular matrix remodelling. Various matrix metalloproteinases are secreted by or expressed on the surface of cells during cell proliferation, tissue remodelling, angiogenesis and in metastasis and tumour cell invasion of tissues, and accordingly amino acid sequences which are known to be sensitive to cleavage by one or more matrix metalloproteinases are also contemplated.

The enzyme-sensitive domain present in the crosslinking group may comprise a naturally occurring amino acid sequence which is a substrate of these enzymes, or it may comprise a synthetic sequence which is recognised and cleaved.

In a particular embodiment, the crosslinking group comprises the amino acid sequence GPQGIWGQ (SEQ ID NO:1) which is recognised by several matrix metalloproteinases and which is rapidly cleaved to GPQG (SEQ ID NO:2) and IWGQ (SEQ ID NO:3) by these enzymes. It will be understood that a substrate sequence of any one of numerous extracellular enzymes may be incorporated into the crosslinking group to allow for the specific cleavage of the crosslinking group and release of the bioactive factor in response to the enzyme. For example, where the releasing species is Cathepsin B, the crosslinking group may comprise the amino acid sequence GLPG (SEQ ID NO:4) (Bettio F et al., (2006) Biomacromolecules 7: 3534-3541, the entire contents of which is incorporated herein by reference). Where the releasing species is a collagenase the crosslinking group may comprise the amino acid sequence GGLGPAGGK (SEQ ID NO:5). Other amino acid sequences which act as substrates for collagenase and which may be used in a crosslinking group are disclosed in Netzel-Arnett et al. (1991) J Biol Chem 266: 6747- 6755, the entire contents of which is incorporated by reference. Where the releasing species is an elastase the crosslinking group may comprise the amino acid sequence AAAAAAAAAK (SEQ ID NO:6) (Mann B et al., (2001) Biomaterials 22: 3045-3051, the entire contents of which are incorporated herein by reference).

Where a crosslinking group is an amino acid sequence, it is contemplated that the crosslinking group is at least 8, at least 9 or at least 10 amino acids in length to allow for both crosslinking at two sites on the crosslinking group and for at least the minimum amino acid sequence required to be recognised and cleaved by a cell-produced proteinase. While the maximum length of the amino acid sequence of the crosslinking group is less critical, amino acid sequences longer than about 30 amino acids may reduce the density of the particles which may interfere with the particle's structural integrity. Where the polymer is based on end-functionalized multi-armed PEG-vinylsulfone macromer described by Lutolf and Hubbell (2003, supra), the crosslinking group will typically comprise at least two cysteine residues to provide the potential for crosslinking with the vinyl sulfone groups of the macromer by Michael-type addition reactions. Alternatively the crosslinking group may be functionalized with an amine, alcohol, or any nucleophilic functional group. The crosslinking group may comprise an oligosaccharide or polysaccharide sequence where the releasing species is an enzyme which is directed to polysaccharide cleavage, such as a heparinase, a heparanase, a heparitinase, a chondroitinase or lysozyme. Heparinase I cleaves heparin and heparan sulfate at the linkages between hexosamines and O-sulfated iduronic acids. Heparinase II cleaves heparan sulfate, and to a lesser extent heparin at the 1-4 linkages between hexosamines and uronic acid residues (both glucuronic and iduronic), yielding mainly disaccharides. Heparinase III cleaves at the 1-4 linkages between hexosamine and glucuronic acid residues in heparan sulfate, yielding mainly disaccharides. Heparanase is an endo-β-D- glucuronidase that catalyzes the hydrolytic cleavage of the β-l,4-glycosidic bond between a D- glucuronate and a D-glucosamine. Chondroitinase ABC catalyzes the eliminative degradation of polysaccharides containing 1 ,4-β-D-hexosaminyl and 1,3-β-D-glucuronosyl or 1,3-α-L- iduronosyl linkages to disaccharides containing 4-deoxy-b-D-gluc-4-enuronosyl groups. Lysozyme catalyzes the hydrolysis of 1,4-β-linkages between N-acetylmuramic acid and N- acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Accordingly, the incorporation of relevant saccharide residues in the crosslinking group to render the crosslinking group sensitive to cleavage by any one or more of these enzymes is contemplated.

The microparticles comprise a binding species which is coupled to the polymer by a coupling group. The binding species provides a site to non-covalently releasably bind a bioactive factor, thus allowing the bioactive factor to be incorporated into the particles in a manner which mimics the binding of a bioactive factor under physiological conditions. In a particular embodiment the binding species is heparin or a heparin-like molecule such as a synthetic heparin or a heparan sulphate. The heparin or heparin-like molecule may be bound to one or more of the other components of the crosslinked polymer by covalent or non-covalent methods. On release from the microparticle, the heparin may then carry and present the bioactive factor to a cell in a manner which mimics the heparin-mediated presentation of bioactive factors in situ.

In a convenient embodiment, the heparin may be coupled by functionalizing it with cysteine residues, allowing the heparin to interact with vinyl sulfone functionalization or other suitable functionalization (e.g. acrylate, acrylamide, maleic half ester, maleimido) on the polymer by a Michael-type addition reaction. In another embodiment heparin may be coupled to the polymer by carbodiimide coupling of heparin to amino-terminated PEG. In other embodiments the heparin may be coupled to the polymer via amide linkages on the polymer and -COOH groups on heparin, via activated carbonates, thiocarbonates and so on (e.g. maleimidocarbonates) on the polymer and amine functionalities introduced to heparin, activated carbonates on the heparin and amine groups on the polymer etc. Those skilled in the art will readily identify water compatible coupling reactions that may be used in this context. "Click chemistry" reactions (see for example KoIb et at. (2001) Angewandte Chemie International Edition, 40: 2004 - 2021, the entire contents of which are incorporated herein by reference) may be suitable for coupling the heparin to the polymer. Suitable click chemistry may include for example cycloaddition reactions, such as the Huisgen 1,3 -dipolar cycloaddition, Cu(I) catalyzed azide-acetylene cycloaddition, Diels-Alder reaction, nucleophilic substitution to small strained rings (e.g. epoxy and aziridine rings), formation of ureas and amides and addition reactions to double bonds, e.g. epoxidation, dihydroxylation.

The use of heparin immobilised in a matrix to sequester and deliver bioactive factors, such as growth factors, is described in US patent serial No 6,894,022 {supra).

The use of heparin-like factors as binding species, such as other glycosaminoglycans including heparan sulphate, hyaluronic acid, dermatan sulphate, chondroitin-4 and -6 sulphates, and keratan sulphates, or synthetic heparin as binding species is also contemplated.

Numerous bioactive factors have been demonstrated to bind heparin. These include growth factors such as basic and acidic FGF, members of the TGF-β superfamily including bone morphogenic proteins, interleukin-8, neurotrophin-6, heparin-binding epidermal growth factor, hepatocyte growth factor, connective tissue growth factor, midkine, heparin-binding growth associated molecule and members of the vascular endothelial growth factor (VEGF) family. These factors have demonstrated the potential to enhance healing in many different types of tissue, including vasculature, skin, nerve and liver. Any one or more of these growth factors, or other heparin-binding growth factors may be employed with the particles described herein. It will be understood that a growth factor will be considered to be heparin-binding if it elutes from a heparin-affinity column at NaCl concentrations above physiological levels (greater than or equal to 140 mM). It will also be understood that numerous bioactive factors bind to heparin-like molecules, and accordingly the delivery of bioactive factors which bind heparin-like molecules is also contemplated.

In addition to growth factors which inherently possess heparin-binding activity, it is contemplated that other bioactive factors may be used with the particles described herein. These bioactive factors may be inherently able to bind heparin non-covalently, such as fibronectin, neural cell adhesion molecule and anti thrombin III.

In addition, bioactive factors, including growth factors, may be engineered to become heparin binding, for instance through the addition of heparin-binding peptide sequences. Exemplary heparin binding peptide sequences are described in US patent serial No. 6,894,022 (supra). Heparin binding amino acid sequences derived from anti-thombin III, platelet factor-4, neural cell adhesion molecule, fibronectin, basic or acid fibroblast growth factor or lipoprotein lipase, for example, may be incorporated into a bioactive factor using techniques described in US patent serial No. 6,894,022 (supra), for example by the creation of a fusion protein.

In addition to the advantageous properties of heparin in non-covalently sequestering and delivering bioactive factors, the inventors have observed that incorporation of heparin as a binding agent during the synthesis of the microparticles unexpectedly stabilised the microparticles.

The binding species may also be Type II pro-collagen (which binds TGF-βl and BMP-2), a pentosan polysulfate, phosphorothioate oligodeoxynucleotides or synthetic heparin analogues.

The microparticles may be used to deliver bioactive factors in a wide variety of circumstances. The microparticles may find use in the same applications as other slow release delivery systems in delivery of therapeutic agents, growth factors and the like, and in addition may be used as an alternative to monolithic cell responsive materials. The microparticles may be conveniently incorporated into matrices of biological or synthetic origin for tissue engineering applications, or they may be incorporated onto the surface of implantable materials to regulate the initial interactions between the implantable material and the surrounding tissue.

It will be understood that the bioactive factor may be selected from amongst a wide range of molecules, both naturally occurring and synthetic. These include trophic factors, immunomodulatory factors, cytokines, factors which stimulate or inhibit the formation of scar tissue, factors which stimulate or inhibit new vessel formation, cytotoxic or cytostatic agents directed toward neoplastic cells, adhesion factors and factors which guide cell migration.

The present invention provides two processes for making the microparticles described herein. The first process comprises the steps of: a) preparing a precursor microparticle comprising a hydrophilic polymer crosslinked with a crosslinking group, said crosslinking group being degradable by a releasing species produced in a cell, and a binding species coupled to the hydrophilic polymer by a coupling group, said binding species being capable of binding the bioactive factor; and then b) binding the bioactive factor to the precursor microparticle to produce the microparticles.

Step a) may comprise the following steps: al) providing a first solution comprising the hydrophilic polymer in a first solvent, said hydrophilic polymer having terminal groups capable of reacting with a crosslinker, said crosslinker comprising the crosslinking group; a2) providing a second solution comprising the binding species bound to the coupling species in a second solvent; a3) providing a third solution comprising the crosslinker in a third solvent; a4) combining the third solution with a fourth solvent that is immiscible with the first, second and third solvents to form a dispersion; a5) combining the first solution and the second solution, thereby coupling the hydrophilic polymer to the binding species to form a mixture; a6) combining the mixture and the dispersion to form a reaction mix; and a7) agitating the reaction mix sufficiently to crosslink the hydrophilic polymer and form the precursor microparticles in the fourth solvent.

The hydrophilic polymer, as noted earlier, may be a PEG polymer, e.g. a branched PEG, although other polymers as earlier described may be used. A suitable polymer is a 4-arm PEG. The polymer should be functionalised with, for example, olefinic groups activated towards Michael addition. The first solution may be between about 10 and about 50mg/ml in polymer, e.g. between about 30 and 40mg/ml, depending on the degree of functionalisation and molecular weight of the polymer. The solution is commonly an aqueous solution and may comprise a buffer. The buffer may be sufficient to maintain the pH of the solution at between about 5.5 and about 6.5, e.g. about 6.0. Commonly the solution will also comprise a surfactant. The surfactant may be a non-ionic surfactant. It may be an alkylphenyl ethoxylate surfactant. The surfactant may be present in a concentration of up to about 0.1% by weight, e.g. between about 0.01 and 0.1 %, for example about 0.05% by weight.

The second process comprises the steps of: i) providing a precursor solution comprising a precursor species, said precursor species comprising a hydrophilic polymer, a binding species coupled to the hydrophilic polymer by a coupling group, and the bioactive factor, said bioactive factor being bound to the binding species; and ii) reacting the precursor species with a crosslinker to form the microparticles, said crosslinker comprising a crosslinking group which is degradable by a releasing species produced in a cell.

Step i) may comprise

11) providing a first solution comprising the hydrophilic polymer in a first solvent, said hydrophilic polymer having terminal groups capable of reacting with a crosslinker, said crosslinker comprising the crosslinking group;

12) providing a second solution comprising the binding species bound to the coupling species in a second solvent;

13) combining the second solution with a solution of the bioactive factor in an aqueous solvent to form a first mixture comprising the bioactive factor bound to the binding species, said aqueous solvent being miscible with the second solvent; and i4) combining the first mixture and the first solution, thereby coupling the binding species to the hydrophilic polymer by means of the coupling species to form the precursor species.

In this second process, step ii) may comprise the step of combining the precursor solution with a solution of the crosslinker and agitating the resulting mixture sufficiently to crosslink the precursor species and form the microparticles.

Examples

Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying Figures.

Example 1. Preparation of Cell-Responsive Microparticles

Two methods were employed to prepare the cell-responsive particles with bound growth factor. In the Method 1, growth factor was added to the particles in the final step, i.e. after the particles had been synthesized. In the Method 2, growth factor was added during the particle synthesis process, in the first step where heparin binding of the growth factor was allowed to take place. The sequence of reagent addition for particle synthesis is illustrated diagrammatically in Figure 1 for Method 1 and in Figure 2 for Method 2.

The synthesis of cell-responsive particles according to Method 1 proceeded as follows in a modification of the technique for producing monolithic materials described by Lutolf and

Hubbell (2003, supra). The modified method utilised a different reaction pH, a surfactant and a non-polar solvent to obtain microparticles through micelle formation. In addition, the use of covalently attached heparin stabilised the formation of particles and prevented aggregation. pH control was required to obtain the correct net charge of the reacting molecules. It is also noted that control of concentration, temperature and reaction pH was important to maintain a balance of solubility and aggregation of the microparticles.

The commercial sources of the reagents were as follows: 4-arm PEG (Molecular Weight 20 kDa; OH-terminated, NEKTAR), Divinyl Sulfone (Merck), TEOA (Fluka), L-cysteine monohydrate hydrochloride (Merck), and Heparin Sodium Salt from porcine intestinal mucosa (SIGMA, H-9399).

The cysteine functionalisation of heparin was modified from Bernkop-Schniirch et ah, (2001) J. Control. Release 71: 277. Briefly, a 1 % (w/v) solution of heparin (Sigma; (Ci₄H₃₈NiO₃₆Ss)_n, monomer m/wt 1090.87) was prepared in deionized water. l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC, Merck) was added to the heparin solution in a final concentration of 50 niM and reacted for 45 minutes. An equal volume of 0.5 % (w/v) solution of L-cysteine monohydrate hydrochloride (Merck) was then added dropwise to the stirring mixture and the pH was adjusted to 4.0 with 1 M hydrochloric acid. The mixture was stirred for 2 hours at room temperature before the pH was raised to 6.0 and reacted for a further hour.

Cysteine-heparin was purified by dialyzing (Spectrum Laboratories, MWCO 3500) the mixture against 1 mM HCl for 1 hour at 4 °C. This was followed by dialysis twice against 1 mM HCl containing 1 % (w/v) sodium chloride (NaCl, Merck) for 1 hour each at 4 °C and, finally, overnight against 1 mM HCl at 4°C. The purified product was isolated by lyophilization (VirTis BenchTop 4K Freeze Dryer). This synthesis produced a degree of substitution of between 300 and 700 μmol cysteine/gram of product. Solutions of 4-arm PEG-vinyl sulfone (PEG-VS) (33.2 mg/ml) as synthesized by the method of Lutolf and Hubbell (2003, supra) and cysteine-heparin (8.62 mg/ml) were prepared in triethanolamine (TEOA) buffer (pH 6.0) containing 0.05% (v/v) Triton-X 100 (Sigma).

A solution of the MMP -2 and plasmin-sensitive peptide crosslinker GCRDGPQGIWGQDRCG (SEQ ID NO:7) (66.67 mg/ml, GenScript Corporation) was also prepared in TEOA buffer (pH 6.0). As described in Lutolf and Hubbell (2003 supra), this peptide was chosen due to its sensitivity to MMPs and its rapid degradation kinetics. A scrambled peptide GCRDGDQGIAGFDRCG (SEQ ID NO:8) was used as a control to investigate whether degradation of the particles and the resultant release of growth factors was dependent on specific protease activity, rather than as a result of a non-specific hydrolysis. The reactions between the 4-arm PEG-VS and the peptide cross-linker and cysteine- functionalized heparin are based on Michael addition of thiols (from cysteine) to vinyl sulfone. 24.2 μl of the 4-arm PEG-VS solution were mixed with 11.2 μl of the cysteine-heparin solution, which provided a molar ratio of VS groups in the 4-arm PEG-VS to Cysteine (in cysteine- heparin) of approximately 3.33 to 1. The mixture was then added to a dispersion of 7.5 μl of the peptide cross-linker solution in 500 μl of dichloromethane (providing a molar ratio of VS groups (in 4-arm PEG-VS) to cysteine (in the peptide cross linker) of approximately 1 to 3.56).

The concentrations of reagents used above were determined empirically using a series of varying concentrations of reactants and a model peptide to determine which concentrations produced microparticles. The mixture was vortexed in a sealed microcentrifuge tube at a speed setting of 4 to 5 overnight (Scientific Industries Vortex Genie 2) to form an emulsion.

The organic phase (dichloromethane) was evaporated to isolate the particles which were formed, and 200 μl of deionized water was added to the remaining mixture. The resulting particle dispersion was transferred to a centrifugal filter (Whatman UF 10OkD), and centrifuged at 1000 rpm and 4°C for 30 min. The remaining particles were then dispersed in 200 μl of deionized water, and rinsed by centrifugation under similar conditions. Finally, the isolated particles were dispersed by tituration in 200 μl of deionized water and stored for further use.

The particle dispersion was mixed with a known concentration of a growth factor solution (1, 10 or 100 ng/ml of human Fibroblast Growth Factor-2 (FGF-2, R&D Systems), or 10 or 100 ng/ml of recombinant mouse Stromal Derived Factor- 1 (SDF-I, R&D Systems), and incubated for 1 hour with mild shaking on an orbital shaker to bind growth factor to the particles. As this method allows the incorporation of bioactive factors after synthesis of the microparticles, it allows the homogeneous incorporation of a bioactive factor within the material. This may not be the case with a monolithic material, where diffusion of the bioactive factor may be limited and therefore the post-addition of growth factor may not be as homogeneous or effective. In addition, the post-synthetic addition of the bioactive factor allows the bioactive factor to be added shortly before when the microparticles are intended for use, thereby minimising any loss of bioactivity of the factor due to storage.

In a second method, growth factor was added in the first step of the particle synthesis process where heparin-binding of the growth factor was allowed to take place. The synthesis of cell-responsive particles according to Method 2 proceeded as follows. A solution of 4-arm PEG- VS (33.2 mg/ml) and cysteine-heparin (8.62 mg/ml) were prepared in TEOA buffer (pH 6.0) containing 0.05% (v/v) Triton-X as described for Method 1. A solution of the peptide crosslinker GCRDGPQGIWGQDRCG (66.67 mg/ml) (SEQ ID NO:7) was also prepared in TEOA buffer (pH 6.0) as described for Method 1.

300 μl of 10 ng/ml of FGF-2 was mixed with 150 μl of the cysteine-heparin solution. The mixture was vortexed for 2.5 hours at room temperature. 250 μl of the resulting mixture were then added to 180 μl of the 4-arm PEG-VS solution, and vortexed for another 30 min at room temperature. Finally, 230 μl of the growth factor/cysteine-heparin/4-arm PEG-VS mixture were added to 30 μl of the peptide crosslinker solution, and vortexed overnight at 4°C. The particle dispersion obtained was then stored at -2O⁰C for further use.

The presumed relationship between each of the elements of the particles in the assembled particles is illustrated diagrammatically in Figure 3. Cleavage of proteolytically degradable linkers, in Figure 3 labelled MMP-degradable linker, by a protease allows for the protease- mediated release from the particle of small amounts of particle containing the cys-heparin-growth factor.

Particle size distributions were obtained using a ZetaPALS zeta potential analyzer (Brookhaven Instruments Corporation, New York, NY). Method 1 yielded discrete particles which were 200-400 nm (Figure 4a). Method 2 tended to generate larger aggregates of particles (Figure 4b). To investigate the requirement of cysteine-heparin in the microparticles to bind the bioactive factor, two sets of microparticles were synthesised according to Method 1, with one set of particles synthesised without cysteine-heparin and another with cysteine-heparin. In order to demonstrate the successful binding of growth factor to the particles, 100 μl of particles were synthesised according to Method 1 and were exposed to FGF-2 (2 ng/ml) or SDF-I . The particles were then removed from the solution by filtration. The concentration of FGF-2 and SDF-I remaining in solution was measured using commercially available ELISAs (R&D Systems for FGF-2; RayBiotech for SDF-I). The results of this analysis are presented in Figure 5. The presence of cysteine-heparin in the particles was necessary for the binding of growth factor by the particles, as illustrated in Figure 5. Similarly, the synthesis of microparticles according to Method 2 was assessed to determine the sequestration of bioactive factor into the particles at each of the steps of synthesis. The concentration of free growth factor (FGF-2 or SDF-I) was monitored in the supernatant by ELISA as described above at each step of the reaction to investigate whether the growth factor was bound to the particles formed, thereby resulting in a drop in the detected concentration of free growth factor. The results of this analysis are presented in Table 1.

To confirm the microparticles produced were able to be degraded by a protease, growth factor loaded particles (50μl 100ng/ml FGF-2 mixed with 50μl of particle suspension) produced according to Method 2 were exposed to plasmin from human plasma (7.5 μg/ml, Sigma) in tris- buffered saline pH 7.4 . 5 μl of plasmin solution was incubated with 50 μl of particle suspension for 24 hours at room temperature. The protein factors were released upon plasmin treatment, as detailed in Table 2.

Table 1. Changes in concentrations of FGF-2 and SDF-I as measured by ELISA at various stages of the article s nthesis rocess. Initial concentration for both FGF-2 and SDF-I was 100 ng/mL.

Table 2. Changes in concentration of FGF-2 and SDF-I as measured by ELISA upon addition of plasmin to article sus ension.

Example 2 Pellet Culture of microparticles with human Mesenchymal Stem Cells

Two cell culture configurations were used to investigate the effect of FGF-loaded cell- responsive particles on the proliferation of the mouse fibroblast cell line (NIH-3T3, ATCC) and human mesenchymal stem cells (hMSCs, Cambrex). In a transwell culture configuration, the particles are physically separated from the cells, but diffusion is possible between the cells and particles. This assay is relevant to examine the where the particles were used for therapeutic purposes. A pellet culture system, where the cells and particles are cultured in close proximity, is useful for studying the effect of the particles on tissue development, including spatial effects within the pellet. Such a system is comparable to the "neo-tissues" used for concomitant delivery of growth factors and cells to the brain.

A suspension of 2000 cells and 66.67 μl of particles (particle suspension prepared according to Method 2 and pre-loaded with 0, 1, 10 or 100ng/ml of FGF-2) and media (to a total of 700 μl) were placed into a well in a 24-well plate. A transwell insert (Nunc) was then placed into each well and filled with 400 μl of tissue culture media. Cells were then incubated for 2 to 3 weeks, and cell viability assessed using alamarBlue^® assay. The results are presented in Figure 6.

% reduction in Figure 6 refers to the percentage of the cell viability marker, alamarBlue^®, which was reduced from the blue to the red form, indicating the relative number of viable cells, i.e. the higher the % reduction, the greater the number of viable cells.

In the transwell configuration, a higher proliferation of NIH-3T3 cells cultured was observed with FGF-2 -loaded particles, compared to the control (cells without particles). The effect appeared to be more pronounced at lower FGF-2 concentrations, especially at 1 ng/mL.

At this concentration, the heparin binding sites were presumably not saturated with growth factor, allowing the heparin to bind and present other factors present in the serum, such as TGF- β. Cells were cultured in DMEM with 10% fetal bovine serum (FBS). These other factors could provide more potent proliferative signals compared to the bound FGF-2. hMSCs (passage 5 to 20) were cultured in a pellet assay to investigate the ability of microparticles to present growth factors to cells. hMSCs (passage 5 to 20) were trypsinized and counted. 100 μl of the particle dispersion obtained from Method 2 described in Example 1 pre- loaded with FGF-2 at IOng/ml was transferred to a 15-mL centrifuge tube, and a hMSC suspension containing 1 x 10⁵-l .5x 10⁵ cells was added to the particle dispersion. The cell/particle dispersion was mixed with mild agitation, and the mixture was then centrifuged at 56Og for 5 min to obtain a cell/particle pellet. The supernatant was then removed, leaving a pellet with a volume of 200 μl. 1 rnL of fresh media MSCGM BulletKit (Cambrex) was then carefully added.

This same conclusion was borne out by the experiment where hMSCs were cultured with the particles in pellet culture. Particles presenting heparin alone, tethered via the MMP- degradable sequence, appeared to be the most effective in terms of enhancing hMSC proliferation (Figure 7). These were in fact more effective than when FGF-2 was bound to heparin or when the scrambled peptide was used. The relative ineffectiveness of FGF-2 might have resulted from the sequestering of FGF-2 by heparin in a manner whereby receptor binding and activation were inhibited.

Claims

1. A microparticle for delivery of a bioactive factor, the microparticle comprising: a hydrophilic polymer crosslinked with a crosslinking group, the crosslinking group being degradable by a releasing species produced by a cell; and a binding species coupled to the hydrophilic polymer by a coupling group, the binding species being capable of binding and releasing a bioactive factor and whereby exposure of the microparticle to the releasing species causes release of the binding species.

2. The microparticle of claim 1 wherein the hydrophilic polymer is a polyether.

3. The microparticle of claim 1 or claim 2 wherein the hydrophilic polymer is branched.

4. The microparticle of any one of claims 1 to 3 wherein the hydrophilic polymer is a branched polyethylene glycol.

5. The microparticle of any one of claims 1 to 4 wherein the crosslinking group is an oligopeptide or a polypeptide.

6. The microparticle of claim 5 wherein the crosslinking group is an oligopeptide having from about 8 to about 30 amino acids.

7. The microparticle of any one of claims 1 to 6 wherein the releasing species is a endopeptidase.

8. The microparticle of any one of claims 1 to 7 wherein the coupling group is a peptide group.

9. The microparticle of any one of claims 1 to 7 wherein the coupling group is a cysteine residue.

10. The microparticle of any one of claims 1 to 9 wherein the binding species is capable of preventing or inhibiting degradation of the bioactive factor.

11. The microparticle of any one of claims 1 to 10 wherein the binding species is capable of potentiating the biological activity of the bioactive factor.

12. The microparticle of any one of claims 1 to 11 wherein the binding species is heparin.

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13. The microparticle of any one of claims 1 to 12 wherein the bioactive factor is a growth factor.

14. The microparticle of any one of claims 1 to 13 wherein the bioactive factor is FGF-2 or io SDF-I.

15. The microparticle of any one of claims 1 to 15 wherein the particle has a diameter of between about 200 and 400nm.

I₅ 16. The microparticle of any one of claims 1 to 15 comprising a bioactive factor bound to the binding species.

17. A process for making microparticles for delivery of a bioactive factor, said process comprising: 0 a) preparing a precursor microparticle comprising a hydrophilic polymer crosslinked with a crosslinking group, said crosslinking group being degradable by a releasing species produced in a cell, and a binding species coupled to the hydrophilic polymer by a coupling group, said binding species being capable of binding the bioactive factor; and b) binding the bioactive factor to the precursor microparticle to produce the 5 microparticles.

18. The process of claim 17 wherein step a) comprises: al) providing a first solution comprising the hydrophilic polymer in a first solvent, said hydrophilic polymer having terminal groups capable of reacting with a crosslinker, said 0 crosslinker comprising the crosslinking group; a2) providing a second solution comprising the binding species bound to the coupling species in a second solvent; a3) providing a third solution comprising the crosslinker in a third solvent; a4) combining the third solution with a fourth solvent that is immiscible with the first,₅ second and third solvents to form a dispersion; a5) combining the first solution and the second solution, thereby coupling the hydrophilic polymer to the binding species to form a mixture; a6) combining the mixture and the dispersion to form a reaction mix; and a7) agitating the reaction mix sufficiently to crosslink the hydrophilic polymer and form 5 the precursor microparticles in the fourth solvent.

19. The process of claim 18 additionally comprising: a') separating the precursor particles from the fourth solvent; and a") washing the precursor particles.

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20. The process of any one of claims 17 to 19 wherein step b) comprises: bl) combining a suspension of the precursor particles in an aqueous solvent with a solution of the bioactive factor in a solvent that is miscible with the aqueous solvent to form a binding mixture; and

I₅ b2) allowing the binding mixture to react for sufficient time for the bioactive factor to bind to the precursor particles.

21. A process for making microparticles for delivery of a bioactive factor, said process comprising: 0 i) providing a precursor solution comprising a precursor species, said precursor species comprising a hydrophilic polymer, a binding species coupled to the hydrophilic polymer by a coupling group, and the bioactive factor, said bioactive factor being bound to the binding species; and ii) reacting the precursor species with a crosslinker to form the microparticles, saids crosslinker comprising a crosslinking group which is degradable by a releasing species produced in a cell.

22. The process of claim 21 wherein step i) comprises:

11) providing a first solution comprising the hydrophilic polymer in a first solvent, said0 hydrophilic polymer having terminal groups capable of reacting with a crosslinker, said crosslinker comprising the crosslinking group;

12) providing a second solution comprising the coupling species bound to the binding species in a second solvent; 13) combining the second solution with a solution of the bioactive factor in an aqueous solvent to form a first mixture comprising the bioactive factor bound to the binding species, said aqueous solvent being miscible with the second solvent; and

14) combining the first mixture and the first solution, thereby coupling the binding 5 species to the hydrophilic polymer by means of the coupling species to form the precursor species.

23. The process of claim 21 or claim 22 wherein step ii) comprises: iil) combining the precursor solution with a solution of the crosslinker and agitating the I₀ resulting mixture sufficiently to crosslink the precursor species and form the microparticles.