The present disclosure relates dispersions, in particular to water based dispersions (latexes), and solutions of functionalized oligomers, to their synthesis, and to the use of the latexes or solutions in the formation of coating materials for hydrophobic or hydrophilic surfaces. More especially, the disclosure relates to the use of oligomer solutions or latexes for coating surfaces of articles comprising a substrate, material or device for use on or in the human or animal body, and in particular medical devices, to render such surfaces cell adhesive. Thus, preferred oligomers contain functionality which, without wishing to be bound by theory, the inventors believe provides cell adhesive properties to a coated device. The present disclosure further relates to articles, in particular substrates, materials or devices for use on or in the human or animal body, such as medical devices, having a coating which promotes cell adhesion.

The present disclosure further provides a new synthetic route to functionalised coatings which can provide desired properties to a substrate which otherwise lacks such properties, in particular which can provide cell-adhesive properties to an otherwise non- cell adhesive surface of an article. Examples of such surfaces include surfaces of medical devices. In general terms, the synthetic route may typically involve: preparation of a polymer having main chain unsaturation, preferably by emulsion polymerisation of a vinyl monomer with a diene or alkyne; degradation of the polymer to provide oligomers having terminal functional groups, preferably by ozonolysis (optionally, partial ozonolysis) to form an ozonide followed by oxidation or reduction to cleave the ozonide; and reacting the resulting terminal functional groups of the oligomer with one functional group of a bi-functional compound, whereby the other functional group serves to provide the desired properties of the coating, more especially, cell adhesive properties. The coating material may desirably be cross-linked, such as by reaction at main chain unsaturation remaining as a result of the partial ozonolysis. Cross-linking is preferably effected after application of the coating to a surface. Background

A variety of means for producing cell-adhesive surfaces has previously been described. One method is to graft cell-adhesive functionality directly onto articles manufactured from a suitable polymer. Janorkar et al. describe such a procedure, where the bio- absorbable polymer poly (L-lactide) was provided with amine functionality by a sequence of reactions. Firstly, the substrate was functionalised by the photo-induced grafting of 4,4'-diaminobenzophenone, then branched architectures were created by successive reactions with succinic acid and tris(2-aminoethyl amine). The amine functionality conferred upon the poly (L-lactide) enhanced its ability to support fibroblast attachment and viability (J. Biomed. Materials Research Part B: Applied Biomaterials DOI 10.1002.jbmb, page 142-152). Although successful, this method is a lengthy, multistep procedure with all chemical reactions performed directly upon the finished article, making it ill-suited to very sensitive substrates or simple bulk coating practices.

Similarly, scaffolds composed of nanofibres electrospun from polyethersulfone (PES) have been functionalised with amine groups by first photo-grafting poly(acrylic acid) and then further reacting the carboxylic acid functionality with a variety of diamines. The resultant scaffolds were shown to support the proliferation and self-renewal of blood hematopoietic stem/progenitor cells (HSPCs). The degree of success was dependent upon the length of the alkyl chain between the terminal amine groups of the diamine (K.-N. Chua. et al. Experimental Hematology 35 (2007) page 771 -781 ; and K.-N. Chua et al. Biomaterials 27 (2006) page 6043-6051 ). Again, this method is comprised of multiple "wet chemical" steps performed directly upon the finished article.

The modification of extant polymers has also been used to provide surfaces with cell adhesive peptide functionality. Taek Gyoung Kim et al. describe the coupling of RGD peptides to nano-meshes electrospun from poly(D,L-lactic-co-glycolic acid). The cell adhesive peptides were covalently bound to the nano-fibres by grafting to NHS- activated surface functionality. The resultant enhancement of cell proliferation was demonstrated for NIH3T3 fibroblasts (T.G. Kim & T.G. Park, Tissue Engineering 12(2) (2006) pages 221-233). As with all surface modification based procedures, the application of this method involves exposing a biomedical article to lengthy chemical reactions. In addition, such methods may be substrate specific and ill-suited to a variety of substrates by virtue of incompatible surface chemistry or bulk sensitivity to conditions such as elevated temperature. A method reported by Mark J Ernsting incorporates peptides containing RGD and PHSRN sequences into a fluorinated additive which was then blended into a bulk poly- urethane homopolymer. Surface segregation, driven by the low surface energy of the fluorinated modifier, then resulted in enriched levels of accessible peptide functionality and concomitant enhancement of human monocyte adhesion (MJ. Ernsting, R. S. Labow, J. P. Santerre, (J. Biomed. Materials Research Part A, DOI 10.1002.jbma, pages 759-769). This approach enables bioactivity to be conferred upon a material during processing and, because the surface functionality originates from within the bulk, it presumably cannot be permanently removed by abrasion or dissolution. However, it cannot be applied to ready-made articles, non-polymeric materials, nor items made from polymers incompatible with the surface segregation phenomenon. This precludes many implants that are made from metals and composite materials.

Preparation of a material with appropriate functionality that, once synthesized, is suitable for coating onto a variety of substrates is known per se. Such a strategy has previously been used to synthesize peptide-amphiphiles, that is, materials comprised of cell adhesive peptide residues bound to hydrophobic alkyl chains. Such molecules are analogous to the natural lipidated proteins that comprise biological membranes and find use in sensors, diagnostics, and model systems for studying cell-cell binding and receptor-ligand interactions (E. Kokkoli et al. Soft Matter (2006) 2, pages 1015-1024). Block copolymers comprised of linked polypeptide and non-polypeptide domains have also been produced and found to self assemble in solution, forming ordered nanostructures with potential utility in drug delivery (D. W. P. M. Lowik, Chem. Soc. Rev. (2004) 33, pages 234-245).

The inventors have previously described in Biomaterials 28 5319 (2007) that hydrogels which are otherwise non cell-adhesive can be modified to produce cell adhesive surfaces. In the described process, hydrogels having glycidyl ether side chains are modified by amination with ammonia or a diaminoalkane to provide side chains with primary amine terminal groups. The length of the alkyl chain of the diaminoalkane was found to be important in determining whether cell adhesive properties were obtained.

Measurement of Cell Adhesion. A variety of means may be used to measure the promotion of cell adhesion by the oligomers of the present disclosure. Preferred methods are those that permit the direct comparison of coated and uncoated articles, in order that the degree of cell adhesion can be readily discerned.

Cell growth, cell viability and cell counting assays may be used. Favoured cell types for assessing the materials of the invention are those specific to the application of a particular coated article. For example, human endothelial cells would be used to test coated coronary stents. Alternatively, results for broad, comparative purposes may be gained using a "general purpose" cell such as the human fibroblast (the NIH3T3 and MC3T3 lines have previously been found to be suitable).

The number of cells adhered to a sample cultured in a suitable medium may be assessed using a Coulter Counter. Alternatively, optical or electron microscopy of a cultured sample followed by image analysis or manual counting may be used (Janorkar et al. J. Biomedical Mater. Research Part B: Appl. Biomaterials DOI 10.1002/jbmb).

The viability of cells attached to a coated article can be assessed by trypan blue exclusion or a Live/Dead assay, such as that marketed by Molecular Probes lnc (Ernsting et al. J. Biomedical Mater. Research Part A DOI 10.1002/jbm.a). The results of the latter procedure are amenable to quantitative assessment by fluorescence microscopy and image analysis.

A further measure of the degree of cell spreading can also be obtained by image analysis. Coated articles which are exposed to a cell containing medium may be dried and analysed by Scanning Electron Microscopy (SEM). Manual cell counting in combination with image analysis can then provide a value for the surface-area per cell. Such measurements of cell morphology can provide a valuable indication of cell preferences (Ernsting et al. J. Biomedical Mater. Research Part A DOI 10.1002/jbm.a).

Another means of assessing the quantity of viable cells on a coating material is by an MTT assay. This well-known colorimetric test has the benefit of yielding absorption figures that enable ready, quantitative comparison of various material embodiments of the invention.

According to a first aspect of the present disclosure, there is provided an article having a substantially non-cell adhesive surface and comprising on said surface a coating effective to render the article cell adhesive, wherein the coating comprises an oligomer having terminal amine functionality or peptide, peptide analogue or peptide derivative functionality. Preferably, but not essentially, the coating covers the whole of said non- cell adhesive surface. The article may comprise more than one such non-cell adhesive surface, the respective non-cell adhesive surfaces being made from the same materials or from different materials. Preferably, but not essentially, where the article comprises a plurality of non-cell adhesive surfaces, the coating covers all of said surfaces. The coating may cover the whole article, or part of the article. In some preferred embodiments, the coating covers the whole article.

According to a second aspect of the present disclosure there is provided a coating composition adapted to coat an article having a substantially non-cell adhesive surface and to render the surface, when coated, cell adhesive, wherein the coating composition comprises an oligomer having terminal amine, or peptide, peptide analogue or peptide derivative functionality.

In the present disclosure a "non-cell adhesive surface" is defined as a surface which supports less than 30% of the cells per unit area compared to the same type and batch (passage) of cells cultured on tissue culture plastic in cultures that are carried out over the same time period. (As is well known in the art tissue culture plastic is a commercially available polystyrene that has been rendered wettable by oxidation, thereby to increase its adhesiveness for cells).

In the present disclosure an "oligomer" is, in a broader sense, a substance composed of oligomer molecules. An oligomer molecule is broadly defined as a molecule of intermediate relative molecular mass the structure of which comprises a small plurality of units derived, conceptually or actually, from molecules of lower relative molecular mass. Typically, a molecule is regarded as having an intermediate relative molecular mass if it has properties which vary significantly with the removal of one or a few of the units.

More specifically, in the context of the present disclosure an oligomer is defined as a material comprising units (more especially, repeating units) of lower relative molecular mass and with a number average molecular weight of 500 - 10,000 g mol. ^"1 . Particularly useful oligomers are those with number average molecular weights suitable for coalescence of the latex particles into a coherent film at drying temperatures between 15 and 50 ⁰ C. Preferably the coating composition comprises a dispersion or solution of the oligomer.

In preferred embodiments the article is a substrate, material or device for use on or in the human or animal body.

In particular embodiments the device is a medical device selected from surgical repair materials, implants such as hernia repair material, tendon repair material, stents such as coronary stents, peripheral vascular stents, abdominal aortic aneurysm (AAA) devices, biliary stents and catheters, TIPS catheters and stents, vascular grafts and stent grafts, gastro enteral tubes or stents, gastro enteral and vascular anastomotic devices, indwelling metal implants, bronchial tubes and stents, vascular coils, vascular protection devices, tissue and mechanical prosthetic heart valves and rings, arterial- venous shunts, AV access grafts, dental implants, CSF shunts, pacemaker electrodes and leads, suture material, wound healing, ocular implants, hearing aids including cochlear implants, implantable heart support and vascular systems, indwelling vascular access catheters and associated devices, e.g., ports, maxilo fascial implants, orthopaedic implants and screws e.g. joint replacement, trauma management and spine surgery devices, implantable devices for plastic and cosmetic surgery, implantable meshes, e.g., for hernia or for uro- vaginal repair, brain disorders, gastrointestinal ailments, artificial veins and arteries, stents, ocular devices.

In some preferred embodiments the terminal amine functionality is a primary amine or a quaternary amine.

In some preferred embodiments the terminal peptide functionality comprises an oligomer of from 3 to 20 amino acid residues in length. Thus the coating composition of the present disclosure may comprise an oligomeric backbone having terminal groups with peptide functionality. The terminal groups may themselves be oligomeric, being of from 3 to 20 amino acids in length. In various preferred embodiments the terminal peptide functionality comprises an oligomer of from 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues to 14, 15, 16, 17, 18, 19 or 20 amino acid residues in length.

Preferably the peptide, peptide derivative or peptide analogue is selected from those including RGD sequences, PHSRN sequences, GRGDS sequences, GRGDY sequences and YIGSR sequences. In particularly preferred embodiments the oligomer is represented by

"∞ ^"

Formula (1A) where E represents the moiety

G represents the moiety

(n1 +n2+n3+n4) = n = an integer from about 10 to about 100 and ml is between 0 and 0.01 n Q represents A ^A -R-B ^A where

A ^A represents a linking group between R and C ¹ ;

B ^A represents the terminal amine functionality which confers cell adhesive properties to the compound of Formula (1A);

R represents a branched or unbranched, substituted or unsubstituted alkyl chain having from 2 to 12 carbon atoms, optionally interrupted by one or more moieties selected from secondary or tertiary amine moieties, -O-, -S-, -CO-, amide (-CONR"-) and ester (-COOR"-) where R" represents H or Ci to C _i2 alkyl, or a polymeric moiety, preferably a poly(ethylene oxide), poly(propylene oxide), poly(tetramethylene oxide), poly(ethylene oxide-co-propylene oxide) or poly(ethylene oxide-co-tetramethylene oxide) moiety; or Q represents (A ^p ) _k -B ^p where

A ^p represents a linking group between C ¹ and B ^p , k is 0 or 1 and B ^p is a peptide, peptide derivative or peptide analogue which confers cell adhesive properties to the compound of Formula (1A) R ¹ and R ² may each independently represent H, Ci to Ci ₂ alkyl, phenyl, substituted phenyl, -COOR ³ , -OCOR ³ , -CONHR ³ , SO ₂ R ³ where R ³ is Ci to C _i2 alkyl, provided that at least one of R ¹ and R ² is H, X represents substituted or unsubstituted phenyl or COOR ⁴ where R ⁴ represents a Ci to Ci ₂ alkyl group, Y represents H or CH ₃ and

-[-CO-E _n2 -CO-G _mI -CO-E _n3 -Co-]- indicates random placement of the E and G moieties in the main oligomer chain.

In the above embodiment each respective Ci to C ₁₂ alkyl moiety may independently be Ci alkyl, C ₂ alkyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C _i0 alkyl, Cu alkyl, or C ₁₂ alkyl. In particular such alkyl groups may be selected from Ci to C ₄ alkyl, C ₂ to C _i0 alkyl, C ₂ to C ₈ alkyl, C ₄ to C _i0 alkyl and C ₄ to C ₈ alkyl.

In preferred variations of the above embodiment R may represent a branched or unbranched, substituted or unsubstituted alkyl chain having from 2 to 10 carbon atoms, or from 2 to 8 carbon atoms or from 2 to 6 carbon atoms, or from 4 to 12 carbon atoms or from 4 to 10 carbon atoms or from 4 to 8 carbon atoms. In specific variations, R may represent such an alkyl chain having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.

In some preferred embodiments n may be an integer from about 20 to about 80, such as from about 30 to about 70 or about 40 to about 60. In other preferred embodiments n may be an integer from about 10 or 20 to about 60 or from about 10 or 20 to about 40. In further preferred embodiments n may be an integer from about 40 or 50 to about 90 or 100 or from about 40 to about 80. In further preferred embodiments n may be an integer of from about 50 or 60 to about 90 or 100 or from about 50 or 60 to about 70 or 80.

In some preferred embodiments ml is >0. It is advantageous for ml to be >0 where cross linking of the composition is required, since unsaturation is thus present in the oligomer.

In other preferred embodiments ml is 0.

In some preferred embodiments the oligomer is cross linked. Cross linking can improve the retention of the coating on the coated substrate, and the durability of the coating. In preferred embodiments where the oligomer is cross-linked the cross linking is effected by reaction at the unsaturated portion of at least some of the G moieties.

In some preferred embodiments A ^A is an amine residue. More particularly A ^A — R — B ^A may represent -HN-R-B ^A . A ^A — R— B ^A may represent -HN-(CH ₂ ) _P -B ^A where p is an integer from 1 to 8. In typical examples p may be an integer from 2 to 7 or an integer from 3 to 6 or an integer from 1 to 4. In particular p may be selected from 1 , 2, 3, 4, 5, 6, 7 and 8.

In some embodiments B ^A may be selected from -NR ^a R ^b where R ^a and R ^b may be the same or different and are selected from H or C ₂ , C ₃ , C ₄ , C ₆ C ₈ , Ci ₀ or Ci ₂ alkyl. More ppaarrttiiccuullaarrllyy BB ^AA mmaayy bbee --NNRR ^aa RR ^bb wwhheerree R ^a and R ^b are independently H or C ₂ to C ₄ alkyl, such as C ₂ alkyl, C ₃ alkyl or C ₄ alkyl.

One of R ^a and R ^b may be H. In some preferred embodiments both R ^a and R ^b are H.

In further preferred embodiments Q may be (A ^p ) _k B ^p and k is 0.

In other preferred embodiments Q may be (A ^p ) _k B ^p and k is 1.

In some preferred embodiments B ^p represents a peptide, peptide analogue or peptide derivative comprising an oligomer of from 3 to 20 amino acid residues in length. In various preferred embodiments the oligomer may be from 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues to 14, 15, 16, 17, 18, 19 or 20 amino acid residues in length. More especially B ^p may be a peptide, peptide derivative or peptide analogue selected from those including RGD sequences, PHSRN sequences, GRGDS sequences, GRGDY sequences and YIGSR sequences.

According to a third aspect of the present disclosure there is provided a method of coating an article having a non-cell adhesive surface with a coating effective to render the surface cell adhesive, the method comprising applying to the surface a coating composition according to the second aspect of the disclosure.

According to a fourth aspect of the present disclosure there is provided a method of coating an article having a non-cell adhesive surface with a coating effective to render the surface cell adhesive, the method comprising: applying to the surface a coating cross-linkable composition of the second aspect of the disclosure, and cross-linking the oligomer by reaction at the unsaturated portion of at least some of the G moieties.

In preferred embodiments of the third and fourth aspects of the disclosure the article is a substrate, material or device for use on or in the human or animal body.

In some preferred embodiments the coating is applied by dipping the article in the coating composition or by spraying the article with the coating composition.

According to a fifth aspect of the present disclosure there is provided a method of preparing a material of Formula (1A):

Formula (1A) the method comprising: reacting a compound of Formula (2A) -- ^L'

Formula (2A) with a compound of formula A ^A1 — R — B ^A or with a compound of the formula wherein

E represents the moiety

G represents the moiety

(n1 +n2+n3+n4) = n = an integer from about 10 to about 100 and ml is between 0 and 0.01 n Q represents A ^A -R-B ^A where

A ^A represents a linking group between R and C ¹ ; B ^A represents the terminal amine functionality which confers cell adhesive properties to the compound of Formula (1A).

R represents a branched or unbranched, substituted or unsubstituted alkyl chain having from 2 to 12 carbon atoms, optionally interrupted by one or more moieties selected from secondary or tertiary amine moieties, -O-, -S-, -CO-, amide (-CONR"-) and ester (-COOR"-) where R" represents H or Ci to C _i2 alkyl, or a polymeric moiety, preferably a poly(ethylene oxide), poly(propylene oxide), poly(tetramethylene oxide), poly(ethylene oxide-co-propylene oxide) or poly(ethylene oxide-co-tetramethylene oxide) moiety, or Q represents (A ^p ) _k -B ^p where A ^p represents a linking group between C ¹ and B ^p , k is 0 or 1 and B ^p is a peptide, peptide analogue or peptide derivative which confers cell adhesive properties to the compound of Formula (1A)

R ¹ and R ² may each independently represent H, Ci to Ci ₂ alkyl, phenyl, substituted phenyl, -COOR ³ , -OCOR ³ , -CONHR ³ , SO ₂ R ³ where R ³ is Ci to C _i2 alkyl, provided that at least one of R ¹ and R ² is H,

X represents substituted or unsubstituted phenyl or COOR ⁴ where R ⁴ represents a Ci to Ci ₂ alkyl group, Y represents H or CH ₃ and

-[-CO-E _n2 -CO-G _mI -CO-E _n3 -Co-]- indicates random placement of the E and G moieties in the main oligomer chain,

A ^A1 represents a moiety which is a precursor of A ^A and is susceptible to bond forming reaction with C ¹

A ^P1 represents a moiety which is a precursor of A ^p and is susceptible to bond forming reaction with C ¹ , and

L and L' may be the same or different and represent a leaving atom or group which is displaced on reaction of A ^A1 , A ^P1 or (when k is O) reaction of B ^p respectively at C ¹ . he above embodiment each respective Ci to Ci ₂ alkyl moiety may independently be lkyl, C ₂ alkyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C ₇ alkyl, C ₈ alkyl, C ₉ alkyl, C _i0 alkyl, Cu alkyl, or Ci ₂ alkyl. In particular such alkyl groups may be selected from Ci to C ₄ alkyl, C ₂ to C _i0 alkyl, C ₂ to C ₈ alkyl, C ₄ to C _i0 alkyl and C ₄ to C ₈ alkyl.

In preferred variations of the above embodiment R may represent a branched or unbranched, substituted or unsubstituted alkyl chain having from 2 to 10 carbon atoms, or from 2 to 8 carbon atoms or from 2 to 6 carbon atoms, or from 4 to 12 carbon atoms or from 4 to 10 carbon atoms or from 4 to 8 carbon atoms. In specific variations, R may represent such an alkyl chain having 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms.

In some preferred embodiments n may be an integer from about 20 to about 80, such as from about 30 to about 70 or about 40 to about 60. In other preferred embodiments n may be an integer from about 10 or 20 to about 60 or from about 10 or 20 to about 40. In further preferred embodiments n may be an integer from about 40 or 50 to about 90 or 100 or from about 40 to about 80. In further preferred embodiments n may be an integer of from about 50 or 60 to about 90 or 100 or from about 50 or 60 to about 70 or 80.

In some preferred embodiments L and L' may be the same or different and are selected from H, Hal, OH, OR ^L or OHaI where R ^L is Ci, C ₂ , C ₃ or C ₄ alkyl and Hal is halogen, preferably chlorine.

In preferred embodiments the compound of Formula (2A) is prepared by ozonolysis followed by oxidative or reductive work-up of a compound of Formula (3A)

mula (3A)

where X, Y and n are as defined in claim 29 and m is between 0.2n and 0.01 n.

In some preferred embodiments of this fifth aspect of the disclosure ml =0. In other preferred embodiments of this fifth aspect of the disclosure the ozonolysis step is a partial ozonolysis such that m1 >0. Preferably the step of cross-linking the oligomer is effected by reaction at the unsaturated portion of at least some of the G moieties.

In preferred embodiments the compound of Formula (3A) is present in the form of a dispersion, preferably an aqueous dispersion, or a solution.

Preferably the compound of Formula (1A) is produced in the form of a dispersion, preferably an aqueous dispersion, or a solution.

In some preferred embodiments of the fifth aspect of the disclosure A ^A1 is an amine group, preferably a primary amine group. In some embodiments A ^A1 — R — B ^A may represent H ₂ N-R-B ^A . In particular A ^A1 —R—B ^A may represent H ₂ N-(CH ₂ ) _p -B ^A where p is an integer from 1 to 8. In typical examples p may be an integer from 2 to 7 or an integer from 3 to 6 or an integer from 1 to 4. In particular p may be selected from 1 , 2, 3, 4, 5, 6, 7 and 8.

In some preferred embodiments of this aspect A ^A is an amine residue. A ^A — R — B ^A may represent -HN-R-B ^A . A ^A — R— B ^A may represent -HN-(CH ₂ ) _P -B ^A where p is an integer from 1 to 8. In typical examples p may be an integer from 2 to 7 or an integer from 3 to 6 or an integer from 1 to 4. In particular p may be selected from 1 , 2, 3, 4, 5, 6, 7 and 8.

In some preferred embodiments B ^A may be selected from -NR ^a R ^b where R ^a and R ^b may be the same or different and are selected from H or Ci, C ₂ , C3 or C ₄ alkyl. In particular B ^A may be -NR ^a R ^b where R ^a and R ^b are independently H or Ci to C ₄ alkyl.

In some preferred embodiments one of R ^a and R ^b is H. In other preferred embodiments both R ^a and R ^b are H.

In other preferred embodiments of the fifth aspect of the disclosure Q is (A ^p ) _k B ^p and k is O.

In other preferred embodiments of the fifth aspect of the disclosure Q is (A ^p ) _k B ^p and k is 1. Preferably in these latter embodiments B ^p represents a peptide, peptide derivative or peptide analogue comprising an oligomer of from 3 to 20 amino acid residues in length. In various preferred embodiments the oligomer may be from 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues to 14, 15, 16, 17, 18, 19 or 20 amino acid residues in length. Preferably the peptide, peptide derivative or peptide analogue is selected from those including RGD sequences, PHSRN sequences, GRGDS sequences, GRGDY sequences and YIGSR sequences.

Mechanisms of Cell Adhesion

In general terms, there are two mechanisms by which cell adhesion at a surface may be effected. Firstly, binding may occur by direct interaction of a moiety present at the surface (such as part of a coating) with specific binding sites on cells present at the surface. Such a moiety may be the amine or peptide moiety in the present disclosure. Alternatively, a suitable moiety at the surface may bind to adhesive compounds secreted by the cells. This latter, indirect, mode of cell binding is often mediated by proteins of the extracellular matrix such as collagen, fibronectin, vitronectin, and laminin. The binding of such proteins to the coated surface can be non-specific in nature, caused, for example, by charge-density interactions between the protein and the moiety at the surface.

Amine functionalities, particularly primary amines, have been found to be effective in the oligomer of the disclosure. Without wishing to be bound by theory the applicant believes that amine moieties can be enzymatically active and may promote cell adhesion by acting as lysine-like units. That is, they promote direct binding of the coating to components of the extracellular matrix, which then provides a suitable environment for the cells at the coated surface. However, an alternative explanation of the ability of the oligomers including terminal amine moieties to promote cell growth, is their ability to become positively charged in solution by protonation. This phenomenon can promote electrostatic charge-to-charge interaction with negatively charged antigen proteins on cell surfaces (K. -N. Chau et al. Experimental Hematology 35 (2007) p771- 781 ). Alternatively, the positive charge may selectively enrich protein components within the cell growth medium that contribute to cell proliferation and expansion (K. -N. Chau et al. Biomaterials 27 (2006) p6043-6051 ). Cell adhesion may also be promoted by the selection of quaternary ammonium salts as the terminal amine moiety. Quaternary ammonium salts are thought to promote the non-specific binding of cells and their proteins by virtue of electrostatic interactions with areas of charge density. Quaternary ammonium salts may hence be favoured in applications where indirect, non-specific binding of cells is desired.

The terminal functionality of the oligomers of the present disclosure may alternatively comprise a biological molecule, most especially a peptide, peptide analogue or peptide derivative.

Favoured peptides are preferably oligomers of between 3 and 20 amino acid residues in length. Peptides particularly suited to application within the present disclosure comprise sequences that are capable of direct and specific interaction with receptors of cells at the coated surface. Sequences known per se to promote cell adhesion include the RGD and PHSRN sequences. These sequences promote the adhesion and spreading of numerous cell types, including monocytes and macrophages (Ernsting et al. J. Biomedical Mater. Research Part A DOI 10.1002/jbm.a). Other cell adhesive sequences known in the art include GRGDS, GRGDY and YIGSR.

The choice of a particular sequence will depend, amongst other considerations, upon the type of cell that the user desires to promote adhesion of in their particular application. For example, RGD sequences are particularly useful where the desired mode of cell attachment occurs in vivo via extracellular matrix proteins that possess said cell recognition motif (Kim and Park, Tissue Engineering, 12(2) (2006), p221 -233).

For a better understanding of the invention and to show how the same may be carried into effect, reference will be made to the following drawings in which:

Figure 1 which shows schematically the steps in preparing a coating material and applying the coating to a substrate in accordance with the present disclosure;

Figure 2 shows schematically steps in the formation of a coating from the oligomer latex according to the present disclosure;

Figure 3 illustrates schematically the formation of a cross-linked coating; Figure 4 shows particle sizes for the three oligo(BMA) diamines produced from the reactions with oligo(BMA)-2COOH and selected diamines: n =2, ethylenediamine, n=4, 1 ,4-diaminobutane and n=6, 1 ,6-diaminohexane. Means ± SEM (for 3 replicates) are reported;

Figure 5 shows MTT results to assess the viability of fibroblast cells growing on compressed layered non-coated and coated meshes and tissue culture plastic (TCP). Means ± SEM (for 3 replicates) are reported; and

_TM Figure 6 shows confocal images of cells stained with Red CellTracker" ^vl coated with oligo(BMA) diamine: (a) n=2 coated; (b) n=4; (c) n=6. Scale bar = 20μm.

Referring now to Figure 1 , the first stage in the synthesis of the oligomer of the present disclosure is a polymerisation, preferably an emulsion polymerisation, which provides a material of Formula (3A), preferably in the form of a latex, as illustrated generally by Scheme 1 .

Formula 3.1 Formula 3.2 Formula (3A)

Scheme 1

where Formula 3.1 represents generically a suitable diene where R ¹ and R ² may each independently represent H, alkyl, phenyl, substituted phenyl, -COOR ³ , -OCOR ³ , - CONHR ³ , SO ₂ R ³ where R ³ is alkyl, provided that at least one of R ¹ and R ² is H, X represents substituted or unsubstituted phenyl or COOR ⁴ where R ⁴ represents an alkyl group, Ci to Ci ₂ , Y represents H or CH ₃ , n represents an integer from 10 to 100 and m is between 0.2n and 0.01 n.

In a second stage, the polymer latex of Formula (3A.1 ) resulting from the first stage is subject to ozonolysis to form an intermediate ozonide followed by a work-up to cleave the double bond. The work-up may be an oxidation or a reduction of the ozonide depending on the desired final product (the oligomer of Formula 2.1 ), preferably in the form of a latex. This is shown generally by Scheme 2 in which for the convenience in this example, R ¹ = R ² = H and the work up step is an oxidation. Oxidative work up is generally preferred in the present disclosure and will tend to give a carboxylic product, that is where L and L' are OH in Formula 2.1. Reductive work-up conditions can yield aldehydes or ketones, depending on the substitution of the starting alkene.

Formula (3A.1 ) Scheme 2

The resultant oligomer of Formula 2.1 thus includes terminal groups

and ^cr

\r ^ o where L and L' may be the same or different and independently represent a leaving group or atom which allows for further functionalisation of the oligomer in the subsequent reaction stage. L and L' may, for example be the same or different and may be selected from H, Hal, OH, 0R ^L or OHaI where R ^L is Ci, C ₂ , C ₃ or C ₄ alkyl and Hal is halogen, preferably chlorine.

Suitable conditions for performing the polymerisations and the ozonolysis and oxidation steps can be seen in J. Polymer Science 35 3255-3262 (1997) and will be known to the person skilled in the art. For example, for the preparation of terminal carboxylic acid groups, an oxidative work-up step may be effected by treatment with SeO ₂ ZH ₂ O ₂ .

In Macromol. Rapid Comms 18 723 (1997) there is described the modification of an oligomer of Formula 2.1 in which the terminal groups are a carboxylic acid by amidation with a mono-functional amine. The amidation causes the oligomer dispersion to coagulate into a form which cannot easily be re-dispersed in water. In the present disclosure, the material of Formula 2.1 is modified by a bi-functional compound to form the oligomer of Formula (3A). The resulting product may be have colloidal stability, that is, the product is maintained in (preferably aqueous) dispersion, or, the product may be allowed to precipitate from the reaction mixture and then dissolved in a suitable solvent.

Thus, in a third stage, in one variation of the present disclosure, the oligomer of Formula 2.1 , preferably in the form of a latex, is reacted with a bi-functional compound A ^A1 -R-B ^A which, in the particular example shown below in Scheme 3, is a diamine H ₂ NRNH ₂ . Again, for the purpose of this example, R ¹ = R ² = H and L = L' = OH.

Formula 1.1

In a typical reaction according to Scheme 3 the ingredients (the oligomer, preferably in the form of a latex, diamine and EDC) are added to the reaction vessel with vigorous stirring. The reaction medium is continuously agitated by mechanical stirring at room temperature until all of the carboxylic acid groups have been reacted. Typically, reaction times range from about 4 to about 24 hours.

In another variation, the oligomer of Formula 2.1 , preferably in the form of a latex, is reacted with a peptide, peptide analogue B ^p or with a modified peptide or peptide analogue or a peptide derivative A ^p -B ^p .

Typically R may represent a branched or unbranched, substituted or unsubstituted alkyl chain having from 2 to 12 carbon atoms, optionally interrupted by one or more moieties selected from secondary or tertiary amine moieties, -O-, -S-, -CO-, amide (-CONR"-) and ester (-COOR"-) where R" represents H or alkyl and in particular R is Ci to Cs alkyl. Alternatively R may be a polymeric moiety, in particular a poly(ethylene oxide), poly(propylene oxide), poly(tetramethylene oxide), poly(ethylene oxide-co-propylene oxide) or poly(ethylene oxide-co-tetramethylene oxide) moiety.

Thus in one variation of the present disclosure the oligomer, preferably in latex form, has primary amine functionality at each terminal of the oligomer chain. The inventors believe that this amine functionality promotes cell adhesion to a coating comprising the oligomer. In an alternative variation, the oligomer, also preferably in latex form, has a peptide, peptide analogue or peptide derivative at the respective ends of the oligomer chain. The inventors also believe that the peptide, peptide derivative or peptide analogue promotes cell adhesion to a coating comprising the oligomer.

The oligomer of the present disclosure, such as that of Formula (1A), preferably formulated as a dispersion or latex (in particular an aqueous dispersion) or as a solution, is used to coat articles, in particular the surfaces of medical devices. In this way, an article having a surface which is otherwise non-cell adhesive can, by virtue of its coating, be made such that cell-adhesion is promoted. The properties of the oligomer can be varied, by varying the oligomer composition, in accordance with the nature of the surface to be coated, to promote effective coating, or in accordance with a particular end use. For example the properties of the oligomer of Formula (1A) can be adapted by changing the identity of one or more of X, Y, R, A ^A , A ^p , k, B ^A , B ^p , n and ml .

Thus, the nature of the binding between adhered cells and the oligomer coating at the surface depends upon the choice of amine or peptide moiety. It will readily be appreciated that certain applications of the oligomers of the present disclosure will favour the employment of terminal moieties that favour a particular binding mechanism. This may be done in order to promote the adhesion of a distinct cell type or cell product, at the expense of others that may be present in the environment local to the coated surface. A specific example of this is the acknowledged need for surfaces that promote the adhesion of fibronectin at the expense of albumin, a competing protein that can passivate surfaces against subsequent cell adhesion.

For example, where Formula 3.2 represents butyl methacrylate (Formula 3.2.1 )

Formula 3.2.1 the resultant oligomer has good coating adhesion to poly(glycolide-lactide) fibres such as used in Vicryl™ (polygalactin 910) mesh from the Ethicon division of Johnson & Johnson which is used in hernia repair.

In other examples, for adhesion to materials such as steel, the oligomer of the present disclosure, such as the oligomer of Formula (1A), may be cross-linked, preferably by means of remaining main-chain unsaturation as a result of partial ozonolysis. Typically the cross-linking may be effected by gamma or electron beam irradiation or by treatment with sources of free radicals in appropriate reaction conditions such as heat or uv light.

Application of the oligomer of the present disclosure to a material to be coated may be achieved by various means. In particular, the material to be coated may be spray coated or dip coated. Figure 1 illustrates schematically both these alternatives. In general, the dip coating process is preferred. Following the coating stage, the coated article is allowed to dry naturally by evaporation of the continuous phase (water) from the latex, or (where the oligomer is provided as a solution) by evaporation of the solvent. Alternatively, drying may be promoted by applying a heat source to the coated article, such as an infra red lamp.

As noted, the oligomer can be provided as a coating material in the form of a latex, where a colloidally stable formulation can be prepared. The incorporation of hydrophilic groups such as amine and peptides to the oligomer of the disclosure can assist in conferring stability to the latex. Where the oligomer does not form a stable colloid, the oligomer can be separated from the continuous phase and dissolved in a suitable solvent. Generally, colloidally stable formulations are preferred. The ability of preferred embodiments of the invention to form stable colloids in water is advantageous for a number of reasons. Latexes may be easily coated onto substrates by a variety of low- cost means such as dipping and spraying. Mild conditions, in particular using water as the continuous phase, render the method of the disclosure particularly suited to sensitive substrates as any process chemistry which might potentially have disadvantageous effects on the substrate is performed "off-line", that is, prior to the coating of the article.

The composition and morphology of the small oligomeric particles (preferably 50 to 800 nm in diameter) that comprise the coatings of the disclosure in latex form are of further benefit because they readily coalesce and form coherent films on substrates which films are free of defects and pinholes (as compared to high molecular weight polymer coatings).

Thus, on coating of the material or article to be coated, the oligomers (in particular when present as a latex) tend to coalesce easily, in contrast to higher molecular weight polymers, thereby forming an effective coating. Aspects of the coating process are shown schematically in Figure 2. It is noted that the oligomers of the present disclosure (such as those of Figure (1A)) are primarily hydrophobic but that the terminal functional groups (amine groups or peptides, peptide analogues or peptide derivatives) which promote cell adhesion are not. Thus, the terminal functional groups tend to migrate towards the free surface of the coated article, thereby providing a surface which promotes cell adhesion.

For articles or materials where coating adhesion is otherwise insufficient, or where a greater longevity or durability of the coating is desired, the oligomer can be selected to be cross-linkable. Thus, after the material or article has been coated, the coating is subjected to a cross-linking step, as shown schematically in Figure 3. Preferably, cross- linkable properties can be imparted to the oligomer of Formula (1A) by effecting only partial ozonolysis as described above. However, other means of incorporating cross- linkable functionality, such as by introducing alternative groups (such as pendant cross- linkable groups) into the main oligomer chain are not excluded. In such cases, however, care need to be taken to ensure that cross-linking reactions do not modify or otherwise impair the function of the terminal amine groups or peptide, peptide analogue or peptide derivative groups of the oligomer.

In other applications, a coating stage as such may not be required. For example, the oligomer of the present disclosure may be blended with components of the final article during the manufacturing process. This is, in particular, applicable to the manufacture of fibres from which medical devices are made, or to moulding processes for the formation of medical devices.

Examples

Example 1 - Synthesis of oligo(butyl methacrylate) diamines

A poly(butyl methacrylate-co-butadiene ) ["poly(BMA-co-BD)"] latex was prepared by a starve fed monomer emulsion polymerisation:-

Copolymers of butyl methacrylate and butadiene were synthesized. The experimental outline is described below and the typical quantities used are indicated in Table 1. D0WFAX2A1 (available from The Dow Chemical Company), potassium hydrogen phosphate and β cyclodextrin were added to a beaker containing distilled water (450ml) and a magnetic stirrer bar. These components were stirred until all the potassium hydrogen phosphate and cyclodextrin had dissolved. This aqueous solution was then charged to a reactor, and to deoxygenate, nitrogen was bubbled through with agitation for 1 hour. At the same time the contents of the reactor were heated to 7O ⁰ C ± 1 ⁰ C by circulating hot water through the jacket of the reactor. Separately, butyl methacrylate was added to a 2-neck round bottom flask and again with agitation was deoxygenated with nitrogen for 1 hour. Potassium persulphate was dissolved in distilled water (50ml) and purged with nitrogen for 1 hour. Once the aqueous solution in the reactor had reached 7O ⁰ C, the aqueous solution of Potassium persulphate was added to the reactor. Monomer addition was then started: deoxygenated butyl methacrylate and butadiene were simultaneously added to the contents of the reactor at a constant rate. Upon consumption of the monomers the resulting white latex was removed from the reactor and stored in the fridge at 4 ⁰ C.

Material Quantity Function

D0WFAX2A1 3.6 g Surfactant

Water 500ml Dispersing Medium

Potassium Hydrogen

0.4g pH Buffer Phosphate

Potassium Persulphate 1.2g Initiator

Butadiene 7.4g (« 4000ml) Monomer

Butyl methacrylate 95g (= 100ml) Comonomer 3.6g Butadiene Solubility Aid β Cyclodextrin

Table 1 : Monomer Starve-fed emulsion polymerization reaction formulations β cyclodextrin is added in order to increase the water solubility of butadiene in water. This obviates the need to carry out the reaction under pressures that are higher than 12 atmosphere.

Preparation of oligo(BMA)-2COOH latex (Formula 2.1.2) by ozonolysis

The poly (BMA-co-BD) latex (200ml) was added to a 1 L 3- neck round bottom flask, with a magnetic stirrer bar. To this was added an equal volume of distilled water (200ml) to dilute the latex. Toluene (50ml) was then added via a dropping funnel over a period of 4 hours; with magnetic stirring at room temperature. The swollen latex was left to stir at room temperature for a period of 24 hours before ozonolysis was carried out. A reflux condenser was attached to the round bottom flask, and the flask submerged into an ice bath. An inlet was added for introduction of ozone, and the condenser attached to an outlet for ozone. Ozone was generated, by passing oxygen through an electrical discharge generator (Type BA, Wallace & Tiernan, UK), at a rate of 1.74g of ozone h ^"1 ; and was bubbled through the latex for a period of 6 hours, at which point, the ozone inlet was substituted for a nitrogen inlet, and the reaction mixture purged with nitrogen for about 1 hour to expel any residual ozone present in the flask. Workup to carboxylic acid end groups was then carried out by addition of selenium dioxide (4g) and hydrogen peroxide solution (35% v/v) (100ml) to the colloidally stable latex. This was then heated to 8O ⁰ C and refluxed for a further 24 hours. Amberlite IRA 400(Cl) (ion exchange resin, available from, for example, Sigma Aldrich) was placed into a conical flask; the latex (100ml) was then added. The flask was placed onto an orbital shaker with a very low setting and gently shaken. After about 5 hours the latex was decanted into a second conical flask that contained Amberlite IRA 400 (Cl) and this was gently shaken overnight. The latex was then removed and placed into dialysis visking tubing and placed into distilled water. Twice daily water changes occurred over a period of one week. The latexes were then removed from the dialysis tubing and any toluene present was removed at reduced pressure. The water was then azeotropically removed by addition of ethanol and then removing by reduced pressure.

The oligo(BMA)-2COOH latex (Formula 2.1.2) had a solids content of 16 wt% and the final monomer conversion was approximately 95 mol.%. The poly(BMA-co-BD) had an Mn = 52400 g mol ^"1 and Mw/Mn = 2.0, Zeta potential = -19.53 mV, pH = 3.77. The oligo(BMA)-2COOH had Mn = 2690 and Mw/.Mn = 1.9, Zeta potential = -78.4 mV, pH = 1.56

Preparation of Diamine (Formula 1.1.1 )

Oligo(BMA)-2COOH latex (75ml) was added to a 3-neck round bottom flask, equipped with a condenser and magnetic stirrer bar. DOWFAX 2A1 was then added to the flask and vigorous stirring was started. At the same time 1-(3-dimethyl-aminopropyl)-3- ethylcarbodiimide hydrochloride (EDC) was weighed out and dissolved in 45ml of water. The stirring solution of latex and surfactant was then submerged into an ice bath. The aqueous solution of EDC was then added slowly and, upon completion of the addition, a stable latex remained. A diamine as indicated in Table 2 was then added to the stirring latex slowly over a period of at least an hour. A substantial exotherm was observed at this stage. The reaction was left to warm to room temperature and continued to stir for 24 hours. The quantities used for each reaction are shown in Table 2 and the particle sizes of the resultant dispersions is shown in Figure 4. Diamine Quantity EDC DOWFAX 2A1

Ethylenediamine 4.63g 3.9308g 1.121 1 g

1 ,4 - 1.2531 g

6.79g 3.9583g diaminobutane

1 ,6-diaminohexane 9.0648g 3.683Og 1.2101 g

Table 2: Reaction formulations used to generate oligo(BMA) diamines.

1 , 6-diaminohexane is a solid and so in this case an aqueous solution was prepared in 90ml of water, and this was added to the reaction mixture. The resultant dispersions were removed, placed into dialysis visking tubing and placed into distilled water for 2 weeks, with daily changes of water taking place.

Example 2 Coating of a commercially available device-Ethicon Vicryl TM Mesh

Approximately 1.5cm ² pieces of the Ethicon VicrylTM mesh were cut and then placed into a Petri dish. An amine ended oligomer latex of Example 1 (~ 10 ml) was added to the petri dish. The mesh pieces were left covered by the latex solutions for a period of about 96 hours until evaporation of the continuous phase of the latex had occurred.

Figure 5 shows cell viability data, which can be taken as an indicator of cell numbers, from the cells cultured on tissue culture plastic, three coated meshes and a non-coated mesh. The data show a clear and significant (p<0.05) increase in cell numbers on the coated meshes compared to the non-coated mesh.

Human dermal fibroblasts loaded with Red Cell Tracke™ were then seeded onto the meshes. The images in Figure 6 clearly show the cells as bright features attached to the fibres of the mesh.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.