# Patent

## Title
Medical device with predefined activated cellular integration

## Abstract

A medical device includes a substrate having at least a portion thereof functionalized with at least one reactive member and a chemotactic agent functionalized with at least one complementary reactive member. The at least one reactive member and the at least one complementary reactive member are covalently bonded, adhering the chemotactic agent to the substrate.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Stage Application filed under 35 U.S.C. §371(a) of International Application No. PCT/IB2011/002260 filed Jun. 30, 2011, which claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 61/360,691 filed Jul. 1, 2010, the entire contents of which are incorporated by reference herein.


BACKGROUND
1. Technical Field
The present disclosure relates to methods of making implantable medical devices having a coating that produces a specific chemotactic response.
2. Background of Related Art
Techniques for repairing damaged or diseased tissue are widespread in medicine. Wound closure devices such as sutures, staples and other repair devices such as mesh or patch reinforcements are frequently used for repair. Surgical adhesives have been used to augment and, in some cases, replace sutures and staples in wound closure. For example, in the case of hernias, techniques involving the use of a mesh or patch to reinforce the abdominal wall are being used. The mesh or patch can generally be held in place by suturing or stapling to the surrounding tissue. Unfortunately, the use of such sutures or staples may increase the patient's discomfort and, in certain instances, there may be a risk of weakening thin or delicate tissue where they are attached. Certain techniques involve placing a mesh or patch against the repair site without suturing or stapling, e.g., allowing the pressure of the peritoneum to hold the patch against the posterior side of the abdominal wall. However, fixation of the mesh or patch is generally preferred in order to avoid folding, shrinkage, and migration of the mesh or patch. Surgical adhesives such as cyanoacrylates and fibrin glues have been used as fixatives in lieu of, or in addition to, suturing or stapling the mesh or patch. However, fibrin adhesives can be difficult to prepare and store. Cyanoacrylates may cause irritation at the point of application and may not provide a sufficient degree of elasticity. In addition, surgical adhesives can tend to form a physical barrier between the item or items being attached to biological tissue, thus interfering with tissue ingrowth into the item when ingrowth is desired.
Click chemistry is a popular term for reliable reactions that make it possible for certain chemical building blocks to “click” together and form an irreversible linkage. Since its recent introduction, click chemistry has been used for ligation in biological and medical technology. In the case of azide-alkyne click chemistry, the reactions may be catalyzed or uncatalyzed, as illustrated in US Patent Publication. No. 2005/0222427 and PCT P2006/050262, respectively. Copper-free click chemistry was recently developed by Bertozzi and colleagues using difluorinated cyclooctyne or DIM, that reacts with azides rapidly at physiological temperatures without the need for a toxic catalyst as disclosed in Baskin et al., “Copper Free Click Chemistry for Dynamic In Vivo Imaging,” PNAS, vol. 104, no. 43, 16793-16797 (Oct. 23, 2007). The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote a [3+2] dipolar cycloaddition with azides as disclosed in US Patent Publication No. 2006/0110782 and “Codelli et al., Second Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry,” J. Am. Chem. Soc., vol. 130, no. 34, 11486-11493 (2008). Another suitable cyclooctyne is 6,7-dimethoxyazacyclooct-4-yne (DIMAC) as disclosed in Sletton and Bertozzi, “A Hydrophilic Azacyclooctyne for Cu-free Click Chemistry,” Org. Lett. (2008) 10 (14), 3097-3099. Other click chemistry reactions include Diels-Alder reactions, thiol-alkene reactions, and maleimide-thiol reactions.
Implantable medical devices are formed from a variety of different biodegradable and non-biodegradable materials. Non-biodegradable devices offer increased strength and support, however, some medical devices do not fully integrate within the tissue due poor cell seeding within the devices. Accordingly, it would be beneficial to provide a medical device which includes a coating that encourages cell attachment thereto.
SUMMARY
Implantable medical devices are described herein which include a substrate and a coating disposed on a surface thereof. The coating includes one or more chemotactic agents, which are useful for attracting cells, modulating cell attachment, guiding cellular ingrowth, including cell proliferation and/or differentiation.
In particular, the present disclosure relates to tissue-engineered medical devices, and to cell therapy which restores, maintains and/or improves tissue functions. Such devices may include a three-dimensional substrate (e.g., scaffold) that acts as a substrate for cell attachment and recruitment. Cells seeded in the substrate recreate the in vivo microenvironment, thereby facilitating cell-to-cell interactions and expression of differentiated functions. To construct such a complex structure, the efficiency of the cell seeding process can be important to the overall performance of the scaffold construct. Moreover during in vivo implantation the optimization of the scaffold implant that can specifically interact with cells within the device is also envisioned. The efficiency of the cell seeding process or cells colonization in vivo may also be adjusted by the porosity, the nature and material composition of the scaffold itself.
The present disclosure relates to the functionalization of the materials used in the preparation of scaffolds for regenerative medicine to maximize cellular integration. This may be accomplished via chemical reactions, including but not limited to, “click chemistry” involving azide or alkyne groups. The chemical reaction of click chemistry may include reaction with or without catalysts. Click chemistry for ex vivo or in vivo reactions provides a number of advantages over other reactions because each functional group is substantially inert against the chemical environment of the biological components used for tissue-engineering.
In embodiments, the present disclosure provides for polymer functionalization to immobilize chemotactic agent on the substrate by click chemistry so that it may be used to attract cells, modulate cell attachment, morphology, proliferation and/or differentiation to guarantee cell density and finally guide cellular ingrowth. This concept may be applied to tissue-engineered scaffolds and also to scaffolds used for stem cell therapy. In embodiments, the substrate may be formed in specific structure so that functionality is exposed as the substrate degrades to aid in guiding and orienting of cellular infiltration and ingrowths.
Methods of making such medical devices are described herein which include reacting a functionalized surface of the substrate with a chemotactic agent compound also having a functionalized portion. In embodiments, the surface of the substrate and the functionalized portion of the chemotactic agent are functionalized with reactive members involved in click chemistry.
In embodiments, a medical device may include a substrate having at least a portion thereof functionalized with at least one reactive member and a chemotactic agent functionalized with at least one complementary reactive member, wherein the at least one reactive member and the at least one complementary reactive member are covalently bonded, adhering the chemotactic agent to the substrate.
In embodiments, the substrate is formed from a polymer selected from the group consisting of polycarbonates, polyolefins, polymethacrylates, polystyrenes, polyamides, polyurethanes, polyethylene terephthalate, poly (lactic acid), poly (glycolic acid), poly(hydroxbutyrate), dioxanones (e.g., 1,4-dioxanone), δ-valerolactone, 1,dioxepanones (e.g., 1,4-dioxepan-2-one and 1,5-dioxepan-2-one), poly(phosphazine), polyesters, polyethylene glycol, polyethylene oxides, polyacrylamides, cellulose esters, fluoropolymers, vinyl polymers, silk, collagen, alginate, chitin, chitosan, hyaluronic acid, chondroitin sufate, polyhydroxyethylmethylacrylate, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacetate, polycaprolactone, polypropylene, glycerols, poly(amino acids), copoly (ether-esters), polyalkylene oxalates, polyamides, poly (iminocarbonates), polyalkylene oxalates, polyoxaesters, polyorthoesters, polyphosphazenes, polypeptides and copolymers, block copolymers, homoploymers, blends and combinations thereof.
In embodiments, the reactive member and the complementary reactive member bind to one another via a reaction selected from the group consisting of Huisgen cycloaddition reaction, a Diels-Alder reaction and a thiol-ene reaction. The Huisgen cycloaddition reaction may include an alkyne and an azide and is catalyzed by copper. The Huisgen cycloaddition reaction may involve a cyclooctyne reagent and an azide.
In embodiments, the reactive members and the complementary reactive members are thiols and alkenes.
In embodiments, the reactive members and the complementary reactive members are alkynes and azides. For example, the reactive member is an alkyne and the complementary reactive member is an azide. In embodiments, the reactive member is an azide and the complementary reactive member is an alkyne.
In embodiments, the reactive members and the complementary reactive members are dienes and alkenes.
In embodiments, the substrate is selected from the group consisting of a mesh, a patch, a scaffold, a suture, a ligature, a sling, a pellicle, a film, a barrier, and a foam.
In other embodiments, a method of manufacturing a medical device may include functionalizing a substrate to form a plurality of reactive members of a specific binding pair attached on a surface of the substrate, functionalizing at least one chemotactic agent to form a plurality of complementary reactive members of the specific binding pair, and contacting the substrate and the at least one chemotactic agent, wherein the plurality of reactive members and the plurality of complementary reactive member are covalently bonded, adhering the at least one chemotactic agent to the substrate.
In embodiments, the functionalizing of the substrate includes functionalizing the substrate with an azide. In embodiments, the functionalizing of the at least one chemotactic agent includes functionalizing the at least one chemotactic agent with an alkyne.
In further embodiments, a method of manufacturing a medical device may include contacting a functionalized substrate with a plurality of reactive members of a specific binding pair with at least one functionalized chemotactic agent with a plurality of complementary reactive members of the specific binding pair, wherein the plurality of reactive members and the plurality of complementary reactive member are covalently bonded, adhering the at least one functionalized chemotactic agent to the functionalized substrate. In embodiments, the method further comprises functionalizing the substrate with an azide. In embodiments, the method further comprises functionalizing the at least one chemotactic agent with an alkyne.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawing, which is incorporated in and constitutes a part of this specification, illustrates embodiments of the disclosure and, together with a general description of the disclosure given above, and the detailed description of the embodiments given below, serves to explain the principles of the disclosure.
The FIGURE schematically illustrates a process for forming a medical device in accordance with an embodiment of the present disclosure.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Implantable medical devices in accordance with the present disclosure include a substrate having a surface and a cellular growth promoting coating containing a chemotactic agent which is covalently bound to a surface of the substrate. At least a portion of a surface of the substrate is functionalized with a first reactive member and a portion of the chemotactic agent is functionalized with a second reactive member that is reactive with the first reactive member on the surface of the substrate. The first and second reactive members react to covalently bond the chemotactic agent to the surface of the substrate.
In embodiments, the surface of the substrate is functionalized with a first click-reactive member and the chemotactic agent is functionalized with a second click-reactive member complementary to the first click-reactive member. The “click-reactive members” are meant to include those reactive members used in the processes known to those skilled in the art as click chemistry.
Click chemistry refers to a collection of reactive members having a high chemical potential energy capable of producing highly selective, high yield reactions. The reactive members react to form extremely reliable molecular connections in most solvents, including physiologic fluids, and often do not interfere with other reagents and reactions. Examples of click chemistry reactions include Huisgen cycloaddition, Diels-Alder reactions, thiol-alkene reactions, and maleimide-thiol reactions.
With reference to the FIGURE, a medical device is shown. The medical device includes a substrate shown as a three-dimensional tissue scaffold that promotes cell attachment and growth. In embodiments, the substrate of the medical devices described herein may be made from any polymer as discussed in further detail below. The polymer may be a homopolymer or a copolymer, including random copolymer, block copolymer, or graft copolymer. The polymer may be a linear polymer, a branched polymer, or a dendrimer. The polymer may be of natural or synthetic origin.
The substrate may be fabricated into any desired physical form. Some non-limiting examples include monofilaments, multifilaments, surgical meshes, ligatures, sutures, scaffolds, staples, patches, slings, foams, pellicles, films, barriers, and the like. The substrate may be fabricated for example, by spinning, casting, molding or any other fabrication technique known to those skilled in the art. The substrate may be made into any shape, such as, for example, a fiber, sheet, rod, staple, clip, needle, tube, foam, or any other configuration suitable for a medical device. Where the substrate is in the form of a fiber, the fiber may be formed into a textile using any known technique including, but not limited to, knitting, weaving, tatting and the like. It is further contemplated that the substrate may be a non-woven fibrous structure.
The chemostactic agent may be any chemoattractant or chemorepellents that induce chemotaxis within the cells surrounding the medical device. Suitable chemoattractants include, by way of example: glycyl-histidyl-lysine, alanyl-glycyl-seryl-glutamine, f-methionyl-leucyl-phenylalanine, glycyl-histidyl-glycine, and valinyl-glycyl-seryl-glutamine from Serva Fine Biochemicals, Garden City Park, N.Y.; collagen type I from Collagen Corp., Palo Alto, Calif.; epidermal growth factor from Collaborative Research, Waltham, Mass.; endotoxin standard, prostaglandin E, and histamine from Sigma Biochemicals, St. Louis, Mo., Tuftsin from Calbiochem, La Jolla, Calif., and heparin from Elkin-Sinn.
In order to covalently bond the chemotactic agent to the surface of the substrate, at least a portion of the surface of the substrate is functionalized with a first reactive member and a portion of the chemotactic agent is functionalized with a second reactive member. The first and second reactive members are complementary. By “complementary” it is meant that the first and second reactive members are able to specifically interact together to covalently bond the chemotactic agent to the functionalized polymer.
In embodiments, the surface of the substrate and the chemotactic agent are functionalized to include a first click-reactive member which is an alkyne and a second click-reactive member which is an azide, respectively. In embodiments, the surface of the substrate and the chemotactic agent are functionalized to include a first click-reactive member which is an azide and a second click-reactive member which is an alkyne, respectively. As those skilled in the art will recognize, reaction times between the azide and alkyne members can be reduced from about 24 hours at room temperature to mere seconds at room temperature by the presence of catalysts, such as transition metal ions (e.g., copper ions) as discussed in more detail below.
The first and second click-reactive members are intended to react and covalently bond the chemotactic agent to the functionalized surface of the substrate at a neutral pH. However, in some embodiments, the first and second click-reactive members may react quicker or more completely following the addition of a catalyst, including but not limited to, a pH modifier, a metal ion catalyst, such as transition metal ions (e.g., copper ions), or the introduction of heat or radiation, e.g., UV radiation, may enhance the formation of a covalent bond between the first and second click-reactive members.
The first and second reactive members may be positioned on the substrate and chemotactic agent using any variety of suitable chemical processes. With respect to the first reactive members on the substrate, it is contemplated that a plurality of first reactive members may be present and may be terminally located, or alternatively located along the length of the polymer chain.
In embodiments, the monomers from which the substrate is made can be functionalized so that the reactive members appear along the length of the polymer. The monomers can be initially functionalized with a member such as a halogen to provide a reactive site at which the desired first reactive member can be attached after polymerization. For example, the halogenated polymer can be reacted with sodium azide to provide azide groups along the polymer chain or with propagyl alcohol to provide alkyne groups along the polymer chain. Alternatively, a pre-formed polyester can be halogenated by reaction with a non-nucleophilic strong base, such as lithium diisopropylamide, followed by electrophilic substitution with iodine chloride. The halogenated polyester is then reacted with sodium azide or propagyl alcohol to provide azide or alkyne groups, respectively.
In embodiments, pendant functional groups can also be incorporated into the polymer backbone by, e.g., copolymerization with functionalized monomer such as lactones, cyclic carbonates and morpholine-2,5-diones. The azido group, N3 is a nucleophilic group that exchanges with other nucleophilic groups, e.g., —OH, —NH2 and halogens (Br, Cl, or I). For example, 1,3-dipolar compounds may be conjugated to aliphatic polyesters, by copolymerizing ε-caprolactone and α-chloro-ε-caprolactone and then substituting an azide group for the Cl atom. Polyesters can incorporate pendant dipolarophiles, e.g., propargyl groups, by copolymerization of ε-caprolactone and α-propargyl-δ-valerolactone. Copolymers of L-lactide containing propargyl groups may be prepared by ring opening copolymerization of 5-methyl-5-propargyloxycarbonyl-1,3-dioxanone with L-lactide at a molar ratio of about 90:10 with ZnEt2 as a catalyst as disclosed in Shi et al., Biomaterials, 29 (2008)1118-1126. Azide functionalized polystyrene is synthesized using atom transfer radical polymerization and subsequent modification with azidotrimethylsilane and tetrabutylammonium fluoride as disclosed in Dirks, et al., Chem. Comm., (2005) 4172-4174. Azides may be incorporated onto methacrylates, e.g., 3 azidopropyl methacrylate which is copolymerized to a block copolymer. Diels-Alder functionalities and thiol-ene functionalities are likewise incorporated into polymers herein.
In other embodiments, the substrate is functionalized after it has been fabricated into the desired form. For example, polymeric fibers can be functionalized after the spinning process. In embodiments, the fibers are surface treated and then activated with the first reactive member (optionally with a coupling agent, e.g., a silane coupling agent, being used). Surface activation of and biocompatible aliphatic polyesters can be achieved by acid or base hydrolysis, treatment by means of cold plasma, by chemical reactions or electromagnetic radiations. It is contemplated that such surface activation can be performed before or after the fibers are made into a textile structure.
Hydrolysis can be conducted in the presence of an aqueous solution of a base or an acid to accelerate surface reaction, inasmuch as excessively long processes of activation can induce a reduction in molecular weight and thus in the mechanical properties of the material. Suitable bases for obtaining watery solutions suited to the aim are, for example, strong alkalis, such as LiOH, Ba(OH)2, Mg(OH)2, NaOH, KOH, Na2CO3, Ca(OH)2 and the weak bases, such as for example NH4OH and the ammines such as methylamine, ethylamine, diethylamine and dimethylamine. Acids suitable for surface hydrolysis treatments can be chosen, for example, from among HCl, HClO3, HClO4, H2SO3, H2SO4, H3PO3, H3PO4, HI, HIO3, HBr, lactic acid, glicolic acid. Surface activation by means of hydrolysis can be conducted at temperatures preferably comprised between 0° C. and the material softening temperature. Plasma treatment can be carried out both in the presence of a reactive gas, for example air, Ar, O2 with the formation of surface activation of oxygenate type, such as —OH, —CHO, —COOH.
Surface treatment, whether hydrolytic or with plasma, can remain unaltered or can be followed by further chemical modifications to provide the first reactive members on the substrate. Thus, for example, the COONa members generated by a base hydrolysis can be subsequently converted into COOH members by treatment with strong mineral acids. Further, the surface freeing of alcoholic members by means of a hydrolysis process can be followed by reaction by means of the addition of a compound provided with functional member or members able to react with surface alcoholic members, such as for example by means of the addition of an anhydride such as succinic anhydride, with the conversion of —OH members into —O—CO—CH2—CH2—COOH members. Suitable surface activation techniques are disclosed in U.S. Pat. No. 6,107,453, the entire disclosure of which is incorporated herein by this reference.
With respect to the chemotactic agent, it is contemplated that one or more than one second reactive members can be provided thereon. Chemotactic agent is provided with reactive members of a specific binding pair by conjugation to various components thereof. In one embodiment, the reactive members or complementary reactive members are attached directly to the chemotactic agent. In another embodiment, the reactive members or complementary reactive members are attached to the chemotactic agent via a linker. In either case, situating the reactive members or complementary reactive members on the chemotactic agent can be accomplished by suspending the reactive members or complementary reactive members in a solution or suspension and adding the chemotactic agent to the solution or suspension such that the reactive member binds thereto.
In embodiments, the surface of the substrate and the chemotactic agent are functionalized with electrophilic or nucleophilic functional members, such that, for example, a nucleophilic functional member on the surface of the substrate may react with an electrophilic functional member on the chemotactic agent to form a covalent bond.
Virtually any nucleophilic member can be used to functionalize the surface of the substrate, so long as reaction can occur with the electrophilic member on the chemotactic agent. Analogously, virtually any electrophilic member can be used to functionalize the chemotactic agent, so long as reaction can take place with the nucleophilic member on the surface of the substrate. In embodiments, the reactions of the complementary members may be complete in under 60 minutes, in embodiments under 30 minutes, in yet other embodiments, the reaction occurs in about 5 to 15 minutes or less.
Non-limiting examples of nucleophilic members include, but are not limited to, —NH2, —NHR, —N(R)2, —SH, —OH, —COOH, —C6H4—OH, —PH2, —PHR, —P(R)2, —NH—NH2, —CO—NH—NH2, —C5H4N, and the like, wherein R is hydrocarbyl, typically C1-C4 alkyl or monocyclic aryl. Organometallic moieties are also useful nucleophilic members for the purposes of this disclosure, particularly those that act as carbanion donors. Examples of organometallic moieties include: Grignard functionalities —RMgHal wherein R is a carbon atom (substituted or unsubstituted), and Hal is halo, typically bromo, iodo or chloro; and lithium-containing functionalities, typically alkyllithium members; sodium-containing functionalities.
It will be appreciated by those of ordinary skill in the art that certain nucleophilic members may be activated with a base so as to be capable of reaction with an electrophile. In embodiments, when there are nucleophilic sulfhydryl and hydroxyl members on the surface of the substrate, the composition may be admixed with an aqueous base in order to remove a proton and provide an —S− or —O− species to enable reaction with an electrophile. Unless it is desirable for the base to participate in the reaction, a non-nucleophilic base is used. In some embodiments, the base may be present as a component of a buffer solution.
The selection of electrophilic members provided on the chemotactic agent is made so that reaction is possible with the specific nucleophilic members on the surface of the substrate. Thus, when the surface of the substrate is functionalized with amino members, the chemotactic agent is functionalized with members selected so as to react with amino members. Analogously, when the surface of the substrate is functionalized with sulhydryl moieties, the corresponding electrophilic members are sulfhydryl-reactive members, and the like.
By way of example, when the surface of the substrate is functionalized with amino members (generally although not necessarily primary amino members), the electrophilic members present on the chemotactic agent are amino reactive members such as, but not limited to: carboxylic acid esters, including cyclic esters and “activated” esters; acid chloride members (—CO—Cl); anhydrides (—(CO)—O—(CO)—R); ketones and aldehydes, including α,β-unsaturated aldehydes and ketones such as —CH═CH—CH═O and —CH═CH—C(CH3)═O; (5) halides; isocyanate (—N═C═O); isothiocyanate (—N═C═S); epoxides; activated hydroxyl members (e.g., activated with conventional activating agents such as carbonyldiimidazole or sulfonyl chloride); and olefins, including conjugated olefins, such as ethenesulfonyl (—SO2CH═CH2) and analogous functional members, including acrylate (—CO2—C═CH2), methacrylate (—CO2—C(CH3)═CH2)), ethyl acrylate (—CO2—C(CH2CH3)═CH2), and ethyleneimino (—CH═CH—C═NH). Since a carboxylic acid member is not susceptible to react with a nucleophilic amine, components containing carboxylic acid members must be activated so as to be amine-reactive. Activation may be accomplished in a variety of ways, such as reaction with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). A carboxylic acid can be reacted with an alkoxy-substituted N-hydroxy-succinimide or N-hydroxysulfosuccinimide in the presence of DCC to form reactive electrophilic members, the N-hydroxysuccinimide ester and the N-hydroxysulfosuccinimide ester, respectively. Carboxylic acids may also be activated by reaction with an acyl halide such as an acyl chloride (e.g., acetyl chloride), to provide a reactive anhydride member. In a further example, a carboxylic acid may be converted to an acid chloride member using, e.g., thionyl chloride or an acyl chloride capable of an exchange reaction. Specific reagents and procedures used to carry out such activation reactions will be known to those of ordinary skill in the art and are described in the pertinent texts and literature.
Analogously, when the surface of the substrate is functionalized with sulfhydryl, the electrophilic members present on the chemotactic agent are members that react with a sulfhydryl moiety. Such reactive members include those that form thioester linkages upon reaction with a sulfhydryl member, such as those described in PCT Publication No. WO 00/62827 to Wallace et al., entire disclosure of which is incorporated herein by reference. As explained in detail therein, such “sulfhydryl reactive” members include, but are not limited to: mixed anhydrides; ester derivatives of phosphorus; ester derivatives of p-nitrophenol, p-nitrothiophenol and pentafluorophenol; esters of substituted hydroxylamines, including N-hydroxyphthalimide esters, N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, and N-hydroxyglutarinide esters; esters of 1-hydroxybenzotriazole; 3-hydroxy-3,4-dihydro-benzotriazin-4-one; 3-hydroxy-3,4-dihydro-quinazoline-4-one; carbonylimidazole derivatives; acid chlorides; ketenes; and isocyanates. With these sulfhydryl reactive members, auxiliary reagents can also be used to facilitate bond formation, e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide can be used to facilitate coupling of sulfhydryl members to carboxyl-containing members.
In addition to the sulfhydryl reactive members that form thioester linkages, various other sulfydryl reactive functionalities can be utilized that form other types of linkages. For example, compounds that contain methyl imidate derivatives form imido-thioester linkages with sulfhydryl members. Alternatively, sulthydryl reactive members can be employed that form disulfide bonds with sulthydryl members, such members generally have the structure —S—S—Ar where Ar is a substituted or unsubstituted nitrogen-containing heteroaromatic moiety or a non-heterocyclic aromatic member substituted with an electron-withdrawing moiety, such that Ar may be, for example, 4-pyridinyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2-nitro-4-benzoic acid, 2-nitro-4-pyridinyl, etc. In such instances, auxiliary reagents, i.e., mild oxidizing agents such as hydrogen peroxide, can be used to facilitate disulfide bond formation.
Yet another class of sulfhydryl reactive members forms thioether bonds with sulfhydryl members. Such members include, inter alia, maleimido, substituted maleimido, haloalkyl, epoxy, imino, and aziridino, as well as olefins (including conjugated olefins) such as ethenesulfonyl, etheneimino, acrylate, methacrylate, and α,β-unsaturated aldehydes and ketones.
When the surface of the substrate is functionalized with —OH, the electrophilic functional members on the chemotactic agent must react with hydroxyl members. The hydroxyl member may be activated as described above with respect to carboxylic acid members, or it may react directly in the presence of base with a sufficiently reactive electrophile such as an epoxide member, an aziridine member, an acyl halide, an anhydride, and the like. When the surface of the substrate is functionalized with an organometallic nucleophile such as a Grignard functionality or an alkyllithium member, suitable electrophilic functional members for reaction therewith are those containing carbonyl members, including, by way of example, ketones and aldehydes.
It will also be appreciated that certain functional members can react as nucleophiles or as electrophiles, depending on the selected reaction partner and/or the reaction conditions. For example, a carboxylic acid member can act as a nucleophile in the presence of a fairly strong base, but generally acts as an electrophile allowing nucleophilic attack at the carbonyl carbon and concomitant replacement of the hydroxyl member with the incoming nucleophile.
Table 1, below illustrates, solely by way of example, representative complementary pairs of electrophilic and nucleophilic functional members that may be employed in functionalizing the substrate (e.g., R1 in Table 1) and the chemotactic agent (e.g., R2 in Table 1).









TABLE 1





REPRESENTATIVE




NUCLEOPHILIC
REPRESENTATIVE



COMPONENT
ELECTROPHILIC COMPONENT



(A, FNNU)
(B, FNEL)
RESULTING LINKAGE







R1—NH2
R2—O—(CO)—O—N—(COCH2)
R1—NH—(CO)—O—R2



(succinimidyl carbonate terminus)



R1—SH
R2—O—(CO)—O—N—(COCH2)
R1—S—(CO)—O—R2


R1—OH
R2—O—(CO)—O—N—(COCH2)
R1—S—(CO)—R2


R1—NH2
R2—O(CO)—CH═CH2
R1—NH—CH2CH2—(CO)—O—R2



(acrylate terminus)



R1—SH
R2—O—(CO)—CH═CH2
R1—S—CH2CH2—(CO)—O—R2


R1—OH
R2—O—(CO)—CH═CH2
R1—O—CH2CH2—(CO)—O—R2


R1—NH2
R2—O(CO)—(CH2)3—CO2N(COCH2)
R1—NH—(CO)—(CH2)3—(CO)—OR2



(succinimidyl glutarate terminus)



R1—SH
R2—O(CO)—(CH2)3—CO2—N(COCH2)
R1—S—(CO)—(CH2)3—(CO)—OR2


R1—OH
R2—O(CO)—(CH2)3—CO2—N(COCH2)
R1—O—(CO)—(CH2)3—(CO)—OR2


R1—NH2
R2—O—CH2—CO2—N(COCH2)
R1—NH—(CO)—CH2—OR2



(succinimidyl acetate terminus)



R1—SH
R2—O—CH2—CO2—N(COCH2)
R1—S—(CO)—CH2—OR2


R1—OH
R2—O—CH2—CO2—N(COCH2)
R1—O—(CO)—CH2—OR2


R1—NH2
R2—O—NH(CO)—(CH2)2—CO2—N(COCH2)
R1—NH—(CO)—(CH2)2—(CO)—NH—OR2



(succinimidyl succinamide terminus)



R1—SH
R2—O—NH(CO)—(CH2)2—CO2—N(COCH2)
R1—S—(CO)—(CH2)2—(CO)—NH—OR2


R1—OH
R2—O—NH(CO)—(CH2)2—CO2—N(COCH2)
R1—O—(CO)—(CH2)2—(CO)—NH—OR2


R1—NH2
R2—O—(CH2)2—CHO
R1—NH—(CO)—(CH2)2—OR2



(propionaldehyde terminus)



 


R1—NH2



   (glycidyl ether terminus)
R1—NH—CH2—CH(OH)—CH2—OR2 and R1—N[CH2—CH(OH)—CH2—OR2]2


 


R1—NH2
R2—O—(CH2)2—N═C═O
R1—NH—(CO)—NH—CH2—OR2



(isocyanate terminus)



R1—NH2
R2—SO2—CH═CH2
R1—NH—CH2CH2—SO2—R2



(vinyl sulfone terminus)



R1—SH
R2—SO2—CH═CH2
R1—S—CH2CH2—SO2—R2









In embodiments, Huisgen cycloaddition may also be used in click chemistry. Huisgen cycloaddition is the reaction of a dipolarophile with a 1,3-dipolar compound that leads to 5-membered (hetero)cycles. Examples of dipolarophiles are alkenes and alkynes and molecules that possess related heteroatom functional groups (such as carbonyls and nitriles). 1,3-Dipolar compounds contain one or more heteroatoms and can be described as having at least one mesomeric structure that represents a charged dipole. They include nitril oxides, azides, and diazoalkanes. Metal catalyzed click chemistry is a variant of the Huisgen 1,3-dipolar cycloaddition reaction between alkyl-aryly-sulfonyl azides, C—N triple bonds and C—C triple bonds which is well-suited herein. The results of these reactions are 1,2 oxazoles, 1,2,3 triazoles or tetrazoles. 1,2,3 Triazoles may be formed by a copper catalyzed Huisgen reaction between alkynes and alkyl/aryl azides. Metal catalyzed Huisgen reactions proceed at ambient temperature, are not sensitive to solvents, i.e., nonpolar, polar, semipolar, and are highly tolerant of functional groups. Non-metal Huisgen reactions (also referred to as strain promoted cycloaddition) involving use of a substituted cyclooctyne, are especially well-suited for use herein due to low toxicity since substituted cyclooctyne possess ring strain and electron-withdrawing substituents such as fluorine, that together promote a [3+2] dipolar cycloaddition with azides. Suitable examples of substituted cycloctyne include difluorinated cyclooctyne and 6,7-dimethoxyazacyclooct-4-yne. Reaction of the alkynes and azides is very specific and essentially inert against the chemical environment of biological tissues. One reaction scheme may be represented as:




where R and R′ are a substrate or a chemotactic agent.

In accordance with the disclosure herein, a medical device, such as a surgical patch or mesh is provided with a plurality of reactive members of a specific binding pair attached on the surface of the medical device. When the reactive members of the medical device are contacted with the chemotactic agent containing complementary reactive members of the specific binding pair, covalent attachment occurs, thus adhering the device to the chemotactic agent. In embodiments, the reactive members may be either a dipolarophile or a 1,3 dipolar compound depending on which complement is applied to the chemotactic agent or the medical device. For example, if a dipolarophile is located on the device, the 1,3 dipolar compound will be located on the chemotactic agent. If a dipolarophile is located on the chemotactic agent, the 1,3 dipolar compound will be located on the device. In embodiments, the Diels-Alder members of a specific binding pair may be either a diene and a dienophile depending on which complement is applied to the chemotactic agent or the medical device. For example, if a diene is located on the device, the dienophile can be located on the chemotactic agent. If a diene is located on the chemotactic agent, the dienophile can be located on the device. In embodiments, the thiol-ene members of a specific binding pair may be either a thiol and an alkene depending on which complement is applied to the chemotactic agent or the device. For example, if a thiol is located on the device, the alkene can be located on the chemotactic agent. If a thiol is located on the chemotactic agent, the alkene can be located on the device.
In embodiments, Diels-Alder reaction may also be used in click chemistry. The Diels-Alder reaction combines a diene (a molecule with two alternating double bonds) and a dienophile (an alkene) to make rings and bicyclic compounds. Examples include:




The thiol-alkene (thiol-ene) reaction is a hydrothiolation, i.e., addition of RS—H across a C═C bond. The thiol-ene reaction proceeds via a free-radical chain mechanism. Initiation occurs by radical formation upon UV excitation of a photoinitiator or the thiol itself. Thiol-ene systems form ground state charge transfer complexes and therefore photopolymerize even in the absence of initiators in reasonable polymerization times. However, the addition of UV light increases the speed at which the reaction proceeds. The wavelength of the light can be modulated as needed, depending upon the size and nature of the constituents attached to the thiol or alkene. A general thiol-ene coupling reaction mechanism is represented below:




The substrate may be constructed from any biocompatible synthetic or natural materials, including absorbable polymers or biocompatible non-absorbable polymers. Examples of suitable polymers include polycarbonates; polyolefins such as polyethylene, including ultra high molecular weight polyethylene, (UHMWPE); polymethacrylates; polystyrenes; polyamides; polyurethanes; polyethylene terephthalate; poly(lactic acid); poly(glycolic acid); poly(hydroxbutyrate); dioxanones (e.g., 1,4-dioxanone); δ-valerolactone; 1,dioxepanones, such as 1,4-dioxepan-2-one and 1,5-dioxepan-2-one; poly(phosphazine); polyesters such as polyethylene terepththalate (PET), polyethylene glycol, polyethylene oxides; polyacrylamides; fluorinated polymers such as fluoroethylenes, propylenes, fluoroPEGs, polytetrafluoroethylene; vinyl polymers; natural biological polymers, which may be non-oxidized or oxidized, including but not limited to silk, collagen, gelatin, cellulose, hydroxypropyl cellulose, carboxyethyl cellulose, cellulose esters, alginate, chitin, chitosan, hyaluronic acid, chondroitin sulfate, glycosaminoglycans; polyhydroxyethylmethylacrylate; polyvinylpyrrolidone; polyvinyl alcohol; polyacrylic acid; polyacetate; polycaprolactone; polypropylene; glycerols; poly(amino acids); copoly(ether-esters); polyalkylene oxalates; polyamides; poly(iminocarbonates); polyalkylene oxalates; polyoxaesters; polyorthoesters; polyphosphazenes; nylons; silicones; polybutesters; polyaryletherketone; as well as polypeptides, copolymers, block copolymers, homoploymers, blends and combinations thereof.
In the present application, the term “bioresorbable” and “bioabsorbable” are used interchangeably and are intended to mean the characteristic according to which an implant and/or a material is resorbed by the biological tissues and the surrounding fluids and disappears in vivo after a given period of time, that may vary, for example, from one day to several months, depending on the chemical nature of the implant and/or of the material. Non bioresorbable material—also called permanent material—is not substantially resorbed by tissues and surrounding fluids, after 2 years and more, keeping in particular most (e.g., >80%) of their mechanical properties after such a time. The term “biocompatible” is intended to mean the characteristic according to which an implant and/or a material is well-integrated by the biological tissues and the surrounding fluids without inducing excessive inflammation reaction around the bulk of the material or due to its degradation. The material should avoid also the formation of a fibrous capsule which usually results in the delay of the cellular integration of a porous implant.
In embodiments, the medical device may be a surgical patch. The surgical patch may be selected from any conventional patch type that is suitable for use in tissue reinforcement, e.g., hernia repair, or as an anti-adhesion barrier, hemostatic patch, bandages, pledgets and the like. Many types of patches are currently available and are well known to those skilled in the art. Exemplary polymeric patch materials include nonabsorbable polyester cloth, polyester sheeting, acrylic cloth, polyvinyl sponge or foam, polytetrafluroethylene (PTFE), expanded PTFE, and polyvinyl cloth. Any of the biocompatible polymers listed above may be utilized. In another embodiment, the medical device is a surgical mesh, e.g., polypropylene mesh, nylon mesh, and Dacron mesh. Exemplary absorbable meshes include collagen, polyglycolic acid, polyglactin, polycaprolactone, chitosan, and carbon fiber mesh. It should be understood that any of the above-mentioned biocompatible polymers may be suitable for use herein.
The patch or mesh may be produced from fibers of any biocompatible polymer using any techniques known to those skilled in the art, such as knitting, weaving, tatting, nonwoven techniques, freeze drying, solvent casting and the like. It is envisioned that the patch or mesh may be formed from any permanent biocompatible materials (e.g. polyesters, polypropylene), biodegradable biocompatible materials (e.g. polylactic acid, polyglycolic acid, oxidized cellulose, and chitosan) or with a combination at any proportion of both permanent and biodegradable materials as well as biocompatible polymers listed above. The medical device may, for example, have an openwork three-dimensional (“3D”) structure as disclosed in U.S. Pat. No. 6,451,032, the entire disclosure of which is incorporated herein by reference, e.g., a “honeycomb” structure, and thus a certain thickness which separates the two surfaces of the fabric.
In embodiments, the patch may be formed from a biopolymer foam having openings or pores over at least a portion of a surface thereof. The pores may be in sufficient number and size so as to interconnect across the entire thickness of the porous layer. Alternatively, the pores may not interconnect across the entire thickness of the porous layer. Closed cell foams are illustrative examples of structures in which the pores may not interconnect across the entire thickness of the porous layer. In embodiments, the pores may not extend across the entire thickness of the foam, but rather are present at a portion of the surface thereof. In some embodiments, the openings or pores may be disposed on a portion of the surface of the porous layer, with other portions of the porous layer having a non-porous texture. Those skilled in the art may envision other pore distribution patterns and configurations for the foam.
In embodiments, the foam may be made from non-denatured collagen or collagen which has at least partially lost its helical structure through heating or any other known method, consisting mainly of non-hydrolyzed a chains, and having a molecular weight, in embodiments, of about 100 kDa. The collagen may be native collagen or atelocollagen, which may be obtained via pepsin digestion and/or after moderate heating as defined hereinabove. The collagen may be cured to any desired degree.
Various modifications and variations of the polymers, chemotactic agents, medical devices, click-reactive members and processes described herein will be apparent to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.

## Claims

1. A medical device comprising:
a substrate having a surface functionalized with at least one click reactive member; and
a chemotactic agent functionalized with at least one complementary click reactive member, wherein the at least one click reactive member and the at least one complementary click reactive member are covalently bonded, adhering the chemotactic agent directly to the surface of the substrate, and the surface of the substrate promotes cellular integration.

2. The medical device according to claim 1, wherein the substrate is formed from a polymer selected from the group consisting of polycarbonates, polyolefins, polymethacrylates, polystyrenes, polyamides, polyurethanes, polyethylene terephthalate, poly (lactic acid), poly (glycolic acid), poly (hydroxbutyrate), dioxanones, δ-valerolactone, 1,dioxepanones, poly (phosphazine), polyesters, polyethylene glycol, polyethylene oxides, polyacrylamides, cellulose esters, fluoropolymers, vinyl polymers, silk, collagen, alginate, chitin, chitosan, hyaluronic acid, chondroitin sulfate, polyhydroxyethylmethylacrylate, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacetate, polycaprolactone, polypropylene, glycerols, poly(amino acids), copoly (ether-esters), polyalkylene oxalates, polyamides, poly (iminocarbonates), polyalkylene oxalates, polyoxaesters, polyorthoesters, polyphosphazenes, polypeptides and copolymers, block copolymers, homoploymers, blends and combinations thereof.

3. The medical device according to claim 1, wherein the at least one click reactive member and the at least one complementary click reactive member bind to one another via a reaction selected from the group consisting of Huisgen cycloaddition reaction, a Diels-Alder reaction and a thiol-ene reaction.

4. The medical device according to claim 3, wherein the Huisgen cycloaddition reaction includes an alkyne and an azide and is catalyzed by copper.

5. The medical device according to claim 3, wherein the Huisgen cycloaddition reaction involves a cyclooctyne reagent and an azide.

6. The medical device according to claim 3, wherein the at least one click reactive member and the at least one complementary click reactive member are thiols and alkenes.

7. The medical device according to claim 3, wherein the at least one click reactive member and the at least one complementary click reactive member are alkynes and azides.

8. The medical device according to claim 7, wherein the at least one click reactive member is an alkyne and the at least one complementary click reactive member is an azide.

9. The medical device according to claim 7, wherein the at least one click reactive member is an azide and the at least one complementary click reactive member is an alkyne.

10. The medical device according to claim 3, wherein the at least one click reactive member and the at least one complementary click reactive member are dienes and alkenes.

11. The medical device according to claim 1, wherein the substrate is selected from the group consisting of a mesh, a patch, a scaffold, a suture, a ligature, a sling, a pellicle, a film, a barrier, and a foam.

12. A method of manufacturing a medical device, the method comprising:
functionalizing a substrate to form a plurality of click reactive members of a specific binding pair attached on a surface of the substrate;
functionalizing at least one chemotactic agent to form a plurality of complementary click reactive members of the specific binding pair; and
contacting the substrate and the at least one chemotactic agent, wherein the plurality of click reactive members and the plurality of complementary click reactive member are covalently bonded, adhering the at least one chemotactic agent directly to the surface of the substrate, and the surface of the substrate promotes cellular integration.

13. The method according to claim 12, wherein the functionalizing of the substrate includes functionalizing the substrate with an azide.

14. The method according to claim 13, wherein the functionalizing of the at least one chemotactic agent includes functionalizing the at least one chemotactic agent with an alkyne.

15. A method of manufacturing a medical device, the method comprising:
contacting a functionalized surface of a substrate with a plurality of click reactive members of a specific binding pair with at least one functionalized chemotactic agent with a plurality of complementary click reactive members of the specific binding pair, wherein the plurality of click reactive members and the plurality of complementary click reactive member are covalently bonded, adhering the at least one functionalized chemotactic agent directly to the functionalized surface of the substrate, and the surface of the substrate promotes cellular integration.

16. The method according to claim 15, further comprising functionalizing the substrate with an azide.

17. The method according to claim 16, further comprising functionalizing the at least one chemotactic agent with an alkyne.

18. The medical device according to claim 1, wherein the at least one chemotactic agent is selected from the group consisting of glycyl-histidyl-lysine, alanyl-glycyl-seryl-glutamine, f-methionyl-leucyl-phenylalanine, glycyl-histidyl-glycine, valinyl-glycyl-seryl-glutamine, and Tuftsin.

19. The method according to claim 12, wherein the at least one chemotactic agent is selected from the group consisting of glycyl-histidyl-lysine, alanyl-glycyl-seryl-glutamine, f-methionyl-leucyl-phenylalanine, glycyl-histidyl-glycine, valinyl-glycyl-seryl-glutamine, and Tuftsin.

20. The method according to claim 15, wherein the at least one functionalized chemotactic agent is selected from the group consisting of glycyl-histidyl-lysine, alanyl-glycyl-seryl-glutamine, f-methionyl-leucyl-phenylalanine, glycyl-histidyl-glycine, valinyl-glycyl-seryl-glutamine, and Tuftsin.