Adhesion layer bonded to activated surface

Abstract

A method for coating a surface that is non-reactive or low-reactive with inorganic alkoxides to modify surface properties is disclosed. The surface is activated by oxidation or amination to produce reactive functional groups on the surface, which are then chemically reacted with the inorganic alkoxide to form an inorganic adhesion layer on the surface. This adhesion layer converts the surface into a surface that is receptive to phosphonic acids or can be converted into a surface that can attach bioactive substrates through metal-catalyzed coupling procedures. The phosphonic acids can then be used to impart hydrophobicity or cell adhesion properties to the surface. The adhesion layer can be used to directly bond to other organic substances that are reactive with such metal oxides. Coated surfaces and constructs comprising the coated surfaces are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 592,880, filed November 30, 2017, the entire disclosure of which is incorporated herein by reference for all purposes. Technical Field

[0003] The present invention relates to the field of activating otherwise non-reactive surfaces to render them susceptible to chemical bonding of coatings, wherein the coatings allow modification of surface properties. Such coated surfaces can be used as scaffolds for cell growth, reconstructive medicine, and medical devices. Background Art

[0004] Tissue formation, wound repair, and many disease processes depend on the expression and cell-mediated assembly of appropriate extracellular matrix (ECM) proteins. In particular, oriented ECM fibers are essential for normal tissue development and homeostasis. However, the structure of the ECM can go wrong in many diseases and injury sites, resulting in unaligned collagen fibers that form in scar tissue.

[0005] The goal of regenerative medicine is to promote the formation of new tissue that closely resembles normal tissue in both structure and function. Controlling cell growth in a spatially defined manner enables the regeneration of damaged or diseased tissue, resulting in the correct arrangement of its constituent cells and / or the molecular complexes produced by the cells. In particular, cells direct the alignment of ECM fibrils to correspond to their actin filaments by using cell-surface receptors that indirectly connect to the actin cytoskeleton. Therefore, a major challenge in regenerative medicine is to encourage cells to assemble ECM fibrils, such as collagen, onto scaffold devices into a specific orientation or arrangement in order to produce tissue with desired functional properties.

[0006] Such scaffolds require an appropriate substrate surface on which to attach cells in an environment that stimulates ECM production. Summary of the Invention

[0007] definition

[0008] As used herein, the term "covalent" bond refers to a chemical bond involving the sharing of electron pairs between atoms. The terms "coordinate," "coordinative," or "coordinate covalent" bond refer to a two-center, two-electron covalent bond in which both electrons originate from the same atom, such as the bonding of a metal ion to a ligand. In contrast, an "ionic" bond involves an electrostatic attraction between oppositely charged ions in which electrons are not shared, but one or more electrons are localized on one atom (anion) and removed from another atom (cation).

[0009] As used herein, the term "bonded" means attached or attached, preferably chemically attached without the use of an adhesive. The chemical bond is preferably a covalent or coordinate attachment.

[0010] As used herein, the terms "reactive" and "non-reactive" refer to the ability of a particular functional group to chemically bond with other functional groups (eg, in an inorganic adhesion layer).

[0011] As disclosed herein, multiple numerical ranges are provided. It should be understood that each intermediate value between the upper and lower limits of the range (unless the context clearly stipulates otherwise, to one tenth of a unit of the lower limit) is also explicitly disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is encompassed within the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded by the range, and each range in which one, zero, or two limits are included in the smaller range is also encompassed within the present invention, subject to any explicitly excluded limits in the stated range. Where the stated range includes one or two limits, the range excluding any one or two of those included limits is also encompassed within the present invention. The term "about" generally includes a maximum of plus or minus 10% of the indicated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 20" can mean from 18 to 22. Preferably, "about" includes a maximum of plus or minus 6% of the indicated value. Alternatively, "about" includes a maximum of plus or minus 5% of the indicated value. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" may also mean from 0.5 to 1.4.

[0012] It has now been discovered that suitable substrate surfaces for attaching cells in an environment that stimulates ECM production include polymers, metals, or metalloid surfaces with appropriate surface functional groups, with or without a chemically bonded inorganic oxide adhesion layer. A self-assembled monolayer (SAM) of suitable ligands is chemically bonded directly to the surface functional groups (without the intervening inorganic oxide adhesion layer) or to an attached inorganic oxide adhesion layer. These ligands can have surface modification properties and can also support the attachment of cells, thereby providing a suitable environment for the production of ECM.

[0013] There are a wide class of polymers, metals and other materials that do not have exposed surface functional groups that form covalent or coordinate bonds with the precursor inorganic alkoxides of the bonded adhesion layer in sufficient density or with sufficient reactivity. These classes include various polymers, metals and metalloid surfaces.

[0014] A general method has now been discovered that can be performed rapidly on otherwise non-reactive polymer, metal or metalloid surfaces and provides chemically bonded coatings that enable control of surface properties.

[0015] In some embodiments, the present invention relates to a coating comprising a plurality of coatings, e.g., ...

[0016] Suitable polymers that do not readily react with inorganic alkoxides to form an adhesion layer include polyalkanes or other polymers such as polysiloxanes, polyalkyl aromatics, polyolefins, polythiols, and polyphosphines.

[0017] One aspect of the present invention relates to a construct comprising a coated activated surface comprising an inorganic oxide adhesion layer chemically bonded to the surface, wherein the inorganic oxide is selected from the group consisting of oxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. Preferably, the inorganic adhesion layer of the construct is selected from the group consisting of oxides of Al, Ti, Zr, Si, Mg, and Zn.

[0018] The polymer may be selected from polysiloxanes (such as polydimethylsiloxane (PDMS)), polyalkanes, polyalkylarenes, polyaldehydes, polyolefins, polythiols, and polyphosphines.

[0019] The activated surface coated with inorganic oxide can further include a self-assembled monolayer (SAM) bonded to the adhesion layer, wherein the SAM is selected from an organic compound comprising phosphonic acid, carboxylic acid, sulfonic acid, phosphinic acid, phosphoric acid, sulfinic acid or hydroxamic acid. Preferably, the SAM comprises a phosphonate self-assembled monolayer (SAMP). The phosphonate can be selected from hydrophobic phosphonates, cell adhesive phosphonates and phosphonates that can carry out further metal-catalyzed coupling.

[0020] The phosphonate can be selected from the group consisting of phosphonic acids of the following structures

[0021]

[0022] wherein the R group is selected from optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, wherein heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and heteroarylalkyl contain one or more heteroatoms selected from O, N, and S. Preferably, the hydrophobic phosphonate is selected from R=C3-C 30 Preferably, the cell adhesive phosphonate is selected from R = C3-C 30 More preferably, the cell adhesive phosphonate is selected from C3-C 30 α,ω-diphosphonates.

[0023] Another aspect of the present invention relates to a construct for medical use comprising an activated surface coated with an inorganic oxide, the activated surface further comprising a SAM or SAMP bonded to the inorganic oxide coating. This construct may further comprise other useful moieties covalently bound to the SAM or SAMP, such as alkyne or azide groups, electrochemically active moieties, photochemically active moieties, cell-attracting moieties, cell-adhesive moieties, or anti-infective moieties that allow further refinement using a so-called "click" reaction. Alternatively, the medical construct may further comprise cells attached to the surface coated with the SAM or SAMP. The cells may be selected from fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiomyocytes, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, epithelial cells, stem cells, neural cells, PC12 cells, neural support cells, Schwann cells, radial glial cells, cells that form neurospheres, neural tumor cells, glioblastoma cells, and neuroblastoma cells. The fibroblasts may include NIH 3T3 fibroblasts. The construct may further include an extracellular matrix (ECM). The construct may further be decellularized to leave the attached ECM.

[0024] Yet another aspect of the present invention relates to a method for activating an unactivated surface and coating it with an inorganic oxide adhesion layer, the method comprising the steps of: a) activating the surface of an unactivated substrate; b) providing a coating mixture comprising an organic solvent containing an inorganic compound that reacts with a hydroxyl (-OH), oxy (-O-), oxo (=O), carbonyl (C=O), carboxylic acid (-C(=O)-OH) or carboxylate (-C(=O)-O-) functional group and is dissolved and / or dispersed in the solvent; and c) suspending the activated substrate in the coating solution for a time and temperature sufficient to form an inorganic oxide coating on the activated surface to provide a coated surface, wherein the inorganic compound is selected from the group consisting of alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. The method may further comprise: d) removing the coated substrate from the coating solution; and e) rinsing with a solvent to provide a rinsed coated substrate. The method may still further comprise: f) heating the rinsed coated substrate to 35° C. to 40° C.

[0025] Alternatively, steps b) and c) may be replaced by vapour deposition of an inorganic alkoxide onto the surface, thereby providing an inorganic oxide adhesion layer.

[0026] The inorganic compound of the method can be selected from the alcohol salts of Al, Ti, Zr, Si, Mg and Zn. The alcohol salt can be selected from the group consisting of methoxide, ethoxide, propoxide, isopropoxide, butoxide, isobutoxide, sec-butoxide, and tert-butoxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown are infrared (IR) spectra of a representative oxygen plasma oxidized polydimethylsiloxane (PDMS) construct having a titanium isopropoxide adhesion layer and an octadecylphosphonic acid (ODPA) self-assembled monolayer thereon (Example 1).

[0028] Figure 2 An elemental composition map consistent with the native form of heparin molecules on a stainless steel surface is shown (Example 5), as determined by X-ray photon spectroscopy. DETAILED DESCRIPTION

[0029] The method of the present invention can be used on polymer or metal surfaces that are not reactive with reactive metal alkoxides. Thus, oxidation or amination of polymers and other materials that are not reactive for bonding to the coating (such as phosphonates or siloxanes) can be activated by oxidation methods (including chemical oxidation using chemical oxidants such as permanganate, chlorite, chromic acid, chromates, other chromic acid derivatives, osmium tetroxide, ruthenium tetroxide, iodates, peracids, peroxides, Fenton's reagent (hydrogen peroxide / Fe(II)), lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen. Other methods include oxygen or nitrogen plasma discharge and corona discharge. Oxygen plasma activation can be accomplished as described in U.S. Patent Publication No. 2013 / 0005660 to Dong et al. or as described in U.S. Patent No. 9,655,992 to Clevenger et al., both of which are incorporated herein by reference in their entirety. These chemical reagents and methods are capable of generating hydroxyl (-OH), oxyl (-O-), oxo (=O), carbonyl (C=O), carboxylic acid (-C(=O)-OH) or carboxylate (-C(=O)-O-) functional groups on the surface or, in the case of nitrogen plasma, amino (-NH2). Such functional groups can then react with inorganic alkoxides (such as Zr alkoxides or Ti alkoxides) to form a chemically bonded inorganic adhesion layer. Suitable non-reactive polymers are non-reactive due to the lack of appropriate reactive functional groups on the polymer surface, such as the aforementioned hydroxyl, oxyl, oxo, carbonyl, carboxylic acid or carboxylate functional groups. Suitable non-activated polymers include:

[0030] Polyalkanes or other polymers with terminal alkyl groups, such as polysiloxanes, in which the CH bonds are oxidized

[0031] Reactive C-OH or COOH groups (alcohol or acid) will be generated;

[0032] Polyalkylaromatics or polyaldehydes, in which C-H bond activation occurs oxidatively;

[0033] Polyolefins, where oxidation will produce diols;

[0034] Polythiols, where oxidation produces sulfonic acids; and

[0035] Polyphosphines, where oxidation yields phosphonic, phosphinic or phosphoric acid.

[0036] Non-reactive metal surfaces are those terminated by metal oxides, wherein the metal oxides themselves are not suitable for reaction, such as native oxide layers on titanium (titanium dioxide) or native oxide layers on silicon (silicon dioxide). Other metals in this classification will include chromium and alloys of titanium or chromium, including stainless steel and cobalt chromium. Suitable metalloids for the inventive method include Si, GaAs, GaP, GaN, AlN and perovskites, wherein oxidation will introduce surface-OH or bridging oxygen groups. In addition to native silicon, silicon dioxide and silicon hydride terminated silicon are also suitable for activation using the inventive method.

[0037] Such activated surfaces provide a platform or substrate on which to construct a chemically bonded adhesion layer that can then be used to attach a self-assembled monolayer (SAM), such as a phosphonate self-assembled monolayer (SAMP), which will control the surface properties of the material, for example to make it more or less hydrophobic or to attach other useful moieties, such as alkyne or azide groups (reactive for "click" chemical coupling), electrochemically active moieties, photochemically active moieties, cell-attractive moieties, cell-adhesive moieties, or anti-infective moieties. Suitable anti-infective moieties are disclosed in U.S. Patent Publication No. 2010 / 0215643 to Clevenger et al., U.S. Patent Publication No. 2013 / 0005660 to Dong et al., and U.S. Patent No. 9,655,992 to Clevenger et al., which are incorporated herein by reference in their entireties.

[0038] The surface of the substrate comprising the SAM may be patterned or unpatterned.

[0039] Regarding the adhesion layer (inorganic oxide coating), the non-oxygen inorganic substance preferably has low toxicity in medical applications and can be advantageously selected from Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. Preferably, the inorganic substance is Al, Ti, Zr, Si, Mg, or Zn. More preferably, the inorganic substance is Al, Si, Ti, or Zr. The inorganic substance can be Al. The inorganic substance can be Ti. The inorganic substance can be Zr. The inorganic substance can be Mg. The inorganic substance can be Si. The inorganic substance can be Zn. The inorganic substance can be Mo. The inorganic substance can be Nb. The inorganic substance can be Ta. The inorganic substance can be Sn. The inorganic substance can be W. The inorganic substance can be V.

[0040] By virtue of its method of synthesis from inorganic alkoxides as described herein, the adhesion layer comprises a reactive alkoxide on the surface that can react with appropriate organic compounds to form a SAM or SAMP, but can also react with other organic moieties of interest to chemically bond them directly to the adhesion layer without the intervention of a SAM or SAMP.

[0041] Therefore, the surface of adhesion layer coating can further comprise the self-assembled monolayer (SAM) bonded to adhesion layer, wherein SAM is selected from the organic compound that comprises phosphonic acid group, carboxylic acid group, sulfonic acid group, phosphinic acid group, phosphoric acid group, sulfinic acid group or hydroxamic acid group.Preferably, described SAM comprises phosphonate self-assembled monolayer (SAMP).Phosphonate can be selected from hydrophobic phosphonate, cell adhesive phosphonate and can carry out the phosphonate of further metal catalysis coupling.Suitable phosphonate can be selected from the phosphonic acid with following structure:

[0042]

[0043] wherein the R group is selected from optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, wherein heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and heteroarylalkyl contain one or more heteroatoms selected from O, N, and S. Optional substitutions on the R group may include one or more groups selected from the following: a polyol moiety, a sugar alcohol moiety, a hydroxyl functional group, an amino functional group, a carboxylic acid functional group, a carboxylate functional group, a phosphonic acid functional group, a phosphonate functional group, an ether functional group, an alkyne functional group, an azide functional group, and a thiol functional group. Preferably, the hydrophobic phosphonate is selected from R=C3-C 30 Alkyl. Alkyl can be C5-C 24 Alkyl, or C6-C 20 Alkyl, or C8 to C 18 Alkyl. Alkyl can be C3, C4, C6, C8, C 10 、C 12 、C 14 、C 16 、C 18 , or C 20 Preferably, the cell adhesive phosphonate is selected from R = C3-C 30 Alkyl. Alkyl can be C4-C 24 Alkyl, or C6-C 20 Alkyl, or C8 to C 18 Alkyl. Alkyl can be C3, C4, C6, C8, C 10 、C 12 、C 14 、C 16 、C 18 , or C 20 More preferably, the cell adhesive phosphonate is selected from C3-C 30α,ω-diphosphonate. In this case, the alkylene group can be C3, C4, C6, C8, C 10 、C 12 、C 14 、C 16 、C 18 、C 20 、C 22 、C 24 、C 26 、C 28 , or C 30 Alkylene. α,ω-diphosphonic acid can be C 3-16 Bisphosphonic acid, preferably C 4-12 Bisphosphonic acid, more preferably C4, or C6, or C8, or C 10 , or C 12 The α,ω-diphosphonic acid may be 1,4-butanediphosphonic acid, or 1,6-hexanediphosphonic acid, or 1,8-octanediphosphonic acid, or 1,10-decanediphosphonic acid, or 1,12-dodecanediphosphonic acid, or a mixture of two or more thereof. Preferably, the phosphonate is an alkyne, most preferably a terminal alkyne. The alkyne phosphonate may have R=C3-C 30 Alkynyl. Alkynyl can be C5-C 24 Alkynyl, or C6-C 20 Alkynyl, or C8 to C 18 Alkynyl. Alkynyl can be C3, C4, C6, C8, C 10 、C 12 、C 14 、C 16 、C 18 , or C 20 Preferably, the alkynyl group carries a terminal alkyne.

[0044] Preferably, the SAMP of the coated construct comprises a phosphonic acid covalently attached to the inorganic oxide adhesion layer, the phosphonic acid containing a functional group suitable for cell binding. As noted above, the cell-binding phosphonic acid may comprise one or more functional groups selected from the group consisting of a polyol moiety, a sugar alcohol moiety, a hydroxyl functional group, an amino functional group, a carboxylic acid functional group, a carboxylate functional group, a phosphonic acid functional group, a phosphonate functional group, an ether functional group, an alkyne functional group, an azide functional group, and a thiol functional group. Preferably, the phosphonic acid is a diphosphonic acid, more preferably an α,ω-diphosphonic acid as described above.

[0045] Alkyne and azide functional groups participate in so-called "click reactions." Click chemistry represents a powerful coupling method based on highly specific and efficient bioorthogonal reactions between azide-containing compounds and alkyne-containing compounds to produce cycloaddition products. The small size and reaction specificity of the pre-attached click-reactive groups in SAMPs allow for simple refinement of the SAMP to attach desired moieties, such as electrochemically active moieties, photochemically active moieties, cell-attractive moieties, cell-adhesive moieties, or anti-infective moieties. Thus, Example 3 describes the copper-catalyzed click coupling of a PDMS / adhesion layer / phosphonodec-9-yne SAMP with phenylazide to form a phenyltriazole-terminated SAMP.

[0046] Click chemistry uses a cycloaddition reaction between a 1,3-dipole and a dipolarophile (particularly azide and alkyne) to form a five-membered ring. Azide and alkyne functional moieties are largely inert to biomolecules and aqueous environments. In addition, triazole is similar to the amide moiety that is ubiquitous in nature, but unlike amide, it is not easily cleaved. In addition, triazole is not easily oxidized or reduced. Reports and methods for cycloaddition reactions between 1,3-dipole and dipolarophile are readily available to those of ordinary skill in the art. Relevant literature in the field includes Jewett et al., Chem. Soc. Rev., 2010, 39 (4), 1272-1279 and Schultz et al., Org. Lett. 2010, 12 (10), 2398-2401, all of which are incorporated herein by reference.

[0047] In addition, alkynes can participate in various metal-catalyzed coupling reactions, such as palladium-catalyzed coupling reactions, to introduce additional functional groups on a SAM or SAMP. Reactions such as the Sonogashira reaction are applicable. Thus, Example 2 describes the copper / palladium-catalyzed coupling of a PDMS / adhesion layer / phosphonodec-9-yne SAMP with bromobenzene to form a 10-phenylphosphonodec-9-yne SAMP.

[0048] With respect to the reaction of adhesion layer surface alkoxides directly with other moieties of interest, these moieties can include electrochemically active moieties, photochemically active moieties, cell-attractive moieties, cell-adhesive moieties, anti-infective moieties, or anti-thrombotic moieties, as disclosed herein. Thus, Example 4 describes PDMS coated with an adhesion layer formed from zirconium n-butoxide, which can react directly with glycerol in the absence of a SAM or SAMP.

[0049] Another aspect of the present invention relates to a construct for medical applications, which comprises a coating surface containing SAM or SAMP bonded to an inorganic oxide coating. The construct may further include cells attached to the surface of the SAM or SAMP coating of the construct. The cell may be selected from fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiomyocytes, smooth muscle cells, skeletal muscle cells, tendon cells, ligament cells, epithelial cells, stem cells, nerve cells, PC12 cells, neural support cells, Schwann cells, radial glial cells, cells forming neurospheres, neural tumor cells, glioblastoma cells and neuroblastoma cells. The fibroblasts preferably include NIH 3T3 fibroblasts. The construct may further include extracellular matrix (ECM). ECM is a collection of extracellular molecules secreted by cells that provide structure and biochemical support for surrounding cells. The construct may further be decellularized, leaving the attached ECM.

[0050] Yet another aspect of the present invention relates to a method for activating an unactivated surface and coating it with an inorganic oxide adhesion layer, the method comprising the steps of: a) activating the surface of an unactivated substrate; b) providing a coating mixture comprising an organic solvent containing an inorganic compound that reacts with a hydroxyl (-OH), oxy (-O-), oxo (=O), carbonyl (C=O), carboxylic acid (-C(=O)-OH) or carboxylate (-C(=O)-O-) functional group and is dissolved and / or dispersed in the solvent; and c) suspending the activated substrate in the coating solution for a time and temperature sufficient to form an inorganic oxide coating on the activated surface to provide a coated surface, wherein the inorganic compound is selected from the group consisting of alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. The method may further comprise: d) removing the coated substrate from the coating solution; and e) rinsing with a solvent to provide a rinsed coated substrate. The method may still further comprise: f) heating the rinsed coated substrate to 35° C. to 40° C.

[0051] Alternatively, steps b) and c) may be replaced by vapour deposition of an inorganic alkoxide onto the surface, thereby providing an inorganic oxide adhesion layer.

[0052] The inorganic compound of the method can be selected from the alcohol salts of Al, Ti, Zr, Si, Mg and Zn. The alcohol salt can be selected from the group consisting of methoxide, ethoxide, propoxide, isopropoxide, butoxide, isobutoxide, sec-butoxide, and tert-butoxide.

[0053] Regarding the inorganic alkoxide, the inorganic substance is preferably non-toxic in reconstructive medicine applications and can advantageously be selected from Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. Preferably, the inorganic substance is Al, Ti, Zr, Si, Mg, or Zn. More preferably, the inorganic substance is Al, Si, Ti, or Zr. The inorganic substance may be Al. The inorganic substance may be Ti. The inorganic substance may be Zr. The inorganic substance may be Mg. The inorganic substance may be Si. The inorganic substance may be Zn. The inorganic substance may be Mo. The inorganic substance may be Nb. The inorganic substance may be Ta. The inorganic substance may be Sn. The inorganic substance may be W. The inorganic substance may be V. Preferred alkoxides are selected from methoxide, ethoxide, propoxide, isopropoxide, butoxide, isobutoxide, sec-butoxide, and tert-butoxide.

[0054] One aspect of the present invention relates to a construct comprising a) a surface activated for chemical bonding to an inorganic adhesion layer, the inorganic adhesion layer providing further attachment of portions that modify the overall surface properties, wherein the surface does not contain accessible sufficiently reactive functional groups on the surface, wherein activation comprises generating reactive functional groups on the surface; and b) an inorganic adhesion layer chemically bonded to the activated surface via the reactive functional groups; wherein the functional groups react with an inorganic alkoxide to form the inorganic adhesion layer. The surface of such a construct may comprise a polymer or a metal. The polymer may be selected from polyalkanes, polysiloxanes, polyalkyl aromatics, polyolefins, polythiols, and polyphosphines. The metal may be selected from stainless steel and its various alloys. The surface functional groups of the construct are preferably selected from hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino groups.

[0055] The surface functional groups of the construct can be generated by chemical oxidation using an oxidant such as permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, or oxygen. Alternatively, the surface functional groups can be generated using oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.

[0056] The construct may further comprise a self-assembled monolayer (SAM) bonded to the inorganic adhesion layer, wherein the SAM is selected from an organic compound comprising a phosphonic acid group, a carboxylic acid group, a sulfonic acid group, a phosphinic acid group, a phosphate group, a sulfinic acid group, or a hydroxamic acid group. The SAM may comprise a phosphonate self-assembled monolayer (SAMP), wherein the phosphonate may be selected from a hydrophobic phosphonate, a cell adhesive phosphonate, and a phosphonate capable of further metal-catalyzed coupling. The phosphonate may be selected from a phosphonic acid group having the structure

[0057]

[0058] wherein the R group is selected from optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, wherein heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and heteroarylalkyl contain one or more heteroatoms selected from O, N, and S. The hydrophobic phosphonate of the construct can be selected from R=C3-C 30 alkyl, and the cell adhesive phosphonate is selected from R = C3-C substituted with another phosphonate group 30 The cell adhesive phosphonate can be selected from C3-C 30 α,ω-diphosphonates.

[0059] The construct may have a SAM or SAMP further comprising an anti-infective agent covalently bound thereto. Suitable anti-infective agents include antimicrobial agents selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbicide, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefdroxil, cefazolin, cephalothin, cephalexin, cefaclor, cefhydroxyazole, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin , spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / other Zobactam, ticarcillin / clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfadiazine, sulfamethoxazole, sulfadiazine, silver, sulfadiazine, sulfamethoxazole, sulfamethoxazole Sulfadiazole, sulfadiazine Sulfasalazine, sulfasalazine, sulfamethoxazole Azole, trimethoprim, trimethoprim-sulfamethoxazole The anti-infective agent may be selected from the group consisting of chlorhexidine, biguanide, cationic ammonium compounds, pharmaceutically acceptable salts thereof, and mixtures thereof of two or more thereof. The anti-infective agent may also be selected from the group consisting of cationic ammonium compounds, cationic ammonium dendrimers, silver, copper, cationic substances, and mixtures thereof of two or more thereof. The cationic ammonium compound may be selected from the group consisting of choline and choline derivatives. Alternatively, the anti-infective agent may include polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diol, and other polyols (such as polyvinyl alcohol) and amino alcohols.

[0060] Another aspect of the invention relates to a method for forming a construct of the invention comprising the steps of: a) activating a surface for chemical bonding to an inorganic adhesion layer, the inorganic adhesion layer providing further attachment of portions that modify the overall surface properties, wherein the surface does not contain accessible sufficiently reactive functional groups on the surface, wherein the activation comprises generating reactive functional groups on the surface to provide an activated surface; and b) chemically bonding the inorganic adhesion layer to the functional groups of the activated surface; wherein the functional groups react with an inorganic alkoxide to form the inorganic adhesion layer. The surface of the method may comprise a polymer selected from the group consisting of polyalkanes, polysiloxanes, polyalkyl aromatics, polyolefins, polythiols, and polyphosphines. The surface functional groups of the method may be selected from the group consisting of hydroxyls, oxyls, oxos, carbonyls, carboxylates, carboxylates, and amino groups. The activation step of the method can include chemical oxidation using a chemical oxidant such as permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, or oxygen. Alternatively, the activation step of the method can include oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.

[0061] Another aspect of the present invention relates to a method for activating an unactivated surface and coating it with an inorganic oxide adhesion layer, the method comprising the steps of: a) activating the surface of a substrate that is chemically bonded to an unactivated inorganic adhesion layer; b) providing a coating mixture comprising an organic solvent containing a reactive inorganic compound dissolved and / or dispersed in the solvent; and c) suspending the activated substrate in the coating mixture for a time and at a temperature sufficient to form an inorganic oxide coating on the activated surface to provide a surface coated with the inorganic oxide adhesion layer; wherein the inorganic compound is selected from the group consisting of alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. The method may further comprise d) removing the coated substrate from the coating solution; e) rinsing with a solvent to provide a rinsed coated substrate; and f) heating the rinsed coated substrate to 35° C. to 40° C. Alternatively, steps b) and c) may be replaced by vapor-depositing an inorganic alkoxide onto the surface to provide the inorganic oxide adhesion layer.

[0062] Another aspect of the present invention relates to a construct having a SAMP as disclosed above, wherein the R group is a dodecyl-9-alkynyl group, which is further coupled using a metal-catalyzed reaction on an alkyne. The dodecyl-9-alkynyl R group can also be further coupled using a click reaction on an alkyne.

[0063] Alternatively, one aspect of the present invention relates to a construct coated with an adhesion layer without SAM or SAMP, which further directly reacts with an organic part on an inorganic adhesion layer, the organic part being selected from electrochemically active parts, photochemically active parts, cell attraction parts, cell adhesion parts and anti-infective parts. The anti-infective agent can include polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols and other polyols (such as polyvinyl alcohol) and amino alcohols. Preferably, the anti-infective part is selected from polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, polyols, amino alcohols and a mixture of two or more thereof. Proteins can also be directly attached to the inorganic adhesion layer. Suitable proteins include heparin and heparin derivatives, such as heparin functionalized phosphonates and silane precursor molecules (respectively heparin PUL and heparin silane).

[0064] Thus, one aspect of the present invention relates to a stainless steel (SS) coupon that was oxidized on both sides by oxygen plasma and immersed in a titanium (IV) butoxide solution to form a Ti(IV) butoxide adhesion layer covalently attached to the SS surface. This coated SS surface was functionalized with heparin using heparin-functionalized phosphonate or silane precursor molecules, and the heparin was found to be in its native form (as opposed to denatured) by XPS elemental analysis (see Example 5).

[0065] One aspect of the present invention relates to a construct comprising a) an activated surface comprising chemically accessible reactive functional groups; and b) an inorganic alkoxide adhesion layer chemically bonded to the reactive functional groups of the activated surface; wherein the reactive functional groups react with the inorganic alkoxide to form the inorganic adhesion layer, and the inorganic adhesion layer provides further attachment of additional functional groups that modify part of the overall surface properties. The activated surface of the construct can include a surface that is inherently non-reactive to the chemical bonding of the inorganic adhesion layer, and the inherently non-reactive surface has been treated to produce chemically accessible reactive functional groups on the surface, thereby providing activation. The surface of the construct can include a polymer, stainless steel, or stainless steel alloy that is inherently non-reactive to the chemical bonding of the inorganic adhesion layer. The polymer can be selected from polyalkanes, polysiloxanes, polyalkyl aromatics, polyolefins, polythiols, and polyphosphines.

[0066] The surface reactive functional groups can be selected from hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino. The surface reactive functional groups can be generated by chemical oxidation. The chemical oxidation can include treatment with an oxidant selected from permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen.

[0067] Alternatively, the surface reactive functional groups may be generated by oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.

[0068] The inorganic adhesion layer of the construct may include an inorganic oxide selected from the group consisting of oxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, V, and mixtures of two or more thereof. Preferably, the inorganic oxide adhesion layer is selected from the group consisting of oxides of Al, Ti, Zr, Si, Mg, Zn, and mixtures of two or more thereof.

[0069] Described construct can further comprise the self-assembled monolayer (SAM) of described other functional group bonding with described inorganic adhesion layer, wherein said SAM is selected from the organic compound that comprises phosphonic acid group, carboxylic acid group, sulfonic acid group, phosphinic acid group, phosphate group, sulfinic acid group or hydroxamic acid group.Described SAM preferably comprises phosphonate self-assembled monolayer (SAMP).Described phosphonate can be selected from hydrophobic phosphonate and cell adhesion phosphonate.Described hydrophobic phosphonate and cell adhesion phosphonate can be selected from phosphonic acid of following structure

[0070]

[0071] wherein the R group is selected from optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, wherein the heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and heteroarylalkyl groups contain one or more heteroatoms selected from O, N, and S.

[0072] The R group of the construct can be dodecyl-9-alkynyl, which is further coupled using a metal-catalyzed reaction on the alkyne functional group. The R group of the construct can be dodecyl-9-alkynyl, which is further coupled using a click reaction on the alkyne functional group.

[0073] The hydrophobic phosphonate can be selected from R=C3-C 30 alkyl, and the cell adhesive phosphonate is selected from R = C3-C substituted with another phosphonate group 30 The cell adhesive phosphonate can be selected from C3-C 30 α,ω-diphosphonates.

[0074] The SAM or SAMP of the construct may further include an anti-infective or anti-thrombotic agent covalently bound thereto. The anti-infective agent can be an antimicrobial agent selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbicide, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefdroxil, cefazolin, cephalothin, cephalexin, cefaclor, cefhydroxyazole, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin , spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / other Zobactam, ticarcillin / clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfadiazine, sulfamethoxazole, sulfadiazine, silver, sulfadiazine, sulfamethoxazole, sulfamethoxazole Sulfadiazole, sulfadiazine Sulfasalazine, sulfasalazine, sulfamethoxazole Azole, trimethoprim, trimethoprim-sulfamethoxazole azole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole, mupirocin, plate mycin, quinupristin / dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptable salts thereof, and mixtures of two or more thereof.

[0075] Alternatively, the anti-infective agent may be selected from polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diol, polyol, amino alcohol, and mixtures of two or more thereof.

[0076] The anti-infective agent may be selected from chlorhexidine, biguanide, cationic ammonium compounds, cationic ammonium dendrimers, silver, copper, cationic substances and mixtures of two or more thereof. The cationic ammonium compound may be selected from choline and choline derivatives.

[0077] The antithrombotic agent may be heparin.

[0078] The construct of the present invention may further include cells attached to the coating surface of the construct, wherein the cells are selected from the group consisting of fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiomyocytes, smooth muscle cells, skeletal muscle cells, tenocytes, ligament cells, epithelial cells, stem cells, nerve cells, PC12 cells, neural supporting cells, Schwann cells, radial glial cells, cells that form neurospheres, neural tumor cells, glioblastoma cells, and neuroblastoma cells. The fibroblasts may include NIH 3T3 fibroblasts.

[0079] The construct may further comprise an extracellular matrix (ECM).The construct may also be decellularized to leave the ECM.

[0080] In another aspect, the construct as described above can have an inorganic adhesion layer directly attached to an organic moiety selected from an electrochemically active moiety, a photochemically active moiety, a cell-attractive moiety, a cell-adhesive moiety, and an anti-infective moiety without an intermediate SAM or SAMP layer. The anti-infective moiety can be selected from a polysaccharide, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diol, polyol, amino alcohol, and mixtures of two or more thereof.

[0081] Another aspect of the invention relates to a method of forming a construct as described above, comprising the steps of: a) providing a surface that is inherently non-reactive to chemical bonding of an inorganic adhesion layer; b) activating the surface that is inherently non-reactive to chemical bonding of an inorganic adhesion layer by treating the non-reactive surface to produce reactive functional groups on the surface, thereby providing an activated surface; and c) chemically bonding an inorganic adhesion layer to the reactive functional groups of the activated surface; wherein the reactive functional groups react with an inorganic alkoxide to form the inorganic adhesion layer, and wherein the inorganic adhesion layer provides further attachment of additional functional groups that modify part of the overall surface properties.

[0082] In the above method, the surface can include a polymer that is inherently non-reactive with respect to chemical bonding of the inorganic adhesion layer. The polymer can be selected from polyalkanes, polysiloxanes, polyalkyl aromatics, polyolefins, polythiols, and polyphosphines. The surface functional groups generated by activation can be selected from hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino groups. The activation step can include chemical oxidation. The chemical oxidation can include treatment with an oxidant selected from permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen.

[0083] Alternatively, the activation step may comprise an oxygen plasma discharge, a nitrogen plasma discharge or a corona discharge.

[0084] The inorganic adhesion layer of the method may include an inorganic oxide selected from the group consisting of oxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, V, and mixtures of two or more thereof. Preferably, the inorganic oxide adhesion layer is selected from the group consisting of oxides of Al, Ti, Zr, Si, Mg, Zn, and mixtures of two or more thereof.

[0085] Another aspect of the present invention relates to a method for activating the surface of an unactivated substrate and coating it with an inorganic oxide adhesion layer, comprising the steps of: a) activating the surface of a substrate that is chemically bonded to an inorganic adhesion layer in an unactivated manner by generating reactive functional groups on the surface to form an activated substrate; b) providing a coating mixture comprising an organic solvent containing a reactive inorganic compound dissolved and / or dispersed in the solvent; and c) suspending the activated substrate in the coating mixture for a time and temperature sufficient to react the reactive functional groups and the inorganic compound and form an inorganic oxide coating on the activated surface of the substrate, thereby providing a substrate coated with the inorganic oxide adhesion layer; wherein the inorganic compound is selected from the group consisting of alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V.

[0086] The activation step of the method may comprise chemical oxidation. Chemical oxidation may comprise treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen.

[0087] Steps b) and c) of the method are replaced by vapor depositing an inorganic alkoxide on the surface of the activated substrate and reacting the reactive functional groups with the inorganic alkoxide to form an inorganic oxide adhesion layer bonded thereto.

[0088] Alternatively, the activation step may comprise an oxygen plasma discharge, a nitrogen plasma discharge or a corona discharge.

[0089] The method may further include: d) removing the coated substrate from the coating solution; e) rinsing with a solvent to provide a rinsed coated substrate; and f) heating the rinsed coated substrate to 35°C to 40°C.

[0090] In summary, the methods of the present invention convert originally non-reactive materials into superficially reactive materials. This surface reactivity can be controlled by activation methods and by the properties of the coatings used to activate the surface, enabling cell attachment, proliferation, and ECM formation. Such means of directing the surface properties of materials offer numerous potential applications in medicine and other biomedical and biological fields.

[0091] Example

[0092] Overview. All materials were obtained from commercial sources. Solvents and chemicals included methanol (Sigma Aldrich), 2-propanol (Sigma Aldrich), tert-butyl alcohol (Fisher Scientific), 200 proof ethanol (Pharmco-Aaper), xylene (EMD Millipore Corporation), toluene (EMD Chemical Inc.), hexane (Sigma Aldrich), titanium (IV) isopropoxide (Sigma Aldrich), 1,4-butanediphosphonic acid (Acros Organics), 1,12-dodecanediylbis(phosphonic acid) (Sigma Aldrich), and octadecylphosphonic acid (Alfa Aesar, Sigma Aldrich).

[0093] Example 1. Oxygen plasma oxidation of PDMS with titanium isopropoxide adhesion layer and ODPA self-assembled monolayer

[0094] Polydimethylsiloxane (PDMS) specimens were oxygen plasma oxidized on one side and immersed in a solution of titanium isopropoxide in toluene at a concentration of 10 μL / mL. The samples were immersed in the solution for 15 minutes. The specimens were removed from the solution, and it was noted that the surfaces of these PDMS specimens became slightly less translucent. The specimens were heated at 35°C for 1 min, rinsed with ethanol, and then placed in a solution of octadecylphosphonic acid (ODPA) in toluene at a concentration of 0.5 mg / mL. The specimens were kept in this solution for several hours, removed from the solution, heated at 35°C for 1 min, and rinsed with ethanol. The appearance of the specimen surface did not change during this procedure. Infrared spectroscopy analysis showed the presence of a monolayer of octadecylphosphonate (ODPA) on the PDMS surface. Figure 1 The large peaks can be attributed to the PDMS methyl groups; at 2921 and 2851 cm -1 The peak at is characteristic of a self-assembled monolayer of octadecylphosphonate (ODPA). In contrast, a control PDMS sample that was not pretreated with titanium isopropoxide to form an adhesion layer showed no signs of a phosphonate SAM after a similar ODPA treatment. Similar results were observed using 11-hydroxyundecylphosphonic acid.

[0095] Infrared spectrum. Infrared (IR) spectroscopy can detect functional groups in molecules by identifying unique peaks corresponding to the stretching and bending of chemical bonds. This same technique can be applied to SAMs on both optically transparent (transmission mode) and reflective (grazing angle spectroscopy reflectance mode) substrates. IR can assess successful monolayer preparations and monitor degradation and determine the degree of order in the SAM surface. Antisymmetric and symmetrical methylene stretching are diagnostic peaks based on the monolayer of alkyl groups and appear at 2920 and 2850 cm-1, respectively. -1 The wavenumber of the methylene stretching mode should be understood as diagnostic of whether the chains are present in an all-trans configuration ("ordered" or crystalline state) or in a random configuration ("disordered" and "liquid-like" films). In this work, well-ordered films are defined as being characterized by antisymmetric methylene stretching wavenumbers below 2920 cm -1 And the symmetrical methylene stretching wave number is lower than 2850cm -1 To evaluate the film quality, ATR-FTIR data were acquired using a Nicolet TMiSTM50 FT-IR spectrometer.

[0096] Example 2.A sample of treated PDMS was prepared as described in Example 1 and treated with a solution of zirconium n-butoxide dissolved in toluene at a concentration of 0.5 mg / mL. The sample was heated at 35°C for 1 minute, rinsed with ethanol, and then placed in a solution of phosphonodec-9-yne in toluene at a concentration of 0.5 mg / mL. The sample was kept in this solution for several hours, removed from the solution, heated at 35°C for 1 minute, rinsed with ethanol, and analyzed by IR spectroscopy for the presence of ions at approximately 2135 and 3325 cm -1 A copper / palladium catalyzed coupling reaction, such as with bromobenzene, then produces a 10-phenyl coupled product.

[0097] Example 3. A sample of treated PDMS was prepared as described in Example 1 and treated with a solution of zirconium n-butoxide dissolved in toluene at a concentration of 0.5 mg / mL. The sample was heated at 35°C for 1 minute, rinsed with ethanol, and then placed in a solution of phosphonodec-9-yne in toluene at a concentration of 0.5 mg / mL. The sample was kept in this solution for several hours, removed from the solution, heated at 35°C for 1 minute, rinsed with ethanol, and analyzed by IR spectroscopy for the presence of ions at approximately 2135 and 3325 cm -1 A copper-catalyzed “click” reaction using benzene azide then gave phenyltriazole-terminated phosphonates.

[0098] Example 4. A sample of treated PDMS was prepared as described in Example 1 and treated with a solution of zirconium n-butoxide dissolved in toluene at a concentration of 0.5 mg / mL. The sample was heated at 35°C for 1 minute, rinsed with ethanol, and then placed in a solution of glycerol in ethanol at a concentration of 0.5 mg / mL. The sample was kept in this solution for several hours, removed from the solution, heated at 35°C for 1 minute, rinsed with ethanol, and analyzed by IR spectroscopy for the presence of ions at approximately 1050, 2980, and 3100 cm -1 The characteristic peaks of hydroxyl, ether and aliphatic groups are detected.

[0099] Example 5 Covalently bound heparin on Ti-functionalized stainless steel

[0100] Overview. All materials were obtained from commercial sources. Solvents and chemicals included anhydrous toluene (Sigma Aldrich), tert-butyl alcohol (Fisher Scientific), 200 proof ethanol (Acros Organics), reagent alcohol (Fisher Scientific), titanium (IV) butoxide (Sigma Aldrich), monoamine-functionalized trialkoxysilanes and phosphonates (Gelest, Sikemia), and heparin sodium salt (Sigma Aldrich).

[0101] Stainless steel (SS) coupons were oxygen plasma oxidized on both sides and immersed in a 3% (v / v) solution of titanium (IV) butoxide in toluene. The samples were immersed in the solution for 15 minutes while stirring constantly at 350 rpm. The Ti (IV) butoxide treated coupons were removed from the solution and allowed to dry in a chemical fume hood for 5 minutes. The samples were then placed in a 130°C oven for 10 minutes, then sonicated with reagent alcohol for 10 minutes (twice) and vacuum dried for 10 minutes. When compared to the control SS coupons, the coupons now had a visible gray shade on the surface. The samples were placed in a 1.5-15 mM ethanolic solution of a heparin-functionalized phosphonate or silane precursor (heparin PUL or heparin silane) and kept in this solution overnight at a temperature ranging from 24°C to 37°C. The coupons were rinsed and sonicated in reagent alcohol for 10 minutes (twice) and vacuum dried for 10 minutes. The appearance of the coupon surface did not change during this procedure. X-ray photon spectroscopy analysis revealed an elemental composition consistent with the native form of heparin molecules on the stainless steel surface ( Figure 2 ). Heparin is a sugar dimer that repeats multiple times to form a distribution of different molecular weights. The dimer is composed of five chemical elements. Four of these elements (carbon, oxygen, sulfur, and nitrogen) can be detected by XPS. In addition, the theoretical percentage composition of these elements in heparin can be calculated and associated with the experimentally observed element percentage composition values as determined by XPS. By using non-covalently bound native heparin, a negative control (heat-denatured covalently bound heparin, temperature>100°C), and covalently bound heparin functionalized phosphonates on stainless steel to perform XPS analysis on native heparin and denatured heparin, we found that the element percentage composition diagrams of non-covalently bound native heparin and covalently bound heparin functionalized phosphonates were comparable to the theoretical percentage element composition of the heparin dimer. The largest change in the heparin percentage element composition between the heat-denatured covalently bound heparin group and the covalently bound heparin functionalized phosphonates was the significant loss of element percentage carbon, oxygen, sulfur, and nitrogen.

[0102] X-ray photon spectroscopy.X-ray photon spectroscopy (XPS) is a technique for measuring the percent elemental composition of surface-bound molecules by identifying the presence of specific elements within known functional groups in the molecule within a 10-nm depth profile. In addition to measuring deviations from natural elemental percent composition values, this same technique can also be applied to surface-bound molecules to determine the presence of elements within functional regions. In the present case, due to the different strategies used to bind heparin to stainless steel, XPS can assess successful surface preparation and monitor the degradation of important domains in heparin. In theory, the repeating dimer unit in heparin has an elemental percent composition diagram of carbon (33%), oxygen (55%), sulfur (8.0%), and nitrogen (2.8%). Due to key-lock interactions, the natural elemental composition of heparin supports the interaction of heparin with antithrombin, resulting in structural changes in antithrombin and its activity in preventing thrombosis. It should be understood that significant changes in the elemental percent composition of sulfur are diagnostic of whether the heparin is in its native form or denatured form on the surface.

[0103] Comparison of the elemental percent composition of the covalently bound heparin prepared above with experimentally measured elemental percent composition values for native heparin revealed that the form was native; there were no statistically significant differences, confirming the native covalently bound form.

[0104] Other implementation plans

[0105] From the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0106] All publications cited herein are incorporated by reference in their entirety for all purposes.

Claims

1. A construct for cell growth, reconstructive medicine, and medical devices, comprising: a) oxidizing or aminated polymer surfaces comprising chemically accessible oxygen, oxo, carbonyl, carboxylic acid, carboxylate, and amino reactive functional groups that react with inorganic alkoxides; b) an inorganic oxide adhesion layer chemically bonded to the reactive functional groups on the polymer surface, wherein the inorganic oxide adhesion layer comprises surface alkoxide groups selected from the group consisting of oxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, V, and mixtures of two or more thereof; and c) a self-assembled phosphonate monolayer bonded to the surface alkoxide groups of the inorganic oxide adhesion layer, Prior to the introduction of the functional groups, the polymer surface does not have exposed surface functional groups with sufficient density or sufficient reactivity to form covalent or coordinate bonds with inorganic alkoxides.

2. The construct of claim 1, wherein the reactive functional groups are generated by chemical oxidation, oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.

3. The construct of claim 1 , wherein the polymeric surface comprises a polymer selected from the group consisting of polyalkanes, polysiloxanes, polyalkylaromatics, polyolefins, polythiols, and polyphosphines.

4. The construct of claim 2, wherein the chemical oxidation comprises treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen.

5. The construct of claim 1, wherein the inorganic oxide adhesion layer is selected from oxides of Ti, Zr, Ta, Nb, V, and mixtures of two or more thereof.

6. The construct of claim 1, wherein the phosphonate is selected from the group consisting of a hydrophobic phosphonate and a cell adhesive phosphonate.

7. The construct of claim 6, wherein the hydrophobic phosphonate and the cell adhesive phosphonate are selected from the group consisting of phosphonates of the following structures: wherein the R group is selected from optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, wherein the heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and heteroarylalkyl groups contain one or more heteroatoms selected from O, N, and S.

8. The construct according to claim 7, wherein the hydrophobic phosphonate is selected from R=C3-C 30 alkyl, and the cell adhesive phosphonate is selected from R = C3-C substituted with another phosphonate group 30 alkyl.

9. The construct of claim 8, wherein the cell adhesive phosphonate is selected from the group consisting of C3-C 30 α,ω-diphosphonates.

10. The construct of claim 1, wherein the self-assembled monolayer of the phosphonate further comprises an anti-infective or anti-thrombotic agent covalently bound thereto.

11. The construct of claim 10, wherein the anti-infective agent is an antimicrobial agent selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbicide, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cephalothin, cephalexin, cefaclor, cefadroxil, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefotax ... Sphobutyric acid, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin Amoxicillin, temocillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, ticarcillin / clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfamethoxazole, sulfacetamide, silver, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfasalazine, sulfisoxazole , trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole, mupirocin, plate mycin, quinupristin / dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptable salts thereof, and mixtures of two or more thereof.

12. The construct of claim 10, wherein the anti-infective agent is selected from the group consisting of polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diol, polyols, amino alcohols, and mixtures of two or more thereof.

13. The construct of claim 10, wherein the anti-infective agent is selected from the group consisting of chlorhexidine, biguanide, cationic ammonium compounds, cationic ammonium dendrimers, silver, copper, cationic substances, and mixtures of two or more thereof.

14. The construct of claim 13, wherein the cationic ammonium compound is selected from choline and choline derivatives.

15. The construct according to claim 9, further comprising: Cells attached to the coated surface of the construct, wherein the cells are selected from the group consisting of fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiomyocytes, smooth muscle cells, skeletal muscle cells, tenocytes, ligament cells, epithelial cells, stem cells, neural cells, PC12 cells, neural supporting cells, Schwann cells, radial glial cells, neurosphere-forming cells, neural tumor cells, glioblastoma cells, and neuroblastoma cells.

16. The construct of claim 15, wherein the fibroblasts comprise NIH 3T3 fibroblasts.

17. The construct of claim 15 or 16, further comprising an extracellular matrix.

18. The construct of claim 17, which is decellularized to remove the extracellular matrix.

19. A method of forming a construct according to claim 1 or 2, comprising: a) oxidizing or aminating the polymer surface to form chemically accessible hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino reactive functional groups thereon, thereby providing an activated polymer surface; b) chemically bonding an inorganic alkoxide adhesion layer having surface alkoxide groups to the reactive functional groups on the surface of the activated polymer; and c) bonding a self-assembled phosphonate monolayer to surface alkoxide groups of the inorganic alkoxide adhesion layer, wherein the inorganic alkoxide adhesion layer comprises an inorganic oxide selected from the group consisting of oxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, V, and mixtures of two or more thereof.

20. The method of claim 19, wherein the polymer is selected from the group consisting of polyalkanes, polysiloxanes, polyalkyl aromatics, polyolefins, polythiols, and polyphosphines.

21. The method of claim 19, wherein the oxidizing comprises chemical oxidation.

22. The method of claim 21, wherein the chemical oxidation comprises treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen.

23. The method of claim 19, wherein the oxidizing comprises oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.

24. The method of claim 19, wherein the inorganic oxide is selected from oxides of Ti, Zr, Ta, Nb, V, and mixtures of two or more thereof.

25. A method of activating a substrate polymer surface and coating the activated surface with an inorganic oxide adhesion layer, comprising the steps of: a) activating a polymer surface that is chemically bonded to an inorganic oxide adhesion layer as an unactivated substrate by oxidizing or aminating the polymer surface to produce hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino reactive functional groups on the polymer surface, thereby providing an activated substrate; b) providing a coating mixture comprising an organic solvent containing a reactive inorganic alkoxide compound dissolved and / or dispersed in the solvent; c) suspending the activated substrate in the coating mixture for a time and temperature sufficient to react the reactive functional groups with the reactive inorganic alkoxide compound and form an inorganic oxide coating on the activated polymer surface of the substrate to provide a substrate coated with an inorganic oxide adhesion layer; and d) bonding a self-assembled phosphonate monolayer to the surface alkoxide groups of the inorganic oxide adhesion layer, The inorganic alkoxide compound is selected from the group consisting of alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V.

26. The method of claim 25, wherein the activation comprises chemical oxidation.

27. The method of claim 26, wherein the chemical oxidation comprises treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracid, peroxide, Fenton's reagent, lead tetraacetate, lead tetraacetate / Mn(II), ozone, and oxygen.

28. The method of claim 25, wherein coating steps b) and c) are replaced by vapor depositing an inorganic alkoxide onto the polymer surface of the activated substrate and reacting the reactive functional groups with the inorganic alkoxide to form an inorganic oxide adhesion layer bonded thereto.

29. The method of claim 25, wherein the activation comprises oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.

30. The method according to any one of claims 25 to 27 or 29, further comprising between steps c) and d): i) removing the coated substrate from the coating mixture; ii) rinsing with a solvent to provide a rinsed coated substrate; as well as iii) heating the rinsed coated substrate to 35°C to 40°C.

31. The construct of claim 7, wherein the R group is dodecyl-9-alkynyl.

32. The construct of claim 1 or 2, wherein the inorganic oxide adhesion layer is attached directly to an organic moiety selected from an electrochemically active moiety, a photochemically active moiety, a cell-attractive moiety, a cell-adhesive moiety, and an anti-infective moiety without an intermediate self-assembled monolayer or a self-assembled monolayer of a phosphonate.

33. The construct of claim 32, wherein the anti-infective moiety is selected from the group consisting of polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diol, polyols, amino alcohols, and mixtures of two or more thereof.

34. The construct of claim 10, wherein the antithrombotic agent is heparin.

35. The construct of claim 10, wherein the anti-infective agent is copper.

36. The construct of claim 32, wherein the anti-infective moiety is selected from the group consisting of chlorhexidine, biguanide, cationic ammonium compounds, cationic ammonium dendrimers, polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, polyols, amino alcohols, silver, copper, and mixtures of two or more thereof.

37. A construct for use in cell growth, reconstructive medicine, and medical devices, comprising: a) a polymer surface comprising chemically accessible hydroxyl groups reactive with an inorganic alkoxide; b) an inorganic oxide adhesion layer chemically bonded to the hydroxyl groups on the surface of the polymer, the inorganic oxide adhesion layer comprising surface alkoxide groups; c) a self-assembled phosphonate monolayer bonded to the surface alkoxide groups of the inorganic oxide adhesion layer; and d) An anti-infective agent covalently bonded to the self-assembled monolayer of phosphonate.

38. The construct of claim 37, wherein the inorganic oxide adhesion layer is selected from oxides of Ti, Zr, Ta, Nb, V, and mixtures of two or more thereof.

39. The construct of claim 37, wherein the phosphonate is selected from the group consisting of a hydrophobic phosphonate and a cell adhesive phosphonate.

40. The construct of claim 39, wherein the hydrophobic phosphonate and cell adhesive phosphonate are selected from the group consisting of phosphonates of the following structures: wherein the R group is selected from optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, wherein the heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, and heteroarylalkyl groups contain one or more heteroatoms selected from O, N, and S.

41. The construct of claim 40, wherein the hydrophobic phosphonate is selected from the group consisting of R=C3-C 30 alkyl, and the cell adhesive phosphonate is selected from R = C3-C substituted with another phosphonate group 30 alkyl.

42. The construct of claim 41 , wherein the cell adhesive phosphonate is selected from the group consisting of C3-C 30 α,ω-diphosphonates.

43. The construct of claim 37, wherein the anti-infective agent is an antimicrobial agent selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbicide, loracarbef, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cephalothin, cephalexin, cefaclor, cefadroxil, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cefotaxime, cefotaxime, cefotaxime, ceftazidime, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, ceftriaxone, cefpodoxime, ceftazidime, cefotax ... Sphobutyric acid, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin Amoxicillin, temocillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, ticarcillin / clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfamethoxazole, sulfacetamide, silver, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfasalazine, sulfisoxazole , trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole, mupirocin, plate mycin, quinupristin / dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptable salts thereof, and mixtures of two or more thereof.

44. The construct of claim 37, wherein the anti-infective agent is selected from the group consisting of polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diol, polyols, amino alcohols, and mixtures of two or more thereof.

45. The construct of claim 37, wherein the anti-infective agent is selected from the group consisting of chlorhexidine, biguanide, cationic ammonium compounds, cationic ammonium dendrimers, silver, copper, cationic species, and mixtures of two or more thereof.

46. The construct of claim 45, wherein the cationic ammonium compound is selected from choline and choline derivatives.

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