Patent Application
20200360562
The present invention is related to the field of activation of otherwise unreactive surfaces to make them susceptible to the chemical bonding of coatings, where the coatings allow modification of surface properties. Such coated surfaces have utility as scaffolds for cell growth, reconstructive medicine, and medical devices.
Tissue formation, wound repair, and many disease processes depend on expression and cell-mediated assembly of the appropriate extracellular matrix (ECM) proteins. In particular, oriented ECM fibers are essential for normal tissue development and homeostasis. The organization of the ECM can, however, go awry in many diseases and at sites of injury producing the unaligned collagen fibers that form in scar tissue.
A goal of regenerative medicine is to promote formation of new tissue that closely resembles the normal tissue in organization and function. Controlling cell growth in a spatially defined way enables regeneration of damaged or diseased tissues having the proper alignment of constituent cells and/or alignment of molecular complexes that the cells produce. In particular, cells direct the arrangement of ECM fibrils to correspond to their actin filaments by using cell surface receptors that are indirectly connected to the actin cytoskeleton. Therefore, a major challenge in regenerative medicine is to promote cells to assemble ECM fibrils, such as collagen, into particular orientations or alignments on a scaffold device in order to generate tissues with the required functional properties.
Such a scaffold requires an appropriate substrate surface on which to attach cells in an environment that stimulates the generation of an ECM.
As used herein, the term “covalent” bond refers to a chemical bond that involves the sharing of electron pairs between atoms. The term “coordinate”, “coordinative” or “coordinate covalent” bond denotes a two-center, two-electron covalent bond in which both electrons derive from the same atom, such as the bonding of metal ions to ligands. In contrast “ionic” bonding involves electrostatic attraction between oppositely charged ions, where electrons are not shared, but one or more electrons are localized on one of the atoms (the anion) and removed from the other atom (the cation).
As used herein the term “bonded” means affixed or attached, preferably chemically attached without the use of an adhesive. The chemical bond is preferably a covalent or coordinative attachment.
As used herein the terms “reactive”, and “unreactive” refer to the ability of a specific functional group to chemically bond with other functional groups, for example in an inorganic adhesion layer.
As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. The term “about” generally includes up to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 20” may mean from 18 to 22. Preferably “about” includes up to plus or minus 6% of the indicated value. Alternatively, “about” includes up to plus or minus 5% of the indicated value. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.
It has now been discovered that an appropriate substrate surface on which to attach cells in an environment that stimulates the generation of an ECM comprises a polymer, metal or metalloid surface having appropriate surface functional groups, with or without a chemically bonded inorganic oxide adhesion layer. A self-assembled monolayer (SAM) of appropriate ligands, either chemically bonded directly to the surface functional groups (without the interposition of an inorganic oxide adhesion layer), or chemically bonded to the attached inorganic oxide adhesion layer. These ligands can have surface-modifying properties, and can also support the attachment of cells, providing a suitable environment for generation of an ECM.
There are broad classes of polymers, metals and other materials that do not have exposed surface functional groups in sufficient density or sufficient reactivity to form covalent or coordinative bonds with the precursor inorganic alkoxides of the bonded adhesion layers. These classes include various polymer, metal and metalloid surfaces.
A versatile method has now been discovered that can be effected rapidly on an otherwise unreactive polymer, metal or metalloid surface, which provides a chemically bonded coating that enables control of the surface's properties.
For example, polymers and other materials that are otherwise unreactive or of low reactivity toward the chemical bonding of coatings such as phosphonates or siloxanes, can be activated through oxidation or amination methods, including chemical oxidation using chemical oxidants. Other methods include oxygen or nitrogen plasma discharge and corona discharge. These chemical reagents and methods are able to generate hydroxyl, oxy, oxo, carbonyl, carboxylic acid or carboxylate functional groups on the surface, or, in the case of nitrogen plasma, amino groups. Such functional groups can then react with an inorganic alkoxide (such as a Zr alkoxide or Ti alkoxide) to form a chemically bound inorganic adhesion layer.
Suitable polymers that do not react readily with an inorganic alkoxide to form an adhesion layer include polyalkanes or other polymers such as polysiloxanes, polyalkylarenes, polyolefins, polythiols, and polyphosphines.
One aspect of the invention is directed to a construct comprising a coated activated surface comprising an inorganic oxide adhesion layer chemically bonded to the surface, where the inorganic oxide is selected from the group consisting of the 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 the oxides of Al, Ti, Zr, Si, Mg and Zn.
The polymers can be selected from polysiloxanes (such as polydimethylsiloxane (PDMS)), polyalkanes, polyalkylarenes, polyaldehydes, polyolefins, polythiols, and polyphosphines.
The activated surface coated with an inorganic oxide can further comprise a self-assembled monolayer (SAM) bonded to the adhesion layer, where the SAM is selected from organic compounds comprising a phosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic group. Preferably the SAM comprises a self-assembled monolayer of phosphonates (SAMP). The phosphonates can be selected from the group consisting of hydrophobic phosphonates, cell-adhesive phosphonates and phosphonates capable of further metal-catalyzed coupling.
The phosphonates can be selected from the group consisting of phosphonic acids of structure
where the R group is selected from the group consisting of 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, where heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and heteroarylalkyl contain one or more heteroatoms selected from the group consisting of O, N and S. Preferably the hydrophobic phosphonates are selected from the group consisting of R=C3-C30 alkyl. Preferably the cell-adhesive phosphonates are selected from the group consisting of R=C3-C30 alkyl substituted with a further phosphonate group. More preferably the cell-adhesive phosphonates are selected from the group consisting of C3-C30 α, ω-diphosphonates.
Another aspect of the invention is directed to a construct for medical use comprising the inorganic oxide-coated activated surface further containing a SAM or SAMP bonded to the inorganic oxide coating. This construct can further comprise other useful moieties covalently bound to the SAM or SAMP, such as alkyne or azide groups which allow further elaboration using the so-called “click” reaction, electrochemically active moieties, photochemically active moieties, cell-attractive moieties, cell-adhesive moieties, or anti-infective moieties. Alternatively this medical construct can further comprise cells attached to the SAM- or SAMP-coated surface. The cells are can be selected from the group consisting of fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiac muscle cells, 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 can comprise NIH 3T3 fibroblasts. The construct can further comprise an extracellular matrix (ECM). The construct can be further decelluarized, leaving the ECM attached.
Yet another aspect of the invention is directed to a method of activating and coating an unactivated surface with an inorganic oxide adhesion layer, 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 is reactive with hydroxyl (—OH), oxy (—O—), oxo (═O), carbonyl (C═O), carboxylic acid (—C(═O)—OH) or carboxylate (—C(═O)—O—) functional groups, and is dissolved and/or dispersed in the solvent; and c) suspending the activated substrate in the coating solution for a time and at a temperature sufficient to form an inorganic oxide coating on the activated surface to provide a coated surface, where the inorganic compound is selected from the group consisting of the alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. The method can 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 can still further comprise: f) heating the rinsed coated substrate to 35 to 40° C.
Alternatively, steps b) and c) can be replaced by vapor deposition of an inorganic alkoxide onto the surface, providing the inorganic oxide adhesion layer.
The inorganic compound of the method can be selected from the group consisting of the alkoxides of Al, Ti, Zr, Si, Mg and Zn. The alkoxide can be selected from the group consisting of methoxide, ethoxide, propoxide, iso-propoxide, butoxide, iso-butoxide, sec-butoxide, and tert-butoxide.
The inventive method can be employed on polymer or metal surfaces that are otherwise unreactive with a reactive metal alkoxide. Thus, oxidation or amination of polymers and other materials such as phosphonates or siloxanes, that are otherwise unreactive toward bonding of coatings, can be activated through oxidation methods including chemical oxidation using chemical oxidants such as permanganate, chlorite, chromic acid, chromate, other chromic acid derivatives, osmium tetroxide, ruthenium tetroxide, iodate, 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 US Patent Publication No. 2013/0005660 to Dong et al., or as described in U.S. Pat. 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 able to generate hydroxyl (—OH), oxy (—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 groups (—NH2). Such functional groups can then react with an inorganic alkoxide (such as a Zr alkoxide or Ti alkoxide) to form a chemically bonded inorganic adhesion layer. Suitable unreactive polymers are unreactive by virtue of lacking appropriate reactive functional groups on the polymer surface, such as the aforesaid hydroxyl, oxy, oxo, carbonyl, carboxylic acid or carboxylate functional groups. Suitable unactivated polymers include:
The unreactive metal surfaces are those which are terminated with metallic oxides where the metal oxides themselves are not suitably reactive, such as the native oxide layer on titanium (titanium dioxide) or the native oxide layer on silicon (silicon dioxide). Other metals in this category would include chromium, and alloys of titanium or chromium, including stainless steel and cobalt chrome. Suitable metalloids for use with the inventive method include Si, GaAs, GaP, GaN, AlN and perovskites, where oxidation would introduce surface —OH or bridging oxy groups. In addition to native silicon, silicon dioxide and silicon hydride-terminated silicon are suitable for activation using the present methods.
Such activated surfaces provide a platform or substrate on which to construct a chemically bonded adhesion layer which can then be used to attach a self-assembled monolayer (SAM), such as a self-assembled monolayer of a phosphonate (SAMP), that would 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 the following references, incorporated herein by reference in their entireties: US Patent Publication No. 2010/0215643 to Clevenger et al., US Patent Publication No. 2013/0005660 to Dong et al., and U.S. Pat. No. 9,655,992 to Clevenger et al.
The surface of the substrate comprising a SAM can be patterned or unpatterned.
With regard to the adhesion layer (inorganic oxide coating), the non-oxygen inorganic species preferably has low toxicity in medical applications, and can be advantageously selected from the group consisting of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. Preferably the inorganic species is Al, Ti, Zr, Si, Mg or Zn. More preferably the inorganic species is Al, Si, Ti or Zr. The inorganic species can be Al. The inorganic species can be Ti. The inorganic species can be Zr. The inorganic species can be Mg. The inorganic species can be Si. The inorganic species can be Zn. The inorganic species can be Mo. The inorganic species can be Nb. The inorganic species can be Ta. The inorganic species can be Sn. The inorganic species can be W. The inorganic species can be V.
By virtue of its method of synthesis from an inorganic alkoxide, as described herein, the adhesion layer comprises reactive alkoxides on the surface, which 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.
Thus the adhesion layer-coated surface can further comprise a self-assembled monolayer (SAM) bonded to the adhesion layer, where the SAM is selected from organic compounds comprising a phosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic group. Preferably the SAM comprises a self-assembled monolayer of phosphonates (SAMP). The phosphonates can be selected from hydrophobic phosphonates, cell-adhesive phosphonates and phosphonates capable of further metal-catalyzed coupling. Suitable phosphonates can be selected from the group consisting of phosphonic acids having the structure:
where the R group is selected from the group consisting of 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, where heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and heteroarylalkyl contain one or more heteroatoms selected from the group consisting of O, N and S. The optional substitution on the R group can comprise one or more groups selected from polyol moieties, sugar alcohol moieties, hydroxyl functional groups, amino functional groups, carboxylic acid functional groups, carboxylate ester functional groups, phosphonic acid functional groups, phosphonate functional groups, ether functional groups, alkyne functional groups, azide functional groups and thiol functional groups. Preferably the hydrophobic phosphonates are selected from the group consisting of R=C3-C30 alkyl. The alkyl group can be a C5-C24 alkyl, or a C6-C20 alkyl, or a C8 to C18 alkyl. The alkyl group can be a C3, C4, C6, C8, C10, C12, C14, C16, C18, or C20 alkyl group. Preferably the cell-adhesive phosphonates are selected from the group consisting of R=C3-C30 alkyl substituted with a further phosphonate group. The alkyl group can be a C4-C24 alkyl, or a C6-C20 alkyl, or a C8 to C18 alkyl. The alkyl group can be a C3, C4, C6, C8, C10, C12, C14, C16, C18, or C20 alkyl group. More preferably the cell-adhesive phosphonates are selected from the group consisting of C3-C30 α,ω-diphosphonates. In this case the alkylene group can be a C3, C4, C6, C8, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, or C30 alkylene group. The α,ω-diphosphonic acid can be a C3-16 diphosphonic acid, preferably a C4-12 diphosphonic acid, more preferably a C4, or a C6, or a C8, or a C10, or a C12 diphosphonic acid. The α,ω-diphosphonic acid can 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 mixtures of two or more thereof. Preferably the phosphonates are alkynes, most preferably terminal alkynes. The alkyne phosphonates can have R=C3-C30 alkynyl. The alkynyl group can be a C5-C24 alkynyl, or a C6-C20 alkynyl, or a C8 to C18 alkynyl. The alkynyl group can be a C3, C4, C6, C8, C10, C12, C14, C16, C18, or C20 alkynyl group. Preferably the alkynyl group bears a terminal alkyne.
Preferably the SAMP of the coated construct comprises a phosphonic acid covalently attached to the inorganic oxide adhesion layer, which phosphonic acid contains functionality adapted for cell binding. As noted above, the cell-binding phosphonic acid can comprise one or more functional groups selected from polyol moieties, sugar alcohol moieties, hydroxyl functional groups, amino functional groups, carboxylic acid functional groups, carboxylate ester functional groups, phosphonic acid functional groups, phosphonate functional groups, ether functional groups, alkyne functional groups, azide functional groups and thiol functional groups. Preferably the phosphonic acid is a diphosphonic acid, more preferably an α,ω-diphosphonic acid as described above.
Alkyne and azide functional groups participate in the so-called “click reaction”. Click chemistry represents a powerful coupling approach, based on a highly specific and efficient bio-orthogonal reaction between azido- and alkyne-containing compounds, yielding cycloaddition products. The small size and specificity of reaction of the click-reactive groups pre-attached in a SAMP allow facile elaboration of that SAMP to append such desirable moieties as electrochemically active, photochemically active, cell-attractive, cell-adhesive, or anti-infective moieties. Thus, Example 3 describes a copper-catalyzed click coupling of a PDMS/adhesion layer/SAMP of phosphonodec-9-yne with phenyl azide to form a SAMP of terminated with a phenyltriazole.
Click chemistry employs the cycloaddition reaction between a 1,3-dipole and a dipolarophile (specifically azide and alkyne), to form a five-membered ring. The azide and alkyne functional moieties are largely inert toward biological molecules and aqueous environments. Further, the triazole has similarities to the ubiquitous amide moiety found in nature, but unlike an amide, it is not susceptible to cleavage. Additionally, triazoles are not readily oxidized or reduced. Reports and procedures on cycloaddition reactions between a 1,3-dipole and a dipolarophile are readily available to one 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, the entire disclosures of which are incorporated herein by reference.
Further, alkyne groups can participate in various metal-catalyzed coupling reactions, such as palladium-catalyzed coupling reactions, to introduce additional functionality 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/SAMP of phosphonodec-9-yne with bromobenzene to form a SAMP of 10-phenylphosphonodec-9-yne.
With regard to the reaction of the 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 antithrombogenic moieties, as disclosed herein. Thus, Example 4 describes PDMS coated with an adhesion layer formed from zirconium n-butoxide which can be directly reacted with glycerol, in the absence of a SAM or SAMP.
Another aspect of the invention is directed to a construct for medical applications comprising the coated surface containing a SAM or SAMP bonded to the inorganic oxide coating. The construct can further comprise cells attached to the SAM- or SAMP-coated surface of the construct. The cells can be selected from the group consisting of fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiac muscle cells, 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 preferably comprise NIH 3T3 fibroblasts. The construct can further comprise an extracellular matrix (ECM). The ECM is a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells. The construct can be further decelluarized, leaving the ECM attached.
Yet another aspect of the invention is directed to a method of activating and coating an unactivated surface with an inorganic oxide adhesion layer, 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 is reactive with hydroxyl (—OH), oxy (—O—), oxo (═O), carbonyl (C═O), carboxylic acid (—C(═O)—OH) or carboxylate (—C(═O)—O—) functional groups, 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, where the inorganic compound is selected from the group consisting of the alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. The method can 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 can still further comprise: f) heating the rinsed coated substrate to 35 to 40° C.
Alternatively, steps b) and c) can be replaced by vapor phase deposition of an inorganic alkoxide onto the surface, providing the inorganic oxide adhesion layer.
The inorganic compound of the method can be selected from the group consisting of the alkoxides of Al, Ti, Zr, Si, Mg and Zn. The alkoxide can be selected from the group consisting of methoxide, ethoxide, propoxide, iso-propoxide, butoxide, iso-butoxide, sec-butoxide, and tert-butoxide.
With regard to the inorganic alkoxide, the inorganic species is preferably not toxic in reconstructive medical applications, and can be advantageously selected from the group consisting of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. Preferably the inorganic species is Al, Ti, Zr, Si, Mg or Zn. More preferably the inorganic species is Al, Si, Ti or Zr. The inorganic species can be Al. The inorganic species can be Ti. The inorganic species can be Zr. The inorganic species can be Mg. The inorganic species can be Si. The inorganic species can be Zn. The inorganic species can be Mo. The inorganic species can be Nb. The inorganic species can be Ta. The inorganic species can be Sn. The inorganic species can be W. The inorganic species can be V. Preferred alkoxides are selected from the group consisting of methoxide, ethoxide, propoxide, iso-propoxide, butoxide, iso-butoxide, sec-butoxide, and tert-butoxide.
One aspect of the invention is directed to a construct comprising a) a surface activated to the chemical bonding of an inorganic adhesion layer that provides for further attachment of moieties that modify the overall surface properties, where the surface contains no accessible, sufficiently reactive functional groups on the surface, where activation comprises generation of reactive functional groups on the surface; and b) an inorganic adhesion layer chemically bonded to the activated surface via the reactive functional groups; where the functional groups are reactive with an inorganic alkoxide to form the inorganic adhesion layer. The surface of such a construct can comprise a polymer or a metal. The polymer can be selected from the group consisting of polyalkanes, polysiloxanes, polyalkylarenes, polyolefins, polythiols and polyphosphines. The metal can be selected of stainless steel and various alloys thereof. The surface functional groups of the construct are preferably selected from the group consisting of hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino groups.
The surface functional groups of the construct can be produced by chemical oxidation, using oxidizing agents such as permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracids, peroxides, 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.
The construct can further comprise a self-assembled monolayer (SAM) bonded to the inorganic adhesion layer, where the SAM is selected from organic compounds comprising a phosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic group. The SAM can comprise a self-assembled monolayer of phosphonates (SAMP), where the phosphonates can be selected from the group consisting of hydrophobic phosphonates, cell-adhesive phosphonates and phosphonates capable of further metal-catalyzed coupling. The phosphonates can be selected from the group consisting of phosphonic acids of the structure
where the R group is selected from the group consisting of 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, where heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and heteroarylalkyl contain one or more heteroatoms selected from the group consisting of O, N and S. The hydrophobic phosphonates of the construct can be selected from the group consisting of R=C3-C30 alkyl, and the cell-adhesive phosphonates are selected from the group consisting of R=C3-C30 alkyl substituted with a further phosphonate group. The cell-adhesive phosphonates can be selected from the group consisting of C3-C30 α,ω-diphosphonates.
The construct can have a SAM or SAMP that further comprises an anti-infective agent covalently bound thereto. Suitable anti-infective agents include an antimicrobial selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, 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/tazobactam, ticarcillin/clavulanate, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfonamidochrysoidine, sulfacetamide, sulfadiazine, silver, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilimide, 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, platensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptable salts thereof, and mixtures of two or more thereof. Further, the anti-infective agent can be selected from the group consisting of chlorhexidine, biguanides, cationic ammonium compounds, pharmaceutically acceptable salts thereof, and mixtures of two or more thereof. The anti-infective agent can also be selected from the group consisting of cationic ammonium compounds, cationic ammonium dendrimers, silver, copper, cationic species and mixtures of two or more thereof. The cationic ammonium compounds can be selected from the group consisting of choline and choline derivatives. Alternatively, the anti-infective agent can comprise polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, and other polyols (such as polyvinylalcohol) and aminoalcohols.
Another aspect of the invention is directed to a method of forming an inventive construct comprising the steps of a) activating a surface to chemical bonding of an inorganic adhesion layer that provides for further attachment of moieties that modify the overall surface properties, wherein the surface contains no accessible, sufficiently reactive functional groups on the surface, the activating comprising producing reactive functional groups on the surface to provide an activated surface; and b) chemically bonding an inorganic adhesion layer to the functional groups of the activated surface; where the functional groups are reactive with an inorganic alkoxide to form the inorganic adhesion layer. The surface of the method can comprise a polymer, which can be selected from the group consisting of polyalkanes, polysiloxanes, polyalkylarenes, polyolefins, polythiols and polyphosphines. The surface functional groups of the method can be selected from the group consisting of hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino groups. The activating step of the method can comprise chemical oxidation, using such chemical oxidizing agents as permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracids, peroxides, Fenton's reagent, lead tetraacetate, lead tetraacetate/Mn(II), ozone, of oxygen. Alternatively the activating step of the method can comprise oxygen plasma discharge, nitrogen plasma discharge or corona discharge.
Another aspect of the invention is directed to a method of activating and coating an unactivated surface with an inorganic oxide adhesion layer, comprising the steps of a) activating the surface of an unactivated substrate to the chemical bonding of an inorganic adhesion layer; b) providing a coating mixture comprising an organic solvent containing a reactive inorganic compound that is dissolved and/or dispersed in the solvent; and c) suspending the activated substrate in the coating mixture for a time and temperature sufficient to form an inorganic oxide coating on the activated surface, to provide a surface coated with an inorganic oxide adhesion layer; where the inorganic compound is selected from the group consisting of the alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V. The method can 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 to 40° C. Alternatively, steps b) and c) can be replaced by vapor deposition of an inorganic alkoxide onto the surface, providing the inorganic oxide adhesion layer.
Another aspect of the invention is directed to a construct having a SAMP, as disclosed above, where the R group is dodecyl-9-ynyl, further coupled at the alkyne using a metal-catalyzed reaction. The dodecyl-9-ynyl R group can also be further coupled at the alkyne using a click reaction.
Alternatively, an aspect of the invention is directed to an adhesion layer-coated construct without a SAM or SAMP, further reacted directly at the inorganic adhesion layer with an organic moiety selected from the group consisting of electrochemically active moieties, photochemically active moieties, cell-attractive moieties, cell-adhesive moieties and anti-infective moieties. The anti-infective agent can comprise polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, and other polyols (such as polyvinylalcohol) and aminoalcohols. Preferably the anti-infective moieties are selected from the group consisting of polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, polyols, aminoalcohols and mixtures 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 phosphonate and silane pre-molecule (Heparin PUL and Heparin Silane, respectively).
Thus, one aspect of the invention is directed to stainless steel (SS) coupons which were oxygen plasma-oxidized on both sides and dipped into a solution of titanium (IV) butoxide to form a Ti(IV) butoxide adhesion layer covalently attached to the SS surface. This coated SS surface was functionalized with heparin using either heparin-functionalized phosphonate or silane pre-molecule, and the heparin was found to be in its native form (vs. denatured) by XPS elemental analysis (see Example 5).
One aspect of the invention is directed 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; where the reactive functional groups are reactive with an inorganic alkoxide to form the inorganic adhesion layer, and the inorganic adhesion layer provides additional functional groups for further attachment of moieties that modify the overall surface properties. The activated surface of the construct can comprise an intrinsically unreactive surface towards chemical bonding of an inorganic adhesion layer, which has been treated to produce chemically accessible, reactive functional groups on the surface thereby providing activation. The surface of the construct can comprise a polymer, stainless steel, or a stainless steel alloy that is intrinsically unreactive towards chemical bonding of an inorganic adhesion layer. The polymer can be selected from the group consisting of polyalkanes, polysiloxanes, polyalkylarenes, polyolefins, polythiols and polyphosphines.
The surface reactive functional groups can be selected from the group consisting of hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino groups. The surface reactive functional groups can be produced by chemical oxidation. The chemical oxidation can comprise treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracids, peroxides, Fenton's reagent, lead tetraacetate, lead tetraacetate/Mn(II), ozone, and oxygen.
Alternatively the surface reactive functional groups can be produced by oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.
The inorganic adhesion layer of the construct can comprise an inorganic oxide selected from the group consisting of the 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 the oxides of Al, Ti, Zr, Si, Mg, Zn, and mixtures of two or more thereof.
The construct can further comprise a self-assembled monolayer (SAM) bonded to the additional functional groups of the inorganic adhesion layer, where the SAM is selected from organic compounds comprising a phosphonic, carboxylic, sulfonic, phosphinic, phosphoric, sulfinic, or hydroxamic group. The SAM preferably comprises a self-assembled monolayer of phosphonates (SAMP). The phosphonates can be selected from the group consisting of hydrophobic phosphonates and cell-adhesive phosphonates. The hydrophobic and cell-adhesive phosphonates can be selected from the group consisting of phosphonic acids of structure
where the R group is selected from the group consisting of 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, where heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and heteroarylalkyl contain one or more heteroatoms selected from the group consisting of O, N and S.
The R group of the construct can be dodecyl-9-ynyl, further coupled at the alkyne functional group using a metal-catalyzed reaction. The R group of the construct can be dodecyl-9-ynyl, further coupled at the alkyne functional group using a click reaction.
The hydrophobic phosphonates can be selected from the group consisting of R=C3-C30 alkyl, and the cell-adhesive phosphonates are selected from the group consisting of R=C3-C30 alkyl substituted with a further phosphonate group. The cell-adhesive phosphonates can be selected from the group consisting of C3-C30 α,ω-diphosphonates.
The SAM or SAMP of the construct can further comprise an anti-infective agent or an antithrombogenic agent covalently bound thereto. The anti-infective agent can be an antimicrobial selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, 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/tazobactam, ticarcillin/clavulanate, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfonamidochrysoidine, sulfacetamide, sulfadiazine, silver, sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilimide, 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, platensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptable salts thereof, and mixtures of two or more thereof.
Alternatively, the anti-infective agent can be selected from the group consisting of polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, polyols, aminoalcohols and mixtures of two or more thereof.
The anti-infective agent can be selected from the group consisting of chlorhexidine, biguanides, cationic ammonium compounds, cationic ammonium dendrimers, silver, copper, cationic species and mixtures of two or more thereof. The cationic ammonium compounds can be selected from the group consisting of choline and choline derivatives.
The antithrombogenic agent can be heparin.
The construct of the invention can further comprise cells attached to the coated surface of the construct, where the cells are selected from the group consisting of fibroblasts, endothelial cells, keratinocytes, osteoblasts, chondroblasts, chondrocytes, hepatocytes, macrophages, cardiac muscle cells, 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 can comprise NIH 3T3 fibroblasts.
The construct can further comprise an extracellular matrix (ECM). The construct can also be decelluarized to leave the ECM.
In another aspect, the construct as described above can have the inorganic adhesion layer directly attached to an organic moiety selected from the group consisting of electrochemically active moieties, photochemically active moieties, cell-attractive moieties, cell-adhesive moieties and anti-infective moieties, without an intervening SAM or SAMP layer. The anti-infective moieties can be selected from the group consisting of polysaccharides, chitosan, partially acetylated chitosan, polyglucosamine, chitosan diols, polyols, aminoalcohols and mixtures of two or more thereof.
Another aspect of the invention is directed to a method of forming a construct as described above, the method comprising the steps of a) providing a surface that is intrinsically unreactive towards chemical bonding of an inorganic adhesion layer; b) activating the intrinsically unreactive surface to chemical bonding of an inorganic adhesion layer by treating the unreactive 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; where the reactive functional groups are reactive with an inorganic alkoxide to form the inorganic adhesion layer; and where the inorganic adhesion layer provides additional functional groups for further attachment of moieties that modify the overall surface properties.
In the above method, the surface can comprise a polymer that is intrinsically unreactive towards chemical bonding of an inorganic adhesion layer. The polymer can be selected from the group consisting of polyalkanes, polysiloxanes, polyalkylarenes, polyolefins, polythiols and polyphosphines. The surface functional groups generated by activation can be selected from the group consisting of hydroxyl, oxy, oxo, carbonyl, carboxylic acid, carboxylate, and amino groups. The activating step can comprise chemical oxidation. The chemical oxidation can comprise treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracids, peroxides, Fenton's reagent, lead tetraacetate, lead tetraacetate/Mn(II), ozone, and oxygen.
Alternatively the activating step can comprise oxygen plasma discharge, nitrogen plasma discharge, or corona discharge.
The inorganic adhesion layer of the method can comprise an inorganic oxide selected from the group consisting of the 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 the oxides of Al, Ti, Zr, Si, Mg, Zn, and mixtures of two or more thereof.
Another aspect of the invention is directed to a method of activating an unactivated substrate surface and coating with an inorganic oxide adhesion layer, comprising the steps of: a) activating the surface of an unactivated substrate to chemical bonding of an inorganic adhesion layer by producing 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 that is 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 for the reactive functional groups and the inorganic compound to react and form an inorganic oxide coating on the activated surface of the substrate, providing a substrate coated with an inorganic oxide adhesion layer; where the inorganic compound is selected from the group consisting of the alkoxides of Ti, Zr, Al, Mg, Si, Zn, Mo, Nb, Ta, Sn, W, and V.
The activating step of the method can comprise chemical oxidation. Chemical oxidation can comprise treatment with an oxidizing agent selected from the group consisting of permanganate, chlorite, chromic acid, chromate, osmium tetroxide, ruthenium tetroxide, iodate, peracids, peroxides, Fenton's reagent, lead tetraacetate, lead tetraacetate/Mn(II), ozone, and oxygen.
Steps b) and c) of the method can be replaced by vapor deposition of an inorganic alkoxide onto the surface of the activated substrate and reaction of the reactive functional groups with the inorganic alkoxide to form an inorganic oxide adhesion layer bonded thereto.
Alternatively the activating step can comprise oxygen plasma discharge, nitrogen plasma discharge or corona discharge.
The method can 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 to 40° C.
In summary, the present method converts an otherwise unreactive material into one that is reactive at the surface. This surface reactivity can be controlled by the activation method and by nature of the coating layer of the activated surface, so that cell attachment, spreading and ECM formation are possible. Such means to direct the surface properties of materials provide many possible applications in medicine and other biomedical and biological fields.
General. All materials were procured from commercial sources. Solvents and chemical reagents include methanol (Sigma Aldrich), 2-propanol (Sigma Aldrich), tert-butanol (Fisher Scientific), 200 proof ethanol (Pharmco-Aaper), xylene (EMD Millipore Corporation), toluene (EMD Chemical Inc.), hexanes (Sigma Aldrich), titanium(IV) iso-propoxide (Sigma Aldrich), 1,4-butanediphosphonic acid (Acros Organics), 1,12-dodecanediylbis(phosphonic acid) (Sigma Aldrich), and octadecylphosphonic acid (Alfa Aesar, Sigma Aldrich).
Polydimethylsiloxane (PDMS) coupons were oxygen plasma-oxidized on one side and were dipped into a solution of titanium iso-propoxide in toluene at a concentration of 10 μL/mL. The samples were left submerged in the solution for 15 minutes. The coupons were removed from solution, and it was noted that the surfaces of these PDMS coupons had become slightly less translucent. The coupons 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 coupons were kept in this solution for several hours, removed from solution, heated at 35° C. for 1 min, and rinsed with ethanol. The appearance of the coupon surfaces did not change during this procedure. Infrared spectral analysis showed the presence of a monolayer of octadecylphosphonate on the PDMS surface (
Example 2. A coupon of treated PDMS is prepared as described in Example 1, and is treated with a solution of zirconium n-butoxide dissolved in toluene at a concentration of 0.5 mg/mL. The coupon is heated at 35° C. for 1 min, rinsed with ethanol, and is then placed in a solution of phosphonodec-9-yne in toluene at a concentration of 0.5 mg/mL. The coupon is kept in this solution for several hours, removed from solution, heated at 35° C. for 1 min, rinsed with ethanol, and analyzed by IR spectroscopy for the peaks that are characteristic of a terminal alkynyl group at ca. 2135 and 3325 cm−1. A copper/palladium-catalyzed coupling reaction is then carried out with bromobenzene as an example to yield the 10-phenyl-coupled product.
Example 3. A coupon of treated PDMS is prepared as described in Example 1, and is treated with a solution of zirconium n-butoxide dissolved in toluene at a concentration of 0.5 mg/mL. The coupon is heated at 35° C. for 1 min, rinsed with ethanol, and is then placed in a solution of phosphonodec-9-yne in toluene at a concentration of 0.5 mg/mL. The coupon is kept in this solution for several hours, removed from solution, heated at 35° C. for 1 min, rinsed with ethanol, and analyzed by IR spectroscopy for the peaks that are characteristic of a terminal alkynyl group at ca. 2135 and 3325 cm−1. A copper-catalyzed “click” reaction is then performed using phenyl azide to give the phenyltriazole-terminated phosphonate.
Example 4. A coupon of treated PDMS is prepared as described in Example 1, and is treated with a solution of zirconium n-butoxide dissolved in toluene at a concentration of 0.5 mg/mL. The coupon is heated at 35° C. for 1 min, rinsed with ethanol, and is then placed in a solution of glycerol in ethanol at a concentration of 0.5 mg/mL. The coupon is kept in this solution for several hours, removed from solution, heated at 35° C. for 1 min, rinsed with ethanol, and analyzed by IR spectroscopy for the peaks that are characteristic of hydroxyl, ether and aliphatic groups at around 1050, 2980 and 3100 cm−1.
Example 5. Covalently-Bound Heparin on Ti-Functionalized Stainless Steel General. All materials were procured from commercial sources. Solvents and chemical reagents include anhydrous toluene (Sigma Aldrich), tert-butanol (Fisher Scientific), 200 proof ethanol (Acros Organics), reagent alcohol (Fisher Scientific), titanium(IV) butoxide (Sigma Aldrich), monoamine functionalized trialkoxy silane and phosphonate (Gelest, Sikemia), Heparin sodium salt (Sigma Aldrich).
Stainless Steel (SS) coupons were oxygen plasma-oxidized on both sides and were dipped into a solution of titanium (IV) butoxide in toluene at a concentration of 3% (v/v). The samples were left submerged in the solution for 15 minutes with constant stirring at 350 rpm. The Ti(IV) butoxide treated coupons were taken out of solution and allowed to dry inside a chemical hood for 5 minutes. The samples were then placed in an oven at 130° C. for 10 min, then sonicated with reagent alcohol for 10 min (twice), and vacuum dried for 10 minutes. The coupons at this point had visible shades of gray on the surface when compared to control SS coupons. The samples were placed in a 1.5-15 mM ethanolic solution of heparin-functionalized phosphonate or silane pre-molecule (Heparin PUL or Heparin Silane) and were kept in this solution at a temperature ranging from 24-37° C. overnight. The coupons were rinsed and sonicated in reagent alcohol for 10 minutes (twice) and vacuum dried for 10 minutes. The appearance of the coupon surfaces did not change during this procedure. X-ray photon spectroscopy analysis showed an elemental composition pattern consistent with the native form of the heparin molecule on the stainless-steel surface (
X-ray Photon Spectroscopy. X-ray Photon Spectroscopy (XPS) is technique that measures the percent elemental composition of a surface-bound molecule by identifying the presence of specific elements within known functional groups in a molecule within a 10-nm depth profile. This same technique can be applied to surface-bound molecules to determine the presence of elements within functional regions, in addition to measuring deviations from native elemental percent composition values. In the present case, XPS can evaluate successful surface preparation and monitor degradation of important domains in heparin due to different strategies used to bind heparin to stainless steel. Theoretically, the repeating dimeric unit in heparin has an elemental percent composition pattern of carbon (33%), oxygen (55%), sulfur (8.0%), and nitrogen (2.8%). The native elemental composition of heparin supports the interaction of heparin with anti-thrombin due to the key-lock interaction leading to structural changes on anti-thrombin and its activity to prevent thrombus formation. Significant changes in the elemental percent composition of sulfur are understood to be diagnostic of whether heparin is in its native or denatured form at the surface.
The form of the covalently-bound heparin as prepared above was shown to be native by comparing its elemental percent composition versus the experimentally measured elemental percent composition values of native heparin; there were no statistically significant differences, thereby confirming the native covalently-bound form.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the present claims.
All publications cited herein are incorporated by reference in their entirety for all purposes.
This application claims the benefit of U.S. Provisional Application No. 62/592,880, filed on Nov. 30, 2017, the entire disclosure of which is incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/062958 | 11/29/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62592880 | Nov 2017 | US |
