USOO9056154B2

(12) United States Patent (10) Patent No.: US 9,056,154 B2 Schwartz et al. (45) Date of Patent: Jun. 16, 2015

(54) HIGH-YIELD ACTIVATION OF POLYMER 6,662,805 B2 12/2003 Frondoza et al.

SURFACES FOR COVALENT ATTACHMENT 2004.86 R. 1938 8. Sly,CWaa, ca.al ...... 5.78 OF MOLECULES 2004/0023048 A1 2/2004 Schwartz et al...... 428,472.1 2004/0148002 A1 7/2004 Cheng et al. (75) Inventors: Jeffrey Schwartz, Princeton, NJ (US); 2008/0206443 A1* 8, 2008 Schwartz et al. 427,226 T. Joseph Dennes, Cranbury, NJ (US) 2009, O104474 A1* 4, 2009 Schwartz et al. ... 428,704 2010/0215643 A1* 8/2010 Clevenger et al...... 424,130.1 (73) Assignee: The Trustees of Princeton University, 2011/0093O82 A. 4/2011 Schwartz et al...... 623, 20.14 Princeton, NJ (US) 2012/0208758 A1 8/2012 Maher et al...... 514f13.6 (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 JP 08-295848. A 11, 1996 U.S.C. 154(b) by 344 days. JP 2009-512551 A 3, 2009 WO 2007/050501 A2 5/2007 (21)21) Appl. No.:No 12/939,7369 OTHER PUBLICATIONS (22) Filed: Nov. 4, 2010 Kaim, W. and Schwederski, B., Bioinorganic Chemistry: Inorganic (65) Prior Publication Data th y: Life, John Wiley & Sons LLC, 1994, US 2011 FO1 10986 A1 May 12, 2011 Ignatius, M.J. et al"Bioactive Surface Coatings for Nanoscale Instru ments: Effects on CNSNeurons', J Biomed. Mater. Res., 1998,40(2), Related U.S. Application Data pp. 264-274.* Bradley, D.C. "Metal Alkoxides as Precursors for Electronic and (63) Continuation of application No. 1 1/677.576, filed on Ceramic Materials” Chem. Rev. 1989, 89(6), pp. 1317-1322.* Feb. 21, 2007, now abandoned. VanderKam, S.K. “Zirconium Alkoxide Interfaces for Adhesion (60) Provisional application No. 60/775,127, filed on Feb. that." salysis Thesis (Ph.D.), Princeton Uni 21, 2006, provisional application No. 60/804,633, Balas, Fetal "Surface modification of organic polymers with bioac filed on Jun. 13, 2006. tive titanium oxide without the aidofa silane-coupling agent” JMater Sci: Mater Med, 2007, 18, 1167-1174.* (51) Int. Cl. Samha, et al., Langmuir-Blodgett Films of Pt(II) complexes, A6IL 3L/08 (2006.01) Langmuir (1992); vol. 8, No. 8, pp. 2001-2004. A6L27/30 (2006.01) My al., “Reaction t stility." and C08, 7/04 2006.O1 AI(110)-OH in UHV,” JACS (1995) vol. 117, No. 14, pp. 4037 A6L27/4 3.08: 40441. A6IL 27/28 (2006.01) Dennes, et al., “A nanoscale adhesion layer to promote cell attach ment on PEEK.” JACS (2009), vol. 131, No. 10, pp. 3456-3457 and A6L27/38 (2006.01) S1-S5. A6L 27/50 (2006.01) Dennes, et al., “Controlling cell adhesion on polyurethanes.” Soft A6IL 3L/00 (2006.01) Matter (2008), vol. 4, No. 1, pp. 86-89. C8H L/06 (2006.01) Dennes, et al., “High-yield activation of scaffold polymer surfaces to C09 IS9/04 (2006.01) attach cell adhesion molecules,” JACS (2007), vol. 129, No. 1, pp. (52) U.S. Cl. 96-97. CPC ...... (2013.01); A61L A61L 31/082 27/28 (2013.01);(2013.01). A61LA61L27/30 27/14 cited by examiner (2013.01); A61L 27/38 (2013.01); A61L 27/50 Pri Exami Jon P Web (2013.01);(2013.01); A61L Cosy 31/005 704 (2013.01),(2013.01); CosyC08H 7045 I/06 Assistantrimary traminer-JonExaminer Aaron P weber Kosar (2013.01): C09 IS9/04 (2013.01): COS.J. (74) Attorney, Agent, or Firm – Fox Rothschild LLP: Peter 2379/02 (2013.01) J. Butch III; Robert N. Henrie, II (58) Field of Classification Search (57) ABSTRACT None See application file for complete search history. Polymer Surfaces coated with organometallic layers, wherein the organometallic9. laversy and polypolvmer Surfaces have func (56) References Cited tional groups that react to bond the organometallic layer to the polymer Surface with organometallic functional groups U.S. PATENT DOCUMENTS remaining unreacted for the Subsequent covalent attachment 4,746,366 A * 5/1988 Philipp et al...... 106,287.19 of organic overlayers. Coating methods and coated articles 6,103,255 A 8, 2000 Levene et al. are also disclosed. 6,146,767 A * 1 1/2000 Schwartz ...... 428,457 6,645,644 B1 1 1/2003 Schwartz et al. 27 Claims, 2 Drawing Sheets

U.S. Patent Jun. 16, 2015 Sheet 2 of 2 US 9,056,154 B2

FGS.

(a (b)

US 9,056,154 B2 1. 2 HGH-YELD ACTIVATION OF POLYMER remains a need for polymer Surfaces that Support cell growth SURFACES FOR COVALENT ATTACHMENT as well as the attachment of biologically active molecules and OF MOLECULES other compounds of interest.

CROSS-REFERENCE TO RELATED SUMMARY OF THE INVENTION APPLICATIONS The present invention relates to a coated Substrate having a The present application is a continuation of U.S. applica polymer Surface and an organometallic coating layer formed tion Ser. No. 1 1/677,576, filed Feb. 21, 2007 now abandoned, thereon. The organometallic coating and polymer Surface 10 have co-reactive functional groups that are reacted to bond the which in turn claims priority benefit under 35 U.S.C. S 119(e) coating to the Surface. Preferably, the organometallic coating of U.S. Provisional Patent Application No. 60/775,127, filed has remaining unreacted functional groups that are reacted Feb. 21, 2006, and 60/804,633, filed Jun. 13, 2006. The dis with co-reactive functional groups of a Subsequently applied closures of both applications are incorporated herein by ref compound, oligomer or polymer. CCC. 15 In another embodiment, the present invention relates to a method of coating a polymer Surface, which includes coating STATEMENT REGARDING FEDERALLY an organometallic compound on the polymer Surface, FUNDED RESEARCH wherein the polymer Surface and the organometallic coating have co-reactive functional groups; and reacting the func This invention was made with government Support under tional groups of the organometallic compound with the co Grant No. CHE-0310178 awarded by the National Science reactive functional groups of the polymer Surface to form an Foundation. The U.S. government has certain rights in this organometallic coating on the polymer Surface. Preferably, a invention. compound, oligomer or polymer containing functional groups that react with the remaining unreacted functional BACKGROUND OF THE INVENTION 25 groups of the organometallic compound is applied to the organometallic layer and the functional groups of the com The present invention relates to covalently binding organic pound, oligomer or polymer are reacted with the remaining materials to the surfaces of polymer substrates by function functional groups in the organometallic layer to form an alizing the Surfaces with linker moieties containing transition organic coating on the organometallic coating. metal complexes. In particular, the present invention relates 30 In one embodiment, the present invention provides a novel to modifying polymer Surfaces with organometallic com approach to polymer Surface modification that enables high pounds that have functional groups that react with functional surface density derivatization of polymers with reactive sur groups of the polymer Surface. The modified polymer Surface face moieties containing acidic covalent bonds. The polymers can be further reacted with a compound, polymer or oligomer need not contain pendant functional groups with acidic cova that contains functional groups that are reactive with func 35 lent bonds. For example, polymers with exposed amidefunc tional groups of the organometallic compound that remain tionality contain acidic N-H bonds, which can serve as sites for chemical derivatization when appropriately activated. In after reaction with the polymer surface. The present invention particular, coordination of the carbonyl Group to an appro further relates to functionalizing polymer Surfaces to Support priate metallic center further acidifies the N H bonds and cell growth and the attachment of biologically active mol 40 facilitates derivatization. In addition, acidic C H bonds on ecules and other compounds of interest. polymer Surfaces can also serve as sites for chemical deriva Bioactive polymeric scaffolds are of increasing importance tization when appropriately activated. In particular, coordi for use in tissue regeneration in a variety of clinical applica nation of the carbonyl Group to an appropriate metallic center tions, and a scaffold that Supports cell growth is a critical first further acidifies the CH bonds and facilitates derivatization. step in Such regeneration. Surface wetting properties of many 45 Therefore, according to one aspect of the present invention, polymers used as bioscaffolds are not conducive to biointe a coated Substrate is provided having a polymer Surface with gration, but incorporation of Surface functional groups can exposed reactive functional groups containing acidic cova effect substantial changes in a polymer’s wettability while lent bonds and an organometallic coating layer formed generating reactive sites Suitable for attachment of peptides thereon and covalently bonded thereto, wherein the organo and other biomolecules. 50 metallic coating layer contains transition metal atoms Adjustment of the Surface properties of polymers such as selected from atoms of Group 4. Group 5 and Group 6 of the those from which preformed polymeric therapeutic devices Periodic Chart that have been covalently bonded to the poly are formed, has proven problematic because those polymers mer Surface by reaction of a polyalkoxide or polydialkyla most often used as biomaterials are resistant to specific Sur mide of the transition metal with the reactive functional face treatments. To circumvent this problem, polymer Scaf 55 groups exposed on the polymer Surface. Zirconium and tita fold materials have been prepared by blending, copolymer nium are two examples of such transition metals. Examples of ization, or physical treatment, but these methods can result in reactive functional groups with acidic covalent bonds alteration of the bulk properties of the polymer. Furthermore, include, but are not limited to, hydroxyl groups, phenol these methods or attempts to Surface modify pre-cast poly groups, amide N-H groups, amino groups, imide groups, mers using standard methods of organic synthesis, result only 60 urethane groups, urea groups, thiol groups, carboxylic acid in low Surface coverage by peptides that do not approach groups, carboxylic acid ester groups, carboxylic acid amide those that can be achieved on metallic substrates. groups, Sulfonic acid groups, acidic C-H groups, and the Surface modification has also proven problematic in other like. Each of these groups will covalently bond the organo areas of polymer and polymer Surface customization. There is metallic coating layers. a need to modify polymer Surfaces so as to change the Surface 65 The organometallic layer can then be further bonded to characteristics of the polymer Surface without changing the organic groups or ligands of interest that are reactive with the bulk properties of the polymer. More particularly, there organometallic layer, thereby attaching covalently the US 9,056,154 B2 3 4 organic ligands of interest to the polymer Surface. Therefore, 5 or Group 6 of the Periodic Chart, wherein the dialkylamides according to another aspect of the present invention, a coated or alkoxides are bonded at the transition metal atoms to the Substrate is provided having a polymer Surface with an orga polymer Surface; and nometallic coating layer formed thereon and covalently reacting the transition metal dialkylamide oralkoxide layer bonded thereto, wherein the organometallic coating layer with an organic overlayer comprising a compound, oligomer or polymer capable of reacting with unreacted transition contains transition metal atoms selected from atoms of Group metal dialkylamide oralkoxide groups to covalently bond the 4, Group 5 and Group 6 of the Periodic Chart which have been organic compound, oligomer or polymer to the transition covalently bonded to the polymer surface by reaction of a metals. polyalkoxide or polydialkylamide of the transition metal with The polymer Surface can be provided with the organome the reactive functional groups on the polymer Surface, 10 tallic Surface layer by reacting a polymer Substrate having wherein each transition metal atom additionally has reactive functional groups with acidic covalent Surface bonds covalently bonded to it one or more organic ligands. with a polydialkylamide or polyalkoxide of the Group 4, The present invention provides coated substrates in which Group 5 or Group 6 transition metal having two or more up to 40%, up to 60%, up to 75% or up to 100% of the polymer 15 dialkylamide or alkoxide groups, so that an organometallic Surface has organic groups or ligands bonded thereto. This is surface layer is formed, covalently bonded to the polymer accomplished without changing the bulk material properties Surface, and having at least one unreacted dialkylamide or alkoxide group. of the substrate. The percentage of the polymer surface with The method of the present invention thus provides high organic ligands bonded thereto depends on relationship yield coatings on polymer Surfaces with the adhesion prop between the size of the organic ligand and the size of the metal erties of physical deposition methods under mild reaction complex used to bond the organic ligand to the polymer and conditions. In particular, the coatings of the present invention is readily apparent to one of ordinary skill in the art guided by may be formed at ambient temperatures. the present specification. In addition to the coatings of the present invention and the The present invention thus provides a novel type of inter method by which they are formed, the present invention also face that enables strong adhesion between a polymer Surface 25 provides coated implantable medical devices, methods for and an organic coating. The coating modifies the Surface of improving cellular growth and attachment, tissue in-growth the polymer making it more hydrophilic or hydrophobic as and adhesion to tissue for implantable medical devices using may be desired. The modification can make the polymer the coatings of the invention and the inventive coating meth Surface more or less receptive to Subsequently applied coat ods, and methods for implanting medical devices by first ings, improving or minimizing the adhesion of the polymer to 30 coating them according to the present invention. the Subsequently applied coating. The polymer Surface can be The invention can also be used to make the polymer Surface electrically conductive, semi-conductive or electrically insu a polymer coating on an article made from another material, lating making it useful in organic thin film transistors, light Such as glass, silicon dioxide, metal, or another polymer. The emitting devices and electrolytic capacitors. polymer Surface can also be the Surface of a pre-cast polymer Other features of the present invention will be pointed out article. Examples of suitable polymers with reactive func 35 in the following description that discloses, by way of tional groups include polyamides, such as nylon, silk, and example, the principles of the invention and the best methods collagen, polyacrylamides, polyimides, polyurethanes, poly which have been presently contemplated for carrying them ureas, polysulfonamides, polyesters, polysaccharides, such Out. as haluronic acid, methylcellulose and proteoglycans, and the like, and copolymers of any of these polymers. 40 BRIEF DESCRIPTION OF THE DRAWINGS According to one embodiment of this aspect of the inven tion, the polymer is a biocompatible polymer and the poly FIG. 1 depicts the reaction of N-hexylacetamide and Zir mer-coated or polymer-cast article is a medical implant or conium tetra(tert-butoxide); biological scaffold or porous matrix. The organic ligand can FIG. 2 depicts the reaction of nylon-Zr-amide complex then be a protein, peptide, peptide mimetic, Small molecule 45 with a phosphonic acid and RGDCSEQID NO: 1 coupling: ligand for a cell Surface receptor, or other biologically or FIG.3 depicts the transesterification reaction of nylon-Zr pharmaceutically active compound having utility as a coating amide complex and RGDC ISEQID NO: 1 coupling; and on a medical implant. The organic ligand can thus promote or FIGS. 4a-4c depict fibroblast cell growth on surface prevent cell growth or proliferation, promote or discourage embodiments of the invention according to one embodiment cell adhesion, prevent infection, or prevent or promote blood 50 of a method of the invention. clotting or adhesion. According to another embodiment of this aspect of the DETAILED DESCRIPTION OF PREFERRED invention, the polymer is a fabric formed from a woven or EMBODIMENTS non-woven fiber. The fiber can be a natural fiber with exposed functional groups, such as silk, wool, cotton, linen, collagen 55 The coated Substrates are formed by reacting a polymer and the like. The fiber can also be a synthetic fiber with Surface having functional groups with acidic covalent bonds exposed functional groups, such as nylon. that are reactive with other functional groups, specifically The present invention also provides a method by which transition metal polydialkylamides and polyalkoxides. For organic ligands or groups may be covalently bonded to poly purposes of the present invention, “reactive groups' on a mer Surfaces with reactive functional groups using an organo 60 polymer Surface are defined as functional group with acidic metallic interface. Therefore, according to another aspect of covalent bonds. Examples of Suitable polymer Surface reac the present invention, there is provided a method of forming tive functional groups include those having a reactive N-H an organic layer on a polymer Surface with reactive functional bond Such as amine, amide, imide, urethane and urea groups. groups, which method includes the steps of Examples of other reactive functional groups include providing a Substrate having a polymer Surface covalently 65 hydroxyl, oxy, ether, thiol, carbonyl including keto, ester, free bonded to an organometallic Surface layer of dialkylamides or acid and acid anhydride, Sulfonic acid and acidic —CH alkoxides of transition metals selected from Group 4, Group groups. US 9,056,154 B2 5 6 The polymer can be in the form of a molded article, a rigid covalently bond the transition metal to the reactive functional or flexible film or a coating. Examples of suitable polymers group on the polymer Surface, producing an equivalent quan containing these groups are polyamines such as polyoxy tity of an alkanol or dialkylamine and leaving at least one alkylene polyamines, polyethers such as polyethylene glycol, unreacted alkoxide or dialkylamide Group for Subsequent polyketones such as PEEK, polyamides such as nylon, poly reaction with organic overlayer material. acrylamides, polyimides, polyesters and polyurethanes Such Group 6 transition metals form hexaalkoxides or hexadi as the reaction product of polymeric polyols with polyisocy alkylamides, oxotetra alkoxides or oxotetradialkylamides anates Such as techoflex. The preferred functional groups are and dioxo-dialkoxides or dioxodidialkylamides that are all suitable for use with the present invention. These compounds 10 can also react by proton transfer where possible to covalently bond the transition metal to the reactive functional group on O the polymer Surface, producing an equivalent quantity of an alkanol or dialkylamine and leaving at least one unreacted groups such as those associated with polyamides. Suitable alkoxide or dialkylamide Group for subsequent reaction with polyamides include nylons such 15 as Nylon 6. Nylon 4/6, 15 organic overlayer material. Nylon 6/6, Nylon 6/9, Nylon 6/10, Nylon 6/12, Nylon 12, Advantageously, many of the transition metal alkoxides Nylon 6/66, and the like. dialkylamides suitable for use with the present invention are The reactive functional groups containing acidic covalent commercially available. This includes the preferred zirco bonds may be either pendant to or between monomeric nium tetra (tert-butoxide) and zirconium tetra-diethylamide, repeating units of the polymer, a portion of which are exposed which may be obtained from Strem. However, the transition at the polymer Surface. metal alkoxides and dialkylamides may also be prepared by The organometallic compound used in the practice of the conventional techniques by reacting a halide or oxo-halide of invention is preferably derived from a metal or a metalloid the selected transition metal, depending on the desired num such as selected from Group 3 of the Periodic Chart or a ber of alkoxide or dialkylamide groups, with the correspond transition metal selected from Group 4, 5 and 6 of the Periodic 25 Chart. Preferred metals are aluminum and transition metals ing alkoxide or dialkylamide of a metal selected from Group are selected from Group 4 with titanium and Zirconium being 1 or Group 2 of the Periodic Chart. the most preferred. The organo portion of the organometallic With regard to the preferred metals titanium and zirco compound contains functional groups that are reactive with nium, the alkoxides are titanates and Zirconates. These com the functional groups of the polymer Surface. For purposes of 30 pounds can be reactive simple esters, polymeric forms of the the present invention, “organometallic' compounds are esters and chelates that are relatively stable. Examples of defined as including compounds that do not necessarily con various compounds include tain metal-carbon bonds. Examples of suitable organo groups of the organometallic compound are dialkylamide and alkox a. alkyl ortho esters of titanium and Zirconium having the ide groups containing from 1 to 18, preferably 2 to 8 carbon general formula 25 M(OR), wherein M is selected from Ti atoms. Examples of alkoxide groups include ethoxide, pro 35 and Zr and R is Cls alkyl, poxide, isopropoxide, butoxide, isobutoxide and tert-butox b. polymeric alkyl titanates and zirconates obtainable by ide. Examples of dialkylamide groups include diethylamide, condensation of the alkyl ortho esters of (a), i.e., partially dipropylamide, diisopropylamide, dibutyl amide, diisobutyl hydrolyzed alkyl ortho esters of the general formula RO-M amide and ditert-butyl amide. (OR)2O-R, wherein M and R are as above and x is a Reaction may be by transition metal coordination followed 40 positive integer, by proton transfer to a dialkylamide or alkoxide ligand and loss of an dialkylamine or alkanol, so that an organometallic c. titanium chelates, derived from ortho titanic acid and layer covalently bonds with the polymer surface. The orga polyfunctional alcohols containing one or more additional nometallic layer is believed to consist of a transition metal hydroxyl, keto, carboxyl oramino groups capable of donating layer in which the dialkylamide or alkoxide groups of the 45 electrons to titanium. These chelates have the general formula transition metal are covalently bonded to the polymer at the reactive functional group, Depending upon the position of the transition metal on the Periodic Chart, the transition metal dialkylamide or alkoxide will have from two to six dialkyla wherein a 4-b-c-d; b=4-a-c-d; c=4-a-b-d: d=4-a-b-c; mide oralkoxide groups. Transition metal tetra-alkoxides and 50 R" is H. Ras above or X Y. wherein X is an electron donating tetra-dialkylamides are preferred, with the most preferred group Such as oxygen or nitrogen and Y is an aliphatic radical transition metal tetra-alkoxide and tetra-dialkylamide being having a two or three carbon atom chain Such as zirconium tetra tert-butoxide and zirconium tetra-diethyla i. —CH2CH2—, e.g., of ethanolamine, diethanolamine mide. and triethanolamine, With Group 4 transition metal tetra-alkoxides and tetra dialkylamides, at least one of the alkoxide or amide groups 55 reacts with reactive functional groups on the polymer Surface CHO to form covalent bonds between the reactive functional | || groups and the transition metal. The reaction proceeds by - C - C - O proton transfer to a dialkylamide or alkoxide Group of a H transition metal, producing an equivalent quantity of the cor 60 responding dialkyl amine or alkanol. At least one dialkyla ii. e.g., of lactic acid, mide oralkoxide Group does not react and remains available for Subsequent reaction with the organic overlayer material. Group 5 transition metals form pentaalkoxides or pentadi HC-C-CH=C-CH alkylamides and oxotrialkoxides or oxotridialkylamides that 65 are suitable for use with the present invention. These com O O pounds can also react by proton transfer where possible to US 9,056,154 B2 7 8 iii. e.g., of acetylacetone enol form, and ponents can also enhance the non-fouling characteristics of a Surface so that cells (e.g., from bacteria, Scar tissue, mildew, mold, and other unwanted organisms) do not adhere well to the treated surface. Examples of organophosphorus acids or derivative thereof O-CHCHCH-OH are organophosphoric acids, organophosphonic acids and/or CH organophosphinic acids including derivatives thereof. Examples of derivatives are materials that perform similarly iv. e.g., as in 1.3-octyleneglycol as the acid precursors such as acid salts, acid esters and acid 10 complexes. The organo group of the phosphorus acid may be d. titanium acylates having the general formula Ti(OC a monomeric, oligomeric or polymeric group. Examples of OR) (OR), wherein R is Cls alkyl as above and n is an monomeric phosphorus acids are phosphoric acids, phospho integer of from 1 to 3, and polymeric forms thereof, and nic acids and phosphinic acids including derivatives thereof. e. mixtures thereof. Examples of monomeric phosphoric acids are compounds The organometallic compound is usually dissolved or dis 15 or a mixture of compounds having the following structure: persed in a diluent. Examples of suitable diluents are alcohols Such as methanol, ethanol and propanol, aliphatic hydrocar (RO), P(O)(OR), bons, such as hexane, isooctane and decane, ethers, for wherein X is 1-2, y is 1-2 and X-y–3, R is a radical having a example, tetrahydrofuran and dialkylethers such as diethyl total of 1-30, preferably 6-18 carbons, where R is H, a metal ether. Also, adjuvant materials may be present in the organo Such as an alkali metal, for example, Sodium or potassium or metallic composition. Examples include Surfactants and anti lower alkyl having 1 to 4 carbons, such as methyl or ethyl. static agents. The adjuvants if present are present in amounts Preferably, for a portion of the phosphoric acid compounds in of up to 30 percent by weight based on the non-volatile the overlayer, R' is H. The organic component of the phos content of the composition. phoric acid (R) can be a hydrocarbon and can be aliphatic The concentration of the organometallic compound in the 25 (e.g., alkyl having 2-20, preferably 6-18 carbon atoms) composition is not particularly critical but is usually at least including a Saturated or unsaturated carbon chain (e.g., an 1.0 micromolar, typically from about 1.0 micromolar to about olefin), unsubstituted or substituted aliphatic, such as fluoro 100 millimolar, and more typically from about 1.0 micromo substituted, or can be aryl or aryl-substituted moiety. Substi lar to about 50 millimolar. tution in the omega position is preferred. The organometallic treating composition can be obtained 30 Examples of monomeric phosphonic acids are compounds by mixing all of the components at the same time with low or mixture of compounds having the formula: shear mixing or by combining the ingredients in several steps. The organometallic compounds are reactive with moisture, and care should be taken that moisture is not introduced with (R"), the diluent or adjuvant materials and that mixing is conducted 35 in a Substantially anhydrous atmosphere. (RO), -P(O)-(OR), The organometallic composition is applied to the polymer Surface by conventional means such as dipping or spraying. wherein X is 0-1, y is 1, Z is 1-2 and x-y--Z is 3. Rand R" are The organometallic compound is then exposed to conditions each independently a radical having a total of 1-30, preferably Sufficient to form a polymeric metal oxide coating preferably 40 6-18 carbons. R' is H, a metal, such as an alkali metal, for with unreacted dialkylamide or alkoxide and/or hydroxyl example, Sodium or potassium or lower alkyl having 1-4 groups. This can be accomplished by depositing the film carbons such as methyl or ethyl. Preferably, for at least a under conditions resulting in hydrolysis and self-condensa portion of the phosphonic acid compounds in the overlayer, R' tion of the alkoxide or dialkylamide. These reactions result in is H. The organic component of the phosphonic acid (R and a polymeric coating being formed that provides cohesive 45 R") can be a hydrocarbon and can be aliphatic (e.g., alkyl strength to the film. The conditions necessary for these reac having 2-20, preferably 6-18 carbon atoms) including a satu tions to occur is to deposit the film in the presence of water, rated or unsaturated carbon chain (e.g., an olefin), unsubsti Such as a moisture-containing atmosphere. The resulting film tuted or substitutedaliphatic such as fluoro-substituted, or can preferably has some unreacted dialkylamide or alkoxide be an aryl or aryl-substituted moiety. Substitution in the groups and/or hydroxyl groups for Subsequent reaction and 50 omega position is preferred. possible covalent bonding with reactive groups of an over Examples of monomeric phosphinic acids are compounds layer material. Concurrently with the self-condensation reac or mixture of compounds having the formula: tion, the diluent is evaporated. Depending on the reactivity of the functional groups in the organometallic compound and on the polymer Surface, heating may be required to bond the 55 (R"), organometallic layer to the Substrate. For example, tempera tures of about 50 to about 200° C. may be used. However, for (R), -P(O)-(OR), readily co-reactive groups, ambient temperatures, that is, about 20°C., may be sufficient. wherein X is 0-2, y is 0-2, Z is 1 and x-y--Z is 3. Rand R" are As mentioned above, an overlayer can be applied to the 60 each independently radicals having a total of 1-30, preferably organometallic film. Such an overlayer material can be 6-18 carbons. R' is H, a metal, such as an alkali metal, for derived from a compound, oligomer or polymer that contains example, Sodium or potassium or lower alkyl having 1-4 groups that are reactive with the dialkylamide or alkoxide carbons, such as methyl or ethyl. Preferably, for at least a and/or hydroxyl groups. Preferred overlayers are the layers of portion of the phosphinic acid compounds in the overlayer, R' organic ligands of carboxylic and organophosphorus acids as 65 is H. The organic component of the phosphinic acid (R, R") generally described in U.S. Pat. No. 6,645,644, the disclosure can be a hydrocarbon and can be aliphatic (e.g., alkyl having of which is incorporated by reference. Certain organic com 2-20, preferably 6-18 carbonatoms) including a saturated or US 9,056,154 B2 10 unsaturated carbon chain (e.g., an olefin), an unsubstituted or may be a function of the temperature used, and the tempera Substituted aliphatic Such as fluoro-Substituted, or can be an ture used may be restricted by design considerations and/or aryl or aryl-substituted moiety. Substitution in the omega materials limitations. position is preferred. In a similar aspect, the present invention can be looked at as Examples of organo groups which may comprise RandR" bonding organic ligands or groups to Substrates with polymer include long and short chain aliphatic hydrocarbons, aro Surfaces having functional groups, for example amide groups. matic hydrocarbons and Substituted aliphatic hydrocarbons For purposes of the present invention, "polymer Surfaces and Substituted aromatic hydrocarbons. Examples of Sub having functional groups are defined as polymers with func stituents include carboxyl Such as carboxylic acid, hydroxyl, tional groups either within or between monomeric repeating 10 units, a portion of which are exposed at the polymer Surface, amino, imino, amido, thio, cyano, and halo Such as fluoro. either in the form of a coating or a molded article, and are In addition to the organophosphorus acids mentioned reactive with the organometallic compound, for example, a above, oligomeric or polymeric organophosphorus acids transition metal. Reaction may be by transition metal coordi resulting from self-condensation of the respective organo nation to the functional group. For an amide, this may be phosphorus acid may be used. 15 followed by N H proton transfer to an alkoxide ligand and The overlayer material can further include a suitable sol loss of an alkanol, so that an organometallic layer covalently vent. For example, for organophosphorus materials, solvents bonds with the amide Group on the polymer surface. Such as an alcohol (e.g., ethanol), tetrahydrofuran, dichlo Although not to be bound by any theory, it is believed the romethane, chloroform, 2:1 by volume ethanol:toluene, reaction of the transition metal complex with the polymer acetonitrile and water can be used. The concentration of the substrate proceeds by coordination of the metal to the func overlayer material can range from about 0.1 micromolar to as tional group of the polymer. This may be followed by the high as the upper limit of the solubility of the overlayer transfer of a proton from the polymer functional to the ligand material in a specific solvent, for example, from about 0.1 of the transition metal, forming the corresponding dialky micromolar to about 100 millimolar, from about 0.1 micro lamine or alkanol and bonding the polymer functional group molar to about 10.0 millimolar, for example, about 1.0 milli 25 to the transition metal. In the case of vapor deposition tech molar. niques, once the reaction is complete, the vacuum is main The solution of the overlayer material can be applied to the tained in order to draw off any excess of the transition metal organometallic coating using one or more techniques, and or dialkylamine or alkanol byproduct. allowing the solution to evaporate. For example, the Solution Further, although not to be bound by any theory, it is can be sprayed (e.g., a few microgram per square centimeter) 30 believed the reaction of the transition metal complex with the onto, dropped on, and/or painted on the organometallic coat organic overlayer proceeds by coordination of the functional ing. Alternatively, the substrate with the organometallic coat group on the organic overlayer to the transition metal. This ing can be dipped into the solution. The solution can be may be followed by the transfer of a proton from the organic applied by doctor blade, reverse roll, die coater, wire bar, functional to the ligand of the transition metal, forming the knife and blade coaters. Direct gravure, micro gravure and 35 corresponding dialkylamine or alkanol and bonding the reverse gravure techniques can also be used. Suitable solvents organic overlayer to the transition metal. include solvents in which the organic compound is soluble The organic compound may also be selected from phos including aqueous buffer Solutions, tetrahydrofuran, aceto phoric acid and organophosphonic acids. For purposes of the nitrile, methylene chloride, chloroform and water, and the present invention “phosphoric acid' is defined according to like. The substrate will then be removed from the solution, 40 its well-understood meaning, HPO. “Organophosphonic rinsed with an inert solvent such as water, acetonitrile, metha acids’ refers to compounds having the formula HRPO, nol, tetrahydrofuran or the like and then dried to provide a wherein R is a hydrocarbon ligand with a carbon directly Substrate with a polymer Surface having an organic ligand bonded to phosphorus. surface layer covalently attached thereto. Overlayers based on organophosphorus acids are Besides the techniques mentioned above, the transition 45 covalently bonded as phosphate esters of the organometallic metal amide or alkoxide may be applied to the polymer Sur layer transition metal. The phosphate esters may be hydro face by vapor deposition. Typically, an excess of transition lyzed to form transition metal polyphosphate coatings on the metal amide or alkoxide is employed, and the reaction per Substrate surface. The phosphate and polyphosphate coatings formed at ambient temperature. With vapor deposition, upon are rich in hydroxyl groups that are available for further completion of the reaction, the vacuum should once again be 50 chemical modification. maintained to remove excess transition metal alkoxide and The coated polymer Substrates also include organic ligands alkanol byproduct. or groups covalently bonded to the polymer Surfaces. By Other methods of applying the solution to the organome reacting organic overlayer materials with the transition metal tallic coating are described in US 2004/0023048 and PCT/ dialkylamide or alkoxide layer, organic ligands form as a US/2003/034909, the disclosures of both of which are 15 55 layer on the polymer Surface, covalently bonded at the tran incorporated by reference. Application of the solution may be sition metal to the polymer surface. The ability to react the in one or multiple layers. organic overlayer material with the transition metal dialkyla After the solution of the overlayer material is applied to the mide or alkoxide layer covalently bonded to the polymer organometallic coating and the solvent is allowed to evapo surface at ambient temperature is particularly useful for the rate, the applied layer may optionally be treated to enhance 60 attachment of biologically active ligands such as peptides, bonding directly to the organometallic coating. The applied proteins, or any other ligand which is deactivated under layer can be treated with heat and/or electromagnetic radia extreme conditions. tion, such as microwave radiation (e.g., 2450 MHz or a wave Examples of biologically active ligands that are covalently length of about 12 cm). In some embodiments, the applied attached to the polymer Surface of a Substrate by an organo layer is exposed to radiant and/or induction heating, for 65 metallic layer include integrins, integrin receptors, cell example, to a temperature of 50° C. to about 200° C. (e.g., attachment mediators, such as peptides containing variations about 150° C.) for about 30-120 seconds. The heating time of the Arg-Gly-Asp integrin binding sequence known to US 9,056,154 B2 11 12 enhance cellular attachment, and Substances that enhance or mometasone, fluticaSone, beclomethasone, flunisolide, exclude particular varieties of cellular or tissue ingrowth. budesonide, or the like, or salts thereof, or combinations Such Substances include, for example, osteoinductive Sub thereof. In another embodiment, the active agent can include stances, such as bone morphogenic proteins (BMP), and Sub non-steroidal anti-inflammatory agents including, but not stances that induce cellular growth, proliferation, and/or dif limited to, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, ferentiation such as epidermal growth factor (EGF), naproxen, oxaprozin, diclofenac, etodolac, indomethacin, fibroblast growth factor (FGF), platelet-derived growth factor ketorolac, nabumetone, Sulindac, tolmetin, meclofenamate, (PDGF), insulin-like growth factor (IGF-I and II), TGF-D, mefenamic acid, piroXicam, Suprofen, or the like, or salts vascular endothelial growth factor (VEGF) and the like. thereof, or combinations thereof. Other biologically active ligands include SMAD3, AXIN2, 10 In another embodiment, the active agent can include ID2, HEME Oxygenase-1 and Nell-1. Antibodies, including decongestants including, but not limited to, ephedrine, phe monoclonal antibodies, may also be covalently bound to the nylpropanolamine, pseudoephedrine, phenylephrine, epi polymer Surface. nephrine, ephedrine, desoxyephedrine, naphazoline, When desired, an active agent (or a combination of active oxymetazoline, tetrahydro-Zoline, Xylometa7oline, propyl agents) can be bound to the polymer Surface of a Substrate by 15 hexedrine, or the like, or salts thereof, or combinations the organometallic layer according to the invention in order to thereof. accomplish any of a variety of goals. The particular active In another embodiment, the active agent can include muco agent(s) used, as well as the mechanism to chemically and/or lytics including, but not limited to, acetylcysteine, dornase physically attach the active agent(s) to the derivatized surface, alpha, or the like, or salts thereof, or combinations thereof. will obviously depend upon the chemical and/or physical In another embodiment, the active agent can include anti nature of the derivatization of the Surface, e.g., its reactivity, cholinergics including, but not limited to, ipratropium, atro its functionality, its surface roughness, etc. Nevertheless, the pine, Scopolamine, or the like, or salts thereof, or combina following list of active agents that are suitable for surface tions thereof. immobilization according to the invention is merely exem In another embodiment, the active agent can include non plary and should not be construed as being complete. 25 antibiotic antimicrobials including, but not limited to, tauro In one embodiment, the active agent can include antileu lidine or the like. kotrienes or leukotriene receptor antagonists (e.g., for B4, C4. In another embodiment, the active agent can include mast D4, and/or E4 leukotriene receptors) including, but not lim cell Stabilizers including, but not limited to, cromolyn, ited to, Zafirlukast, montelukast, pranlukast, iralukast, pobi nedocromil, ketotifen, salts thereof (e.g., Sodium), or 10 com lukast, or the like, or sombinations thereof, and/or salts 30 binations thereof. thereof (e.g., Montelukast Sodium, which is commercially In another embodiment, the active agent can include one or available under the tradename SINGULAIRR). more active ingredients such as anti-infective agents, anti In another embodiment, the active agent can include anti inflammatory agents, mucolytic agents, antihistamines, anti histamines including, but not limited to, ethanolamines (e.g., leukotrienes, decongestants, anticholinergics, antifungals, diphenhydramine and/or salts including hydro-chloride, 35 and combinations of these classes of agents. Anti-infective dimenhydrinate, carbinoxamine, clemastine and/or salts such agents contemplated by the present invention include, but are as fumarate, bromodiphenhydramine and/or salts such as not limited to antibiotics, anti-virals, non-antibiotic antimi hydrochloride, phenyloloxamine, doxyl-amine, or the like, or crobials, and antiseptics. Anti-inflammatory agents contem other salts thereof, or combinations thereof), ethylenedi plated by the present invention include, but are not limited to amines (e.g., tripelennamine and/or salts such as hydrochlo 40 steroidal and non-steroidal anti-inflammatory agents, and ride, pyrilamine and/or salts such as maleate, antazoline and/ mast cell inhibitors. Antifungal agents contemplated by the or salts such as phosphate, methapyriline, or the like, or other present invention include, but are not limited to amphotericin salts thereof, or combinations thereof), alkylamines (e.g., B, and azole antifungals. Examples of contemplated antibi chlorpheniramine and/or salts such as maleate, bromphe otics include, but are not limited to cefuroxime, ciprofloxacin, niramine and/or salts such as maleate, dexchlorpheniramine 45 tobramycin, cefoperaZone, erythromycin, and gentamycin. and/or salts such as maleate, dimethindene and/or salts such Exemplary anti-infective agents include, but are not lim as maleate, triprolidine and/or salts such as hydrochloride, ited to, penicillins, cephalo sporins, macrollides, ketolides, pheniramine and/or salts such as maleate, or the like, or other Sulfonamides, quinolones, aminoglycosides, beta lactam salts thereof, or combinations thereof), piper-zines (e.g., antibiotics, and lineZolid. Exemplary non-antibiotic antimi cyclizine and/or salts such as hydrochloride and/or lactate, 50 crobials include taurolidine. Exemplary steroidal anti-in meclizine and/or salts such as hydrochloride, hydroxy Zine flammatory agents include glucocorticoids. Exemplary non and/or salts such as hydrochloride and/or pamoate, buclizine, steroidal anti-inflammatory agents include diclofenac. chlorcyclizine, or the like, or other salts thereof, or combina Exemplary mast cell stabilizers include cromolyn and ned tions thereof), phenothiazines (e.g., promethazine and/or cromil sodium. Exemplary mucolytic agents are acetylcys salts such as hydrochloride, propiomazine, methdilazine, tri 55 teine and dornase alpha. Exemplary decongestants are phe meprazine and/or salts such as tartrate, or the like, or other nylephrine, naphazoline, oxymetazoline, tetrahydrozoline salts thereof, or combinations thereof), and/or miscellaneous and Xylometoazoline. Exemplary antihistamines include others (e.g., cyproheptadine, ketotifen, azatadine and/or salts loratidine. Exemplary antibiotic combinations include Such as maleate, terfenadine, fexofenadine, astemizole, cefuroxime and gentamicin Exemplary anticholinergics diphenylpyraline, phenindamine, or the like, or salts thereof, 60 include ipratropium, atropine and Scopolamine. Exemplary or combinations thereof). antifungals include amphotericin B, itraconazole, flucona In another embodiment, the active agent can include anti Zole, and miconazole. septics including, but not 10 limited to, iodine, chlorhexidine In another embodiment, the active agent can include, but acetate, Sodium hypochlorite, and calcium hydroxide. are not limited to, anti-inflammatory agents (e.g., alclometa In another embodiment, the active agent can include Ste 65 Sone, amcinonide, amlexanox, balsalazide, betamethasone, roidal anti-inflammatory agents including, but not limited to, celecoxib, choline magnesium, trisalicylate, choline salicy betamethasone, triamcinolone, dexamethasone, prednisone, late, chlobetasol, colchicine, cortisone acetate, curcumin, dis US 9,056,154 B2 13 14 unite, dexamethasone, diclofenac, diflunisal, etodolac, feno analogs/derivatives thereof, salts thereof, or combinations profen, fluocinolone, fluometholone, flurandren-olide, thereof anesthetic agents such as lidocaine, bupivacaine, flurandrenolide, flurbiprofen, hydrocortisone, ibuprofen, ropivacaine, or the like, analogs/derivatives thereof, salts indomethacin, ketoprofen, ketorolac, meclofenamate, mefe thereof, or combinations thereof, anti-coagulants such as namic acid, meloxicam, mesalamine, Methylprednisolone, D-Phe-Pro-Arg chloromethyl ketone, an RGD peptide-con nabumetone, naproxen, olSalazine, oxaprozin, piroxicam, taining compound, heparin, hirudin, antithrombin com prednisone, rofecoxib, Salsalate, Sulfasalazine, Sulindac, tol pounds, platelet receptor antagonists, antithrombin antibod metin, triamcinolone, Valdecoxiband, analogs/derivatives ies, anti-platelet receptor antibodies, aspirin, prostaglandin thereof, salts thereof, or combinations thereof), immunosup inhibitors, platelet inhibitors, tick antiplatelet peptides, or the pressants (e.g., azathioprine, basiliximab, cyclosporine, 10 like, analogs/derivatives thereof, salts thereof, or combina daclizumab, leflunomide, lymphocyte immune globulin, tions thereof. Vascular cell growth promoters such as growth methotrexate, muromonab-CD3, myco-phenolate, sirolimus, factors, transcriptional activators, translational promoters, or tacrolimus, thalidomideand, analogs/derivatives thereof, the like, analogs/derivatives thereof, salts thereof, or combi salts thereof, or combinations thereof), anti-cell proliferation nations thereof. Vascular cell growth inhibitors such as agents (e.g., alkylating agents Such as buSulfan, cisplatin, 15 growth factor inhibitors, growth factor receptor antagonists, cyclophosphamide, oxaliplatin, or the like; nitrosourea com transcriptional repressors, translational repressors, replica pounds such as in carmustine, lomustine, or the like; anthra tion inhibitors, inhibitory antibodies, antibodies directed cycline compounds Such as epirubicin, mitoxantrone, or the against growth factors, bifunctional molecules consisting of a like; anti-androgen compounds such as bicalutamide, fluta growth factor and a cytotoxin, bifunctional molecules con mide, nilutamide, or the like; antibiotics Such as bleomycin, sisting of an antibody and a cytotoxin, or the like, analogs/ dactinomycin, mitomycin, or the like; anti-metabolite com derivatives thereof, salts thereof, or combinations thereof; pounds such as cladribine, fluorouracil, gemcitabine, hydrox protein kinase and tyrosine kinase inhibitors such as tyr yurea, methotrexate, or the like; anti-microtubular com phostins, genistein, quinoxalines, or the like, analogs/deriva pounds such as docetaxel, paclitaxel, or the like; aromatase tives thereof, salts thereof, or combinations thereof; prosta inactivators such as anastroZole, exemestane, or the like; hor 25 cyclin analogs; cholesterol-lowering agents; angiopoietins: mones such as estramumustine, megestrol, or the like; mono resveratrol and derivatives thereof antimicrobial agents such clonal antibody compounds such as alemtuzumab, rituximab, as triclosan, cephalosporins, 13-lactams, aminoglycosides, or the like; protein synthesis inhibitors such as asparaginase, nitrofurantoin, or the like, analogs/derivatives thereof, salts pegaspargase, or the like; other compounds such as carbopl thereof, or combinations thereof cytotoxic agents; cytostatic atin, dipyridamole, doxorubin, doxorubicin, etopo-side, ima 30 agents; cell proliferation affectors; vasodilating agents; tinib, misonidazole, mercaptopurine, testolactone, trimetrex agents that interfere with endogenous Vascoactive mecha ate, glucuronate, tiripaZamine, topotecan, Vindesine, nisms; analogs/derivatives thereof salts thereof metabolites Vincristine, analogs/derivatives thereof, salts thereof, or com thereof, or combinations thereof. binations thereof), anti-thromosis, anti-platelet, and/or fibrin Exemplary genetic active agents include, but are not lim olysis agents (e.g., abcimab, antithrombin III, argatroban, 35 ited to, anti-sense DNA and RNA as well as DNA coding for: aspirin, clopidogrel, dipyridamole, eptifibatide, fonda (a) anti-sense RNA, (b) tRNA or rRNA to replace defective or parinux, heparin, low molecular weight heparin, heparin Sul deficient endogenous molecules, (c) angiogenic factors fate, recombinant hirudin such as bivalirudin, lepirudin, or the including growth factors such as acidic and basic fibroblast like, ticlopidine, tissue recombinant plasminogen activators growth factors, vascular endothelial growth factor, epidermal Such as alteplase, reteplase, Streptokinase, tenecteplase, 40 growth factor, transforming growth factor C. and B, platelet urokinase, or the like, tirofibanand, analogs/derivatives derived endothelial growth factor, platelet-derived growth thereof, salts thereof, or combinations thereof), extracellular factor, tumor necrosis factor C, hepatocyte growth factor and matrix mediators (e.g., calprotectin, catechins, Sulfonylated insulin-like growth factor, (d) cell cycle inhibitors including amino acid hydroxamates, tetracycline compounds Such as CD inhibitors, and (e) thymidine kinase (“TK) and other demeclo-cycline, doxycycline, minocycline, oxytetracycline, 45 agents useful for interfering with cell proliferation. Also of tetracycline, or the like, analogs or derivatives thereof, salts interest is DNA encoding for the family of bone morphogenic thereof, or combinations thereof), and the like, and combina proteins (“BMPs'), including BMP-2, BMP-3, BMP-4, tions thereof. BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8, BMP-9, In another embodiment, the active agent can include, but BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, are not limited to, anti-thrombotic agents such as heparin, 50 and BMP-16. Such molecules include any of the "hedgehog heparin derivatives, urokinase, PPack (dextro-phenylalanine proteins, or the DNA’s encoding them. proline arginine chloromethylketone), or the like, analogs/ Vectors of interest for delivery of genetic active agents derivatives thereof, salts thereof, or combinations thereof; include, but are not limited to, (a) plasmids, (b) viral vectors steroidal and non-steroidal anti-inflammatory agents Such as adenovirus, adeno-associated virus, lentivirus, or the (NSAIDs) such as dexamethasone, prednisolone, cortico-ste 55 like, and (c) non-viral vectors such as lipids, liposomes, cat rone, hydrocortisone and budesonide estrogen, SulfaSalazine ionic lipids, or the like. Cells include cells of human origin and mesalamine, Salicylic acid, salicylates, ibuprofen, (autologous or allogenic), including stem cells, or from an naproxen, Sulindac, diclofenac, piroXicam, ketoprofen, animal source (Xenogenic), which can be genetically engi diflunisal, nabumetone, etodolac, oxaprozin, indomethacin, neered if desired to deliver proteins of interest. or the like, analogs/derivatives thereof, salts thereof, or com 60 Non-limiting examples of useful antimicrobial agents binations thereof anti-neoplastic oranti-proliferative oranti include: Antiamebics, e.g., Arsthinol, Bialamicol, Carbar mitotic agents such as paclitaxel, 5-fluorouracil, cisplatin, sone, Cephaeline, Chlorbetamide, Chloroquine, Chlorphe vinblastine, Vincristine, epothilones, endostatin, angiostatin, noxamide, Chlortetracycline, Dehydroemetime, Dibro doxorubicin, metho-trexate, angiopeptin or the like, analogs/ mopropamidine, Diloxanide, DiphetarSone, Emetine, derivatives thereof, salts thereof, or combinations thereof, 65 Fumagillin, Glaucarubin, Glycobiarsol, 8-Hydroxy-7-iodo monoclonal antibodies capable of blocking Smooth muscle 5-quinoline-Sulfonic Acid, Iodochlorhydroxyquin, cell proliferation, thymidine kinase inhibitors, or the like, Iodoquinol, Paromomycin, Phanquinone, Polybenzarsol, US 9,056,154 B2 15 16 Propamidine, Quinfamide, Scenida7ole, Sulfarside, Teclo Furaltadone, Furazolium Chloride, Nifuradene, Nifuratel, Zan, Tetracycline. Thiocarbamizine. ThiocarbarSone, Timi Nifurfoline, Nifur-pirinol, Nifurprazine, Nifurtoinol, Nitro dazole; Antibiotics, e.g. Amino-glycosides (such as Amika furantoin, and the like), Quinolones and Analogs (such as cin, Apramycin, Arbekacin, Bambermycins, Butirosin, Cinoxacin, Ciprofloxacin, Clinafloxacin, Difloxacin, Enoxa Dibekacin, Dihydrostreptomycin, Fortimicin(s), Gentami cin, Fleroxacin, Flumequine, Grepafloxacin, Lomefloxacin, cin, Isepamicin, Kaniamycin, Micronomicin, Neomycin, Miloxacin, Nadifloxacin, Nadilixic Acid, Norflaxacin, Neomycin Undecylenate, Netilmicin, Paromomycin, Ribo Ofloxacin, Oxolinic Acid, PaZufloxacin, Pefloxacin, Pipe stamycin, Sisomicin, Spectinomycin, Streptomycin, Tobra midic Acid, Piromidic Acid, Rosoxacin, Rufloxacin, Spar mycin, Trospectomycin, and the like), Amphenicols (such as floxacin, Temafloxacin, ToSufloxacin, Trovafloxacin, and the AZidamfenicol, Chloramphenicol, Florfenicol, Thiampheni 10 like), Sulfonamides (such as Acetyl Sulfamethoxpyrazine, col, and the like), Ansamycins (such as Rifamide, Rifampin, Benzylsulfamide, Chloramine-B, Chloramine-T, Dichloram Rifamycin, Rifapentine, Rifaximin, and the like), 13-Lac ine T. N2-Formylsulfisomidine, N4-y-D-Glucosylsulfanil tams (e.g., Carbacephems, Loracarbef, Carbapenems (such amide, Mafenide, 4'-(Methylsulfamoyl)sulfanil-anilide, as Biapenem, Imipenem, Meropenem, Panipenem, and the Noprylsulfamide, Phthalylsulfacetamide, Phthalylsulfathiaz like), Cephalosporins (such as Cefaclor, Cefadroxil, Cefa 15 ole, Salazo-sulfadimidine. Succinylsulfathiazole, Sulfaben mandole, Cefatrizine, Cefazedone, Cefazolin, Cefcapene Zamide, Sulfacetamide, Sulfachlor-pyrida7ine, Sulfachrysoi Povoxil, Cefclidin, Cefdinir, Cefditoren, Cefepime, Cefe dine, Sulfacytine, Sulfadiazine, Sulfadicramide, Sulfadi tamet, Cefixime, Cefinenoxine, Cefodizime, Cefonicid, methoxine, Sulfadoxine, Sulfaethidole, Sulfaguanidine, CefoperaZone, Ceforanide, Cefotaxime, Cefotiam, Cefozop Sulfaguanol, Sulfalene, Sulfaloxic, Sulfamerazine, Sulfame ran, Ce?pimizole, Ce?piramide, Ce?pirome, Cefpodoxime ter, Sulfamethazine, Sulfamethizole, Sulfa-methomidine, Proxetil, Cefprozil, Cefroxadine, Cefsulodin, Ceftazidime, Sulfamethoxazole, Sulfamethoxypyridazine, Sulfametrole, Cefiteram, Ceftezole, Ceftibuten, Ceftizoxime, Ceftriaxone, Sulfamido-chrysoidine, Sulfamoxole, Sulfanilamide, 4-Sul Cefuroxime, CefuZonam, Cephacetrile Sodium, Cephalexin, fanilamidosalicylic Acid, N4-Sulfanilyl-sulfanilamide, Sul Cephaloglycin, Cephaloridine, Cephalosporin, Cephalothin, fanily lurea, N-Sulfanilyl-3,4-xylamide, Sulfanitran, Sulfa Cephapirin Sodium, Cephradine, Piveefalexin, and the like), 25 perine, Sulfaphenazole, Sulfaproxyline, Sulfapyrazine, Cepha-mycins (such as CefbuperaZone, Cefinetazole, Cefni Sulfapyri-dine, Sulfasomizole, Sulfa-symazine, Sulfathiaz nox, Cefotetan, Cefoxitin, and the like), Monobactams (such ole, Sulfathiourea; Sulfatolamide, Sulfisomi-dine, Sulfisox as Aztreonam, Carumonam, Tigemonam, and the like), azole, and the like), Sulfones (such as Acedapsone, Acedia Oxacephens (such as Flomoxef, Moxalactam, and the like), Sulfone, Acetosul-fone Sodium, Dapsone, Diathymosulfone, Penicillins (such as Amdinocillin, Amdinocillin Pivoxil, 30 Glucosulfone Sodium, Solasulfone, Succisulfone, Sulf-anilic Amoxicillin, Ampicillin, Apalcillin, Aspoxicillin, AZidocil Acid, p-Sulfanilylbenzylamine, Sulfoxone Sodium, Thiazol lin, AZlocillin, Bacampicillin, Benzylpenicillic Acid, Benzyl sulfone, and the like), Clofoctol, Hexedine, Methenamine, penicillin Sodium, Carbenicillin, Carindacillin, Clometocil Methenamine Anhydromethylenecitrate, Methenamine Hip lin, Cloxacillin, Cyclacillin, Dicloxacillin, Epicillin, purate, Methenamine Mandelate, Methenamine Sulfosalicy Fenbenicillin, Floxacillin, Hetacillin, Lenampicillin, 35 late, Nitroxoline, Taurolidine, Xibomol, and the like; lepros Metampicillin, Methicillin Sodium, Mezlocillin, Naacillin tatic antibacterial agents, such as Acedapsone, Acetosulfone Sodium, Oxacillin, Penamecillin, Penethamate Hydro-io Sodium, Clofazimine, Dapsone, Diathymosulfone, Gluco dide, Penicillin G Benethamine, Penicillin G BenZathine, sulfone Sodium, Hydnocarpic Acid, Solasulfone, Succisul Penicillin G Benzhydryl-amine, Penicillin G Calcium, Peni fone, Sulfoxone Sodium, and the like, antifungal agents such cillin G Hydrabamine, Penicillin G Potassium, Penicillin G 40 as Allyl-amines Butenafine, Naftifine, Terbinafine, Imida Procaine, Penicillin N, Penicillin 0, Penicillin V, Peniclin V Zoles (e.g., Bifonazole, Butoconazole, Cholordantoin, Chlo Benzathine, Penicillin V Hydrabamine, Penimepicycline, rmid-azole, Cloconazole, Clotrimazole, Econazole, Enil Phenethicillin Potassium, Pipera-cillin, Pivampicillin, Propi conazole, Fenticonazole, Flutrimazole, Isoconazole, cillin, Quinacillin, Sulbenicillin, Sultamicillin, Talampicillin, Ketoconazole, Lanoconazole, Miconazole, Omoconazole, Temocillin, Ticarcillin, and the like), Ritipenem, Lincosa 45 Oxiconazole Nitrate, Sertaconazole, Sulconazole, Tiocona mides (such as Clindamycin, Linco-mycin, and the like), Zole, and the like). Thiocarbamates (e.g., Tolcilate, Tolindate, Macrollides (such as Azithromycin, Capbomycin, Clarithro Tolnaftate, and the like), Triazoles (e.g., Fluconazole, Itra mycin, Dirithromycin, Erythromycin, Erythromycin Acis conazole, Saperconazole, Terconazole, and the like). Acrisor trate, Erythromycin Estolate, Erythromycin Glucoheptonate, cin, Amorolfine, Biphenamine, Bromosalicylchloranilide, Erythromycin Lactobionate, Erythromycin Propionate, 50 Buclosamide, Calcium Propionate, Chlorphenesin, Ciclo Erythromycin Stearate, Josamycin, Leucomycins, Mideca pirox, Cloxyquin, Coparaffinate, Diamthazole Dihydrochlo mycins, Miokamycin, Oleandomycin, Primycin, Rokitamy ride. Exalamide, Flucytosine, Halethazole, Hexetidine, Lof cin, Rosaramicin, Roxithromycin, Spiramycin, Troleando lucarban, Nifuratel, Potassium Iodide, Propionic Acid, mycin, and the like), poly-peptides (such as Amphomycin, Pyrithione, Salicylanilide, Sodium Propionate, Sulbentine, Bacitracin, Capreomycin, Colistin, Enduracidin, Envio-my 55 Tenonitrozole, Triacetin, Ujothion, Undecylenic Acid, Zinc cin, Fusafungine, Gramicidin S. Gramicidin(s), Mikamycin, Propionate, etc.; or the like; analogs/derivatives thereof: salts Polymyxin, Pristinamycin, Ristocetin, Teicoplanin, Thios thereof, or combinations thereof. trepton, Tuberactinomycin, Tyrocidine, Tyrothricin, Vanco Other antimicrobial agents useful in the present invention mycin, Viomycin, Virginiamycin, Zinc Bacitracin, and the include, but are not limited to, Q-lactamase inhibitors (e.g. like), Tetracyclines (such as Apicycline, Chlortetracycline, 60 Clavulanic Acid, Sulbactam, Tazobactam, and the like): Clomocycline, Demeclocycline, Doxycycline, Guamecy Chloramphenicols (e.g. AZidamphenicol, Chloramphenicol, cline, Lymecycline, Meclocycline, Methacycline, Minocy Thiaphenicol, and the like); Fusidic Acid; synthetic agents cline, Oxytetracycline, Penimepicycline, Pipacycline, Roli Such as Trimethoprim, (optionally in combin-ation with Sul tetracycline, Sancycline, Tetracycline, and the like), Cyclo fonamides) Nitroimidazoles (e.g., Metronidazole, Tinida serine, Mupirocin, Tuberin; synthetic antibacterial agents, 65 Zole, Nimor-azole, and the like), and the like: Antimycobac e.g. 2,4-Diaminopyrimi-dines (such as Brodimoprim, Tex terial agents (e.g., Capreomycin, Clofazimine, Dapsone, troxoprim, Trimethoprim, and the like), Nitrofurans (such as Ethambutol, Isoniazid, Pyrazinamide, Rifabutin, Rifampicin, US 9,056,154 B2 17 18 Streptomycin, Thioamides, and the like); Antiviral agents and derivatives, or the like, or analogs/derivatives thereof, (e.g., Acryclovir, Amanta-dine, AZidothymidine, Ganciclo salts thereof, and/or combinations thereof. vir, Idoxuridine, Tribavirin, Trifluridine, Vidarabine, and the In another embodiment, the active agent can include anti like); Interferons; antiseptic agents (e.g., Chlorhexidine, microbial agents, analgesics, antiinflammatory agents, Gentian violet, Octenidine, Povidone Iodine, Quaternary counter irritants coagulation modifying agents, diuretics, ammonium compounds, Silver Sulfadi-azine, Triclosan, and sympatho-mimetics, anorexics, antacids and other gas the like); or the like; analogs/derivatives thereof: salts thereof; trointestinal agents, antiparasitics, antidepressants, antihy or combinations thereof. pertensives, anticholinergics, stimulants, antihormones, cen In some embodiments, the active agent may include, but is tral and respiratory stimu-lants, drug antagonists, lipid 10 regulating agents, uricoSurics, cardiac glycosides, not limited to, collagen (e.g., Type 1), osteonectin, bonesia electrolytes, ergot and derivatives thereof, expectorants, hyp loproteins (Bsp), alpha-2HS-glycoproteins, bone Glaprotein notics and sedatives, antidiabetic agents, dopaminergic (Bgp), matrix Gla-protein, bone phosphoglycoprotein, bone agents, antiemetics, muscle relaxants, para-sympathomimet phosphor-protein, bone proteoglycan, protolipids, bone mor ics, anticonvul-sants, antihist-amines, beta-blockers, purga phogenic proteins (e.g., BMP-1, -2A, -2B, -3, -3b, -4, -5,-6, 15 tives, antiarrhytmics, contrast materials, radio-pharmaceuti -7, -8, -8b, -9, -10, -11, -12, -13, -14, -15), cartilage induction cals, antiallergic agents, tranquilizers, vasodilators, antiviral factor, platelet derived growth factor (PDGF-1, -2), endothe agents, and anti-neoplastic or cytostatic agents or other agents lial cell growth factors (ECGF-1, -2a, -2b), skeletal growth with anticancer properties, vitamins (including micro- and factor (SKF=IGF-2), insulin-like growth factors (IGF-1, macro-nutrients), or a combination thereof. IGF-2), fibroblast growth factor (ODGF-1, -2, -3, -4, -5, -6, In another embodiment, the active agent includes an anti -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, muscle spasm agent, anti-spasmodic, bone resorption inhibi -21, -22, -23), colony stimulating factor, transforming growth tor, Smooth muscle contractile agent, calcium absorption factor (e.g., TGF-a, TGF-(3, or the like), vascular endothelial enhancer, muscle relaxant, or a mixture thereof. Suitable growth factors (VEGF), growth/differentiation factors (GDF anti-muscle spasm agents include, but are not limited to, 1, -3, -5, -6, -7, -8, -9, -9B, -10, -11, -15, -16), osteogenic 25 baclofen, botulinum toxin, carisoprodol, chlorphenesin, proteins (OP-1=BMP-7, OP-2=BMP-8, OP-3=BMP-8b), chlorZoxaZone, cyclobenzaprine, dantrolene, diazepam, bone growth hormone, parathyroid hormone (PTH), insulin, metaxalone, methocarbamol, orphenadrine, tizani-dine, and calcitonin, and the like, and combinations thereof. Addition mixtures thereof. Suitable anti-spasmodics include, but are ally or alternately, the active agents may include proteins not limited to, atropine, baclofen, dicyclomine, hyoscine, associated with cartilage, such as chondrocalcining protein; 30 propatheline, oxybutynin, S-oxybutynin, tizanidine, cevime proteins associated with dentin, such as phosphosphoryn, line, chlordiazepoxide, hydrochloride, dicyclomine, hyos glycoproteins and Gla proteins; proteins associated with cine, hyoscyamine, glycopyrrolate, and mixtures thereof. enamel Such as amelognin and enamelin; structural proteins Suitable bone resorption inhibitors include, but are not lim Such as fibrin, fibrinogen, keratin, tubulin, elastin, and the ited to alendronate, ibandronate, minodronate, risedronate, like; blood proteins, whether in plasma or serum, e.g., serum 35 etidronate, tiludronate, and mixtures thereof. A suitable albumin; non-protein growth factors such as prostaglandins Smooth muscle contractile agent includes, but is not limited and statins (e.g., Simvastatin, Lovastatin, or the like); or the to, hyoscine, and mixtures thereof. Suitable calcium absorp like: analogs/derivatives thereof: salts thereof; or combina tion enhancers include, but are not limited to, alfacalcidol. tions thereof. calcitriol, and mixtures thereof. Suitable muscle relaxants In another embodiment, the active agent can includeamino 40 include, but are not limited to, baclofen, carisoprodol, chlor acids, anabolics, analgesics and antagonists, anesthetics, phenesin, chlorZoxaZone, cyclobenzaprine, dantrolene, diaz angiogenesis agents, anti-angiogenetic agents, antihelm epam, metaxalone, methocarbamol, orphenadrine, and mix intics, anti-adrenergic agents, anti-asthmatics, anti-athero tures thereof. Sclerotics, antibacterials, anticholesterolics, anticholinergics, In another embodiment, the active agent includes an anti anti-coagulants, antidepressants, antidotes, anti-emetics, 45 diuretic, anti-muscle spasm agent, anti-spasmodic, agent for anti-epileptic drugs, anti-fibrinolytics, antihistamines, anti treating urinary incontinence, anti-diarrheal agent, agent for inflammatory agents, antihypertensives, antimetabolites, treating nausea and/or vomiting, Smooth muscle contractile antimigraine agents, antimycotics, antinauseants, antine agent, anti-secretory agent, enzyme, anti-ulcerant, bile acid oplastics, anti-obesity agents, anti-Parkinson agents, antipro replacement and/or gallstone solubilizing drug, or a mixture toZoals, antipsychotics, antirheumatics, antiseptics, antiver 50 thereof. Suitable anti-diuretics include, but are not limited to, tigo agents, antivirals, appetite stimulants, bacterial vaccines, acetazolamide, benzthiazide, bendroflumethazide, bumet bioflavonoids, calcium channel blockers, capillary stabilizing anide, chlorthali-done, chlorothiazide, ethacrynic acid, furo agents, coagulants, corticosteroids, detoxifying agents for semide, hydrochlorothiazide, hydroflume-thiazide, methy cytostatic treatment, diagnostic agents (like contrast media clothiazide, polythiazide, quinethaZone, Spironolactone, and radioisotopes), drugs for treatment of chronic alcohol 55 triamterene, torsemide, trichlomethiazide, desmopressin, ism, drugs targeting dopaminergic pathways, electrolytes, oxytocin, and mixtures thereof. Suitable anti-muscle spasm enzymes, enzyme inhibitors, ferments, ferment inhibitors, agents include, but are not limited to, baclofen, botulinum gangliosides and ganglioside derivatives, hemostatics, hor toxin, carisoprodol, chlorphenesin, chlorZoxaZone, mones, hormone antagonists, hypnotics, immuno-modula cyclobenzaprine, dantrolene, diazepam, metaxalone, metho tors, immunostimulants, immuno-Suppressants, minerals, 60 carbamol, orphenadrine, tizanidine, and mixtures thereof. muscle relaxants, neuron-modulators, neurotransmitters and Suitable anti-spasmodics include, but are not limited to, atro neurotropics, osmotic diuretics, parasympatholytics, para pine, baclofen, dicyclomine, hyoscine, propatheline, oxybu sympathomimetics, peptides, proteins, psychoStimulants, tynin, S-oxybutynin, tizanidine, and mixtures thereof. Suit respiratory stimulants, sedatives, serum lipid reducing able agents for treating urinary incontinence include, but are agents, Smooth muscle relaxants, sympatholytics, sympatho 65 not limited to, darifenacin, Vamic-amide, detrol, ditropan, mimetics, vasodilators, vaso-protectives, vectors for gene imipramine, and mixtures thereof. Suitable anti-diarrheal therapy, viral vaccines, viruses, vitamins, oligonucleotides agents include, but are not limited to, Ondansetron, palno US 9,056,154 B2 19 20 setron, tropisetron, attapulgite, atropine, bismuth, diphe beta 1B. Suitable glucose production inhibitors include, but noxylate, loperamide, and mixtures thereof. Suitable agents are not limited to, acarbose, acetohexamide, chlorpropamide, for treating nausea and/or vomiting include, but are not lim glipizide, glyburide, metformin, miglitol, nateglinide, piogli ited to, alosetron, dolasetron, granisetron, meclizine, meto taZone, rosiglitaZone, tolbutamide, and tolaZamide. Suitable clopramide, ondansetron, palnosetron, prochloperazine, insulin enhancing agents include, but are not limited to, acam promethazine, trimethobenzamiode, tropisetron, and mix prosate, miglitol, troglitaZone, chlorpropamide, glimepiride, tures thereof. A Suitable Smooth muscle contractile agent glipizide, glyburide, and repaglinide. A Suitable insulin sen includes, but is not limited to, hyoscine. Suitable anti-secre sitizer includes, but is not limited to, is BRL 49653. Suitable tory agents include, but are not limited to, esomeprazole, cytokines include, but are not limited to, darbepoetin alfa, lansoprazole, omeprazole, pantoprazole, rabeprazole, teneto 10 epoetin alpha, erythropoietin, and NESP. Suitable metabolic pra-Zole, ecabet, misoprostol, teprenone, and mixtures regulators include, but are not limited to, allopurinol and thereof. Suitable enzymes include, but are not limited to, oxypurinol. A Suitable eosinophil and/or mast cell antago alpha-galactosidase, alpha-L-iduronidase, imiglucerase/al nists includes, but is not limited to, nedocromil. Suitable glucerase, amylase, lipase, protease, pancreatin, olsalazine, anti-inflammatory drugs include, but are not limited to, alos and mixtures thereof. Suitable anti-ulcerants include, but are 15 etron, anakinra, beclomethasone, betamethasone, budes not limited to, cimetidine, ranitidine, famotidine, misopros onide, clobetasol, celecoxib, cromolyn, desoximetaSone, tol. Sucralfate, pantopra-Zole, lanSoprazole, omepra-Zole, and dexamethasone, epinastic, etanercept, etoricoxib, flunisolide, mixtures thereof. A suitable bile acid replacement and/or fluocinonide, fluticaSone, formoterol, hydrocortisone, gallstone solubilizing drug includes, but is not limited to, hydroxychloroquine, ibudilast, ketotifen, meloxicam, ursodiol. mesalamine, methotrexate, methylprednisolone, mometa In another embodiment, the active agent includes an endo Sone, montelukast, nedocromil, olsalazine, prednisone, rama crine modulator, glucose production inhibitor, agent for treat troban, rofecoxib, Salsalate, terbutaline, triamcinolone, Val ment of type II diabetes, anti-secretory agent, glycolipid, decoxib, and Zafirlukast. Suitable anti-obesity drugs include, glycoprotein, anti-hyperthyroid agent, thyroid hormone, or a but are not limited to, dexedrine, diethylpropion, mazindol, mixture thereof. Suitable endocrine modulators include, but 25 oleoyl-estrone, phentermine, phendimetrazine, and Sibutra are not limited to, methimazole, Voglibose, finasteride, mine. A suitable COX and/or LO inhibitor includes, but is not GI198745, liothyronine, glyburide, metformin, nateglinide, limited to, is ML-3000. ioglitaZone, pegvisomant, minoxidil, and mixtures thereof. In another embodiment, the active agent includes an anti Suitable glucose production inhibitors include, but are not arrhythmic, anti-hypertensive, heart regulator, cardiovascular limited to, acarbose, acetohexamide, chlorpropamide, glipiz 30 agent, plaque stabilization agent, vasodilator, antianginal, ide, glyburide, metformin, miglitol, nateglinide, pioglitA anti-coagulant, anti-hypotensive, anti-thrombotic, drug for Zone, rosiglitazone, tolbutamide, tolazamide, and mixtures treating congestive heart failure, p-FOX (fatty acid oxidation) thereof. Suitable agents for treatment of type II diabetes inhibitor, or a mixture thereof. Suitable antiarrhythmics include, but are not limited to, acarbose, acetohex-amide, include, but are not limited to, adenosine, amioda-rone, bepri chlorpropamide, glipizide, glyburide, metformin, miglitol, 35 dil, bretylium, digitoxin, digoxin, diltiazem, disopyramide, nateglinide, pioglit-aZone, rosiglitaZone, tolbutamide, tolaza dofetilide, D-sotolol, flecamide, lidocaine, mexiletine, mil mide, and mixtures thereof. Suitable anti-secretory agents rinone, phenyloin, pilsicamide, procain-amide, propafenone, include, but are not limited to, esomeprazole, lanSoprazole, propranolol, quinidine, tocamide, dolfetilide, and mixtures omeprazole, pantoprazole, rabeprazole, tenetoprazole, eca thereof. Suitable anti-hypertensives include, but are not lim bet, misoprostol, teprenone, and mixtures thereof. Suitable 40 ited to, acebutolol, alfuZosin, amlodipine, atenolol, amlo glycolipids include, but are not limited to imigulcerase, van dipine/benazepril, barnidipine benazepril, bepridil, betax comycin, Vevesca (OGT918), GMK vaccine, and mixtures olol, bisoprolol, bosentan, candesartan, captopril, cariporide, thereof. Suitable glycoproteins include, but are not limited to, carvedilol, celiprolol, cilaZapril, clonidine, diltiazem, dox staphvax, bimosiamose (TBC1269), GCS-100, heparin, and aZosin, enalapril, eplerenone, eprosartan, esmolol, felo mixtures thereof. Suitable anti-hyperthyroid agents include, 45 dipine, fenoldopam, fosinopril, guanfacine, imidapril, irbe but are not limited to, methimazol, propylthiouracil, and mix Sartan, isradipine, labetalol, lercanidipine, lisinopril, tures thereof. losartan, manidipine, methyldopa, metoprolol, moxonidine, In another embodiment, the active agent includes a choles nadolol, nicardipine, nicorandal, nifedipine, nitrendipine, terol-lowering agent, aldosterone antagonist, triglyceride noSoldipine, omapatrilat, perindopril erbumine, pindolol. lowering agent, leukotriene receptor antagonist, immuno 50 prazosin, propranolol, quinapril, ramipri, Sotalol, spirapril, modulator or immunogen, glucose production inhibitor, tamsulosin, telmisartan, teraZosin, torsemide, trandolapril, agent for treatment of type II diabetes, bone resorption inhibi Valsartan, Vatanidipine, midodrine, and mixtures thereof. tor, calcium absorption enhancer, insulin enhancing agent, Suitable heart regulators include, but are not limited to, insulin sensitizer, cytokine, metabolic regulator, mast cell digoxin, digitoxin, dobut-amine, and mixtures thereof. Suit mediator, eosinophil and/or mast cell antagonist, glycolipid, 55 able cardiovascular agents include, but are not limited to, glycoprotein, anti-inflammatory drug, anti-obesity drug, edaravone, iloprost, levosimendan, molsidomine, tezosentan, COX (cyclooxygenase) and/or LO (lipoxygenase) inhibitor, tirilazad, YMO87, adenosine, avasimibe, fenofibrate, and or a mixture thereof. Suitable cholesterol-lowering agents mixtures thereof. A Suitable plaque stabilization agent include, but are not limited to, atorvastatin, benzofibrate, includes, but is not limited to, avasimibe. Suitable vasodila bezafibrate, cerivastatin, cholestyramine, ciprofibrate, clofi 60 tors include, but are not limited to, buflomedil, cilostazol, brate, colesevelam, colestipol, eZetimibe, fluvastatin, gemfi dipyridamole, diaZOxide, hydralazine, minoxidil, naftidrofu brozil, lovastatin, niacin/lovastatin, pravastatin, probucol, ryl, nicorandil, nitroprusside, alprostadil, apomorphine, rosuvastatin, and simvastatin. A Suitable aldosterone antago phentolamine mesylate, sildenafil, tadalafil. Vardenifil, and nist includes, but is not limited to, spironolactone. A Suitable mixtures thereof. Suitable anti-anginals include, but are not triglyceride-lowering agent includes, but is not limited to, 65 limited to, amilodipine, amyl nitrite, atenolol, bepridil, dilt fenofibrate. Suitable immunomodulators or immunogens iazem, erythrity1 tetranitrate, felodipine, isosorbide dinitrate, include, but are not limited to, interferon beta 1A, interferon isradipine, metoprolol, nadolol, nicardipine, nifedipine, US 9,056,154 B2 21 22 nimodipine, pentaerythritol tetranitrate, propranolol, and razepate, cisatracurium, cyclobenzaprine, eperisone, esopi mixtures thereof. Suitable anti-coagulants include, but are not clone, hydroxy Zine, mirtazapine, mivacurium, pagoclone, limited to, abciximab, ardeparin, argatroban, bivalirudin, clo Sulperide, Zaleplon, and Zopiclone. Suitable dopamine pidogrel, dalteparin, danaparoid, desirudin, dipyridamole, metabolism inhibitors include, but are not limited to, entaca enoxaparin, eptifibatide, fondaparinux, H376/95, lepirudin, pone, lazebemide, selegiline, and tolcapone. Suitable agents melagatran, nadroparine, nafamo.stat mesilate, pentosan, pen to treat post stroke sequelae include, but are not limited to, toxifylline, reviparin, sarpogrelate, SNAC/SNAD-heparin, glatiramer, interferon beta 1A, interferon beta 1B, estradiol, ticlopidine, tinzaparin, tirofiban, warfarin, and mixtures and progesterone. Suitable neuron-protectants include, but thereof. Suitable anti-hypotensives include, but are not lim are not limited to, donepezil, memainine, nimodipine, rilu ited to, midodrine, dobutamine, fludrocortisone, and mixtures 10 thereof. Suitable anti-thrombotics include, but are not limited Zole, rivastigmine, tacrine, TAK147, and Xaliproden. Suitable to, aspirin, abciximab, enoxaparin, integrelin, ticlopidine, agents to treat Alzheimer's disease include, but are not lim and mixtures thereof. Suitable drugs for treating congestive ited to, carbidopa, levodopa, tacrine, doneZepil, rivastigmine, heart failure include, but are not limited to, aminone, and galantamine. Suitable neurotransmitters include, but are benazepril, bumetanide, captopril, digitoxin, digoxin, dob 15 not limited to, acetylcholine, serotonin, 5-hydroxytryptamine utamine, dopamine, enalapril, ethacrynic acid, foSino-pril, (5-HT), GABA, glutamate, aspartate, glycine, histamine, epi furosemide, hydralazine, lisinopril, milrinone, minoxidil, nephrine, norpinephrine, dopamine, adenosine, ATP, and moexipril, quinapril, ramipril, torsemide, and mixtures nitric oxide. Suitable neuron-transmitter agonists include, but thereof. A suitable p FOX inhibitor includes, but is not limited are not limited to, almotriptan, aniracetam, atomoxetine, to, ranolazine. benserazide, bromocriptine, bupropion, cabergoline, citalo In another embodiment, the active agent includes an aldos pram, clomipramine, desipramine, diazepam, dihydroergota terone antagonist, immunomodulator or immunogen, immu mine, doxepin dulloxetine, eletriptan, escitalopram, fluvox nosuppressant, cytokine, leukotriene receptor antagonist, amine, gabapentin, imipramine, moclobemide, naratriptan, mast cell mediator, eosinophil and/or mast cell antagonist, nefazodone, nefiracetam acamprosate, nicergoline, nortryp mucolytic, glucocorticoid, glycolipid, or a mixture thereof. A 25 tiline, paroxetine, pergolide, pramipexole, rizatriptan, ropin Suitable aldosterone antagonist includes, but is not limited to, irole, Sertraline, Sibutramine, Sumatriptan, tiagabine, traZ spironolactone. Suitable immuno-Suppressants include, but odone, Venlafaxine, and Zolmitriptan. Suitable sedatives are not limited to, azathioprine, cyclophosphamide, include, but are not limited to, dexmedetomidine, esZopi cyclosporine, ERL 080, enlimomab, methotrexate, mitoxan clone, indiplon, Zolpidem, and Zaleplon. Suitable agents for trone, mycophenolate, mofetil, sirolimus, tacrolimus (FK 30 treating attention deficit disorder include, but are not limited 506), and mixtures thereof. Suitable mucolytics for use in the to, amphetamine, dextroamphetamine, methyl-phenidate, buccal sprays of the invention include, but are not limited to, and pemoline. Suitable agents for treating narcolepsy ambroXol, bromhexin, fudostein, acetylcestine, and mixtures include, but are not limited to, modafinil and mazindol. A thereof. Suitable central adregenic antagonist includes, but is not lim In another embodiment, the active compound is a p-FOX 35 ited to, mesoridazine Suitable anti-depression agents include, (fatty acid oxidation) inhibitor, acetylcholinesterase inhibi but are not limited to, amitriptyline, amoxapine, bupropion, tor, nerve impulse inhibitor, anti-cholinergic, anti-convul clomipramine, clomipramine, clorgyline, desipramine, dox sant, anti-psychotic, anxiolytic agent, dopamine metabolism epin, fluoxetine, imipramine, isocarboxazid, maprotiline, inhibitor, agent to treat post stroke sequelae, neuroprotectant, mirtazapine, nefazodone, nortriptyline, paroxetine, agent to treat Alzheimer's disease, neurotransmitter, neu 40 phenelZine, protriptyline, Sertraline, tranylcypromine, traZ rotransmitter agonist, sedative, agent for treating attention odone, and Venlafaxine. Suitable agents for treating Parkin deficit disorder, agent for treating narcolepsy, central adrege son's disease include, but are not limited to, amantadine, nic antagonist, anti-depression agent, agent for treating Par bromocriptine, carvidopa, levodopa, pergolide, and sel kinson's disease, benzodiazepine antagonist, stimulant, neu egiline. A suitable benzo-diazepine antagonist includes, but is rotransmitter antagonist, tranquilizer, or a mixture thereof. 45 not limited to, flumazenil. A suitable neuron-transmitter Suitable acetylcholinesterase inhibitors include, but are not antagonist includes, but is not limited, to deramciclane. Suit limited to, galantamine, neostig-mine, physostigmine, and able stimulants include, but are not limited to, amphetamine, edrophonium. Suitable nerve impulse inhibitors include, but dextroamphetamine, dinoprostone, methylphenidate, meth are not limited to, levobupivacaine, lidocaine, prilocalne, ylphenidate, modafinil, and pemoline A Suitable tranquilizer mepivacaine, propofol, rapacuronium bromide, ropivacaine, 50 includes, but is not limited to, mesoridazine. tubocurarine, atracurium, doxaurium, miva-curium, pancuro In another embodiment, the active agent includes a nerve nium, Vercuronium, pipecuronium, and rocuronium. Suitable impulse inhibitor. Suitable nerve impulse inhibitors include, anti-cholinergics for use in the buccal sprays of the invention but are not limited to levobupivacaine, lidocaine, prilocalne, include, but are not limited to, amantadine, ipratropium, mep-ivacaine, propofol, rapacuronium bromide, ropivacaine, oxitropium, and dicycloverine. Suitable anti-convulsants 55 tubocurarine, atracurium, doxacurium, mivacurium, pancu include, but are not limited to, acetazolamide, carbam ronium, Vecuronium, pipecuronium, rocuronium, and mix azepine, clonazepam, diazepam, divalproex (Valproic acid), tures thereof. ethoSuximide, lamotrignine acid, levetriacetam, Oxcarba In another embodiment, the active agent includes an anti Zepine, phenol-barbital, phenyloin, pregabalin, primidone, opioid agent. Suitable anti-opioid agents for use in the buccal remacemide, trimethadione, topiramate, vigabatrin, and 60 sprays of the invention include, but are not limited to, nalox Zonisamide. Suitable antipsychotics include, but are not lim one, nalmefene, naltrexone, cholecystokinin, nociceptin, ited to, amisulpride, aripiprazole bifemelane, bromperidol. neuropeptide FF, oxytocin, vasopressin, and mixtures clozapine, chlorpromazine, haloperidol, illoperidone loperi thereof. done, olanzapine, quetiapine, fluphenazine, fumarate, risperi In another embodiment, the active agent includes an anti done, thiothixene, thioridazine, Sulpride, and Ziprasidone. 65 migraine agent. Suitable anti-migraine agents for use in the Suitable anxiolytic agents include, but are not limited to, buccal sprays of the invention include, but are not limited to, amitryptiline, atracurium, buspirone, chlorZoxazone, clo froVatriptan, Zolmitriptan, rizatriptan, almotriptan, eletriptan, US 9,056,154 B2 23 24 naratriptan, almotriptan, ergotamine, diethylergotamine, pharmaceutically acceptable salts thereof, base forms Sumatriptan, and mixtures thereof. thereof, and any combination of Such opioids and/or their In another embodiment, the active agent includes a pain derivatives. control agent. Suitable pain control agents for use in the In certain embodiments, the opioidagonist includes hydro buccal sprays of the invention include, but are not limited to, codone, morphine, hydromorphone, oxycodone, codeine, non-steroidal anti-inflammatory drugs, alfentanil, butorpha levorphanol, meperidine, methadone, oxymorphone, nol, codeine, dezocine, fentanyl, hydrocodone, hydromor buprenorphine, fentanyl, dipipanone, heroin, tramadol, etor phone, levorphanol, meperidine, methadone, morphine, nal phine, dihydroetorphine, butorphanol, levorphanol, pharma buphine, oxycodone, oxymorphone, propoxyphene, ceutically acceptable salts thereof, base forms thereof, and pentazocine, Sufentanil, tramadol, and mixtures thereof. 10 any and all mixtures thereof. The opioid agonist can, in some In another embodiment, the active agent includes an anes embodiments, include oxycodone, hydrocodone, fentanyl. thetic. Suitable anesthetics for use in the buccal sprays of the buprenorphine, pharmaceutically acceptable salts thereof, invention include, but are not limited to, benzo-natate, bupi base forms thereof, and any and all mixtures thereof. The vacaine, desflurane, enflurane, isoflurane, levobupivacaine, opioid agonist can, in other embodiments, include buprenor lidocaine, mepivacaine, prilocalne, propofol, rapacuronium 15 phine, pharmaceutically acceptable salts thereof, base forms bromide, ropivacaine, sevoflurane, ketamine, and mixtures thereof, fentanyl, pharmaceutically acceptable salts thereof, thereof. base forms thereof, and any combination of Such opioids In another embodiment, the active agent can include, but is and/or their derivatives. not limited to, cyclo sporine, sermorelin, octreotide acetate, General categories of active agents can, in one embodi calcitonin-Salmon, insulin lispro, Sumatriptan Succinate, ment, include, but are not limited to: ACE inhibitors; adeno cloZepine, cyclobenzaprine, dexfenfluramine hydrochloride, hypophyseal hormones; adrenergic neuron blocking agents; glyburide, Zidovudine, erythromycin, ciprofloxacin, adrenocortical steroids; inhibitors of the biosynthesis of ondansetron hydrochloride, dimenhydrinate, cimetidine adrenocortical steroids; alpha-adrenergic agonists; alpha hydro-chloride, famotidine, phenyloin Sodium, phenyloin, 25 adrenergic antagonists; selective alpha-two-adrenergic ago carboprost thromethamine, carboprost, diphenhydramine nists; androgens; anti-addictive agents; antiandrogens; anti hydrochloride, isoproterenol hydrochloride, terbutaline sul infectives, such as antibiotics, antimicrobials, and antiviral fate, terbutaline, theophylline, albuterol sulfate, neutraceuti agents; analgesics and analgesic combinations; anorexics: cals (i.e., nutrients with pharmacological action, e.g., car antihelmintics; antiarthritics; antiasthmatic agents; anticon 30 Vulsants; antidepressants; antidiabetic agents; antidiarrheals; nitine, Valerian, echinacea, and the like), or the like; analogs/ antiemetic and prokinetic agents; antiepileptic agents; anti derivatives thereof, saltsfalternate salts thereof; or estrogens; antifungal agents; antihistamines; antiinflamma combinations thereof. tory agents; antimigraine preparations; anti-muscarinic Any opioid or non-u-opioid, a pharmaceutically accept agents; antinauseants; antineoplastics; antiparasitic agents: able salt thereof, a base form thereof, or mixture of any 35 anti-parkinsonism drugs; antiplatelet agents; antiprogestins: combination of such opioids and/or their derivatives that are antipruritics; antipsychotics; anti-pyretics; antispasmodics; known in the art can be included. Opioids believed to have at anticholinergics; antithyroid agents; antitussives; azaspirode least Some u-opioid receptor agonist activity (and optionally cane-diones; sympathomimetics: Xanthine derivatives; car at least some agonist activity also at one or more of the diovascular preparations, including potassium and calcium K-opioid receptor, the 8-opioid receptor, and the ORL-1 40 channel blockers, alpha blockers, beta blockers, and antiar receptor) include, but are not limited to, alfentanil, allylpro rhythmics; antihypertensives; diuretics and antidiuretics; dine, alphaprodine, anilleridine, benzylmorphine, bezitra vasodilators, including general coronary, peripheral, and mide, buprenorphine, butorphanol, clonitaZene, codeine, cerebral; central nervous system stimulants; vasoconstric desomorphine, dextromoramide, dezocine, diampromide, tors; cough and cold preparations, including decongestants; diamorphone, dihydro-codeine, dihydromorphine, dihydro 45 hormones, such as estradiol and other Steroids, including morphone, dihydroisomorphine, dimenoxadol, dimephep cortico-steroids; hypnotics; immunosuppressives; muscle tanol, dimethylthiambutene, dioxaphetyl butyrate, dipi relaxants; parasympatholytics; psychoStimulants; sedatives; panone, eptazocine, ethoheptazine, ethylmethylthiambutene, tranquilizers; nicotine and acid addition salts thereof; benzo ethylmorphine, etonitaZene, etorphine, dihydroetorphine, diazepines; barbituates; benzothiadiazides; beta-adrenergic fentanyl, heroin, hydrocodone, hydromorphone, hydromor 50 agonists; beta-adrenergic antagonists; selective beta-one-adr phodone, hydroxypethidine, isomethadone, ketobemidone, energic antagonists; selective beta-two-adrenergic antago levorphanol, levophenacylmorphan, lofentanil, meperidine, nists; bile salts; agents affecting Volume and composition of meptazinol, metazocine, methadone, metopon, morphine, body fluids; butyrophenones; agents affecting calcification; myrophine, narceline, nicomorphine, norlevorphanol, catecholamines; cholinergic agonists; cholinesterase reacti normethadone, nalorphine, nalbuphene, normorphine, norpi 55 vators; dermatological agents; diphenylbutyl-piperidines; panone, opium, Oxycodone, oxymorphone, pantopon, papa ergot alkaloids; ganglionic blocking agents; hydantoins; Veretum, paregoric, pentazocine, phenadoxone, phendime agents for control of gastric acidity and treatment of peptic trazine, phendimetraZone, phenomorphan, phenazocine, ulcers; hematopoietic agents; histamines; 5-hydrox phenoperidine, piminodine, piritramide, propheptazine, ytryptamine antagonists; drugs for the treatment of hyper promedol, properidine, propoxyphene, propylhexedrine, 60 lipiproteinemia; laxatives; methylxanthines; moncamine oxi Sufentanil, tilidine, tramadol, and mixtures thereof. Non-L- dase inhibitors; neuronmuscular blocking agents; organic opioids include, but are not limited to, ORL-1-specific opioid nitrates; pancreatic enzymes; phenothiazines; prostaglan agonists, such as nociceptin, deltorphin, and the like, and dins; retinoids; agents for spasticity and acute muscle spasms; mixtures thereof. In a preferred embodiment, the opioid Succinimides; thioxanthines; thrombolytic agents; thyroid includes buprenorphine, pharmaceutically acceptable salts 65 agents; inhibitors of tubular transport of organic compounds; thereof, base forms thereof, fentanyl, pharmaceutically drugs affecting uterine motility; vitamins; and the like; or a acceptable salts thereof, base forms thereof, oxycodone, combination thereof. US 9,056,154 B2 25 26 Alternately or in addition to an opioid agonist, another In another embodiment, the active agent can include, but is active compound may be added including, but not limited to, not limited to, anti-staphylococcal agents (e.g., YSPXTNF fluorogestone acetate, hydroxyprogesterone, hydroxyproges SEQ ID NO: 2), YSPWTNF SEQID NO:3), YSPWTNF terone acetate, hydroxyprogesterone caproate, medroxy NH2 SEQID NO: 4), GENBANK/AF202641 SEQID NO: progesterone acetate, norethindrone, norethindrone acetate, 5), GENBANK/AF205220 SEQ ID NO: 6), GENBANK/ norethisterone, norethynodrel, desogestrel, 3-keto AAG03056 SEQ ID NO: 7), or the like, or combinations desogestrel, gestadene, levonorgestrel, estradiol, estradiol thereof). Other agents that modulate the production or secre benzoate, estradiol Valerate, estradiol cyprionate, estradiol tion of bacterial or microbial toxins or virulence factors may decanoate, estradiol acetate, ethynyl estradiol, estriol, also be used as active agents. For instance, thiolactones and estrone, mestranol, betamethasone, betamethasone acetate, 10 bacterial toxin regulatory proteins such as RNAIII-inhibiting cortisone, hydrocortisone, hydrocortisone acetate, corticos peptides (RIPs) are classes of active agents. See, e.g., Bala terone, fluocinolone acetonide, prednisolone, prednisone, tri ban, N., et al., “Regulation of Staphylococcus aureus patho amcinolone, aldosterone, androsterone, testosterone, methyl genesis via target of RNAIII-activating Protein (TRAP). J. testosterone, or a combination thereof. Biol Chem., 2001 Jan. 26: 276(4): 2658-67, which is incor Alternately or in addition to an opioid agonist, another 15 porated by reference herein in its entirety. active compound may be added including, but not limited to: When an active agent of the present invention is acidic, a) corticosteroids, e.g., cortisone, hydrocortisone, predniso salts may be prepared from pharmaceutically acceptable non lone, beclomethasone propionate, dexamethasone, toxic bases. Salts derived from all stable forms of inorganic betamethasone, flumethasone, triamcinolone, triamcinolone bases include aluminum, ammonium, calcium, copper, iron, acetonide, fluocinolone, fluocinolone acetonide, fluocino lithium, magnesium, manganese, potassium, Sodium, Zinc, lone acetate, clobetasol propionate, or the like, or a combina etc. In one embodiment, the salt includes ammonium, cal tion thereof; b) analgesic anti-inflammatory agents, e.g., cium, magnesium, potassium, or a sodium salt. Salts derived acetaminophen, mefenamic acid, flufenamic acid, from pharmaceutically acceptable organic non-toxic bases indomethacin, diclofenac, diclofenac sodium, alclofenac, include salts of primary, secondary, and tertiary amines, Sub ibufenac, oxyphenbutaZone, phenylbutaZone, ibuprofen, flur 25 stituted amines including naturally occurring Substituted biprofen, ketoprofen, salicylic acid, methylsalicylate, acetyl amines, cyclic amines and basic ion-exchange resins such as salicylic acid, 1-menthol, camphor, slindac, tolmetin Sodium, arginine, betaine, caffeine, choline, N.Ndibenzylethylenedi naproxen, fenbufen, or the like, or a combination thereof; c) amine, diethylamine, 2-diethylaminoethanol, 2-dimethyl hypnotic sedatives, e.g., phenobarbital, amobarbital, cyclo aminoethanol, ethanolamine, ethylene-diamine, N-ethylmor barbital, lorazepam, haloperidol, or the like, or a combination 30 pholine, N-ethylpiperidine, glucamine, glucosamine, thereof; d) tranquilizers, e.g., fulphenazine, thioridazine, histidine, iso-propylamine, lysine, methyl-glucosamine, diazepam, flurazepam, chlorpromazine, or the like, or a com morpholine, piperazine, piperidine, polyamine resins, bination thereof; e) anti-hypertensives, e.g., clonidine, cloni procaine, purine, theobromine, triethylamine, trimethy dine hydrochloride, bopinidol, timolol, pindolol, propra lamine, tripropylamine, etc. nolol, propranolol hydrochloride, bupranolol, indenolol. 35 When an active agent of the present invention is basic, salts bucumolol, nifedipine, bunitrolol, or the like, or a combina may be prepared from pharmaceutically acceptable non-toxic tion thereof: f) hypotensive diuretics, e.g., bendroflumethiaz acids. Such acids include acetic, benzene-Sulfonic, benzoic, ide, poly-thiazide, methylchlorthiazide, trichlor-methiazide, camphorsulfonic, citric, ethane-Sulfonic, fumaric, gluconic, cyclopenthiazide, benzyl hydrochloro-thiazide, hydrochlo glutamic, hydrobromic, hydrochloric, isethionic, lactic, rothiazide, bumetanide, or the like, or a combination thereof; 40 maleic, mandelic, methane-Sulfonic, mucic, nitric, pamoic, g) anti-biotics, e.g., penicillin, tetracycline, oxytetracycline, pantothenic, phosphoric, Succinic, Sulfuric, tartaric, p-tolu metacycline, doxycycline, minocycline, fradiomycin Sulfate, enesulfonic, etc. In one embodiment, the acid includes citric, erythromycin, chloramphenicol, or the like, or a combination hydrobromic, maleic, phosphoric, Sulfuric, and/or tartaric thereof h) anesthetics, e.g., lidocaine, benzocaine, ethylami acids. nobenzoate, or the like, or a combination thereof i) antimi 45 Although some active agents can be bound directly to the crobial agents, e.g., benzalkonium chloride, nitrofuraZone, polymer Surface of a Substrate by the organometallic layer nystatin, Sulfacet-amide, clotriamazole, or the like, or a com according to the invention, many active agents according to bination thereof, j) anti-fungal agents, e.g., penta mycin, the present invention can often be attached to the polymer amphotericin B. pyrrol nitrin, clotrimazole, or the like, or a Surface of a Substrate via a codi-functional linkers or spacers combination thereof, k) vitamins, e.g., vitaminA, ergocalcif 50 bound to the organic overlayer. Such spaces or linkers will erol, cholecalciferol, octotriamine, riboflavin butyric acid often be tailored to the particular active agent(s) used. It is ester, or the like, or a combination thereof: 1) antiepileptics, noted that the a- and co functional groups of the linkers/ e.g., nitrazepam, meprobamate, clonazepam, or the like, or a spacers can be similar or different, and often are different combination thereof; m) antihistamines, e.g., diphenhy particularly where release of the active agent(s) is(are) dramine hydrochloride, chlorpheniramine, diphenylimicla 55 intended (e.g., in vivo). Such attachment (and release) of Zole, or the like, or a combination thereof, n) antitussives, e.g., active agents can be effected by covalent bonding (cleaving), dextromethorphan, terbutaline, ephedrine, ephedrine hydro ionic bonding (dissociation), physical transitions of State chloride, or the like, or a combination thereof; o) sex hor Such as crystallization (melting) or liquid crystalline-type mones, e.g., progesterone, estradiol, estriol, estrone, or the ordering (disordering), hydrogen bonding (dissociation), Van like, or a combination thereof p) antidepressants, e.g., dox 60 der Waals interactions (repulsions), or the like, or any com epin, q) Vaso-dilators, e.g., nitroglycerin, isosorbide nitrate, bination thereof. The linkers/spacers can be of relatively nitroglycol, pentaerythritol tetranitrate, dipyridamole, or the Small molecular weight (e.g., less than about 200 g/mol), like, or a combination thereof, r) other drugs, e.g., 5-fluorou relatively intermediate molecular weight (e.g., from about racil, dihydroergotamine, desmopressin, digoxin, methoclo 200 to about 2000 g/mol), relatively large molecular weight pramide, domperidone, Scopolamine, Scopolamine hydro 65 (e.g., more than about 2000 g/mol), or some combination chloride, or the like, or a combination thereof, or the like; or thereof. Particularly when the linkers/spacers are of relatively a combination thereof. intermediate and/or relatively large molecular weight, they US 9,056,154 B2 27 28 can include, but are not limited to, oligomers, polymers, those skilled in the art. The scaffold structure is typically and/or copolymers described above as bioactive moieties or porous to allow generous cellular ingrowth. The polymer biodegrade-able polymers; additionally or alternately, the scaffolds may also be molded to form external scaffolding for linkers/spacers can include, but are not limited to, oligomers, the support of in vitro culturing of cells for the creation of polymers, and/or copolymers having one or more of the fol external Support organs. lowing types of repeat units: urethanes, ureas, ethers, ketones, Polymers that are suitable for use as scaffold materials are esters, amines, carbonates, amides, saccharides, or the like, or non-toxic, physiologically compatible, and optionally biode combinations thereof. gradable. The polymer must be selected for biocompatibility In other embodiments, polyfunctional linkers/spacers can at the time of implant, and, if biodegradable, the products of be used to attach active agents to the coatings/derivatized 10 its degradation process must also be biocompatible. Addi Surfaces according to the invention. Such poly-functional tional parameters that play an important role include the linkers/spacers can include, but are not limited to, oligomers, mechanical properties of the material, especially its mechani polymers, and/or copolymers that are branched, hyper cal rigidity. Relatively high rigidity is advantageous so that branched, dendritic, star-shaped, brushes, combs, block, the scaffold can withstand the contractile forces exerted by multiblock, or the like, or any combination thereof. 15 cells growing within the scaffold. Also important are the The transition metal phosphate esters can also serve as a thermal properties, such as the glass transition temperature, template for first chemical, then biological growth of bone Tg, and the biodegradation kinetics, if degradable, which tissue hydroxyapatite in the implant Surface. Surface-bound should match the rate of the healing process. transition metal phosphate layers insinuate themselves The scaffold functions to mimic the extracellular matrices directly into bone tissue hydroxylapatite to make a strong (ECM) of the body. The scaffold serves as both a physical composite seal between the implant Surface and the Support and an adhesive Substrate for isolated cells during in hydroxyapatite. Alternatively, the phosphate may be hydro vitro culture and Subsequent implantation. As the trans lyzed to form transition metal polyphosphates having a two planted cell populations grow and the cells function normally, dimensional structure, the layers of which also insinuate they begin to secrete their own ECM support. The scaffold themselves directly into bone tissue hydroxyapatite. 25 polymer preferably degrades as the need for an artificial Sup Essentially any organophosphonic acid capable of forming port diminishes. a thin film on an organo-metallic Surface is suitable for use In the reconstruction of structural tissues like cartilage and with the present invention. The organophosphonic acid will bone, tissue shape is integral to function, requiring the mold have a hydrocarbon ligand that may be saturated or unsatur ing of the polymer scaffold into articles of varying thickness ated, branched or unbranched, substituted or unsubstituted, 30 and shape. Any crevices, apertures or refinements desired in and may be aromatic or non-aromatic. Typical hydrocarbon the three dimensional structure can be created by removing ligands of organophosphonic acids will contain between two portions of the matrix with scissors, a scalpel, a laser beam or and twenty carbon atoms or for example, between three and any other cutting instrument. Scaffold applications include eighteen carbon atoms. Stearylligands, for example, may be the regeneration of tissues such as nervous, musculoskeletal, used. 35 cartilaginous, tendenous, hepatic, pancreatic, ocular, integu A preferred class of organophosphorus and organic car mentary, arteriovenous, urinary or any other tissue forming boxylic acids are those with omega-functionalized organo Solid or hollow organs. groups that can be chemically transformed to react and The scaffold may also be used intransplantation as a matrix covalently bond to the aforementioned biologically active for dissociated cells such as chondrocytes or hepatocytes to and pharmaceutically active compounds. Examples of Such 40 create a three-dimensional tissue or organ. Any type of cell omega functional groups include amino, carboxylate, thiol, can be added to the scaffold for culturing and implantation, hydroxyl, carbonate, ester, carbamate, and amide groups. including cells of the muscular and skeletal systems. Such as A preferred application technique involves a two-stage mesenchymal stem cells, chondrocytes, fibroblasts, osteo vapor deposition process in which the transition metal alkox cytes and osteoblasts, parenchymal cells such as hepatocytes, ide—or dialkylamide—is first vapor deposited on the poly 45 pancreatic cells (including Islet cells), cells of intestinal ori mer Surface. When the reaction is complete, vacuum is gin, and other cells such as exocrine cells, bile duct cells, applied to remove excess transition metal alkoxide and parathyroid cells, thyroid cells, cells of the adrenal-hypo dialkylamine or alkanol by-product, which is then followed thalmic-pituitary axis, heart muscle cells, kidney epithelial by vapor deposition of the organic overlayer material. Upon cells, kidney tubular cells, kidney basement membrane cells, completion of the reaction with the organic compound, the 50 nerve cells, neurons, blood vessel cells, endothelial cells, vacuum is then applied to withdraw excess organic com cells forming bone and cartilage, Smooth muscle cells, skel pound solution and alkanol or dialkylamine byproduct. etal muscle cells, ocular cells, integumentary cells, kerati Suitable substrates with polymer surfaces also include fab nocytes and skin cells, and, either as obtained from donors, rics formed from a woven or non-woven fiber. The fiber can embryonic and non-embryonic stem cells, established cell be a natural fiber with exposed functional groups, such as silk, 55 culture lines, including embryonic and non-embryonic stem wool, cotton, collagen, linen, and the like. The fiber can also cell culture lines, and either before or after genetic engineer be a synthetic fiber with exposed amide groups, such as nylon. ing. Pieces of tissue can also be used, which may provide a The polymer-coated and polymer-cast Substrates of the number of different cell types in the same structure. present invention may be fabricated into scaffolds for tissue The cells are obtained from a suitable donor, or the patient engineering and tissue guided regeneration applications, 60 into which they are to be implanted, dissociated using stan including reconstructive Surgery. The scaffolds may also be dard techniques and seeded onto and into the scaffold. In vitro molded to form external scaffolding for the support of in vitro culturing optionally may be performed prior to implantation. culturing of cells for the creation of external Support organs. Alternatively, the scaffold is implanted, allowed to vascular The scaffolds may also be used in transplantation as a matrix ize, then cells are injected into the scaffold. Methods and for dissociated cells. 65 reagents for culturing cells in vitro and implantation of a Polymer scaffolds and mixed polymer? ceramic scaffolds tissue scaffold are known to those skilled in the art. The for tissue engineering and their manufacture are known to polymer Scaffolds can be utilized in vivo as tissue engineering US 9,056,154 B2 29 30 and tissue guided regeneration scaffold in mammals such as imidopropionic acid N-hydroxySuccinimide ester in dry primates, including humans, sheep, horses, cattle, pigs, dogs, acetonitrile for 16 hours to produce 8. Immersion of 8 in a 0.1 cats, rats and mice, or in vitro. mMaqueous solution of RGDCSEQID NO: 1) at pH 6.5 for The polymer-coated and polymer-cast Substrates of the 24 hours produced 9a. The nylon-Zr surface complex was present invention may also be fabricated from biocompatible derivatized with the succinimide ester of 3-maleimidopropi polymers into articles for medical implant applications. The onic acid directly by transesterification to produce 8, which organic ligand bonded to the article Surface can be a biologi can result in either an RGDC SEQID NO: 1 or DANSYL cally- or pharmaceutically-active compound having utility as Cys-tethered surface (9a or 9b). Complexes 9a and 9b have a a coating on a medical implant. The polymer-coated and 1:2 ratio of zirconium to RGDC SEQ ID NO: 1 or DAN polymer-cast articles are formed into shaped articles Such as 10 SYL-Cys, respectively. vascular grafts and stents, bone plates, Sutures, implantable sensors, barriers for Surgical adhesion prevention, implant Example 3 able drug delivery devices and other therapeutic agent articles. The same issues related to toxicity and tissue com Preparation of DANSYL-Cys-Modified Nylon patibility for tissue scaffold polymers also apply to medical 15 implant polymers. Substrate The following non-limiting examples set forth hereinbe low illustrate certain aspects of the present invention. They Fluorescent molecule-labeled analogues 7b and 9b were are not to be considered limiting as to the scope and nature of prepared as described for 7a and 9a, but a 0.1 mM aqueous the present invention. In the examples which follow, all parts solution of N-(5-(dimethylamino)-1-naphthyl-sulfonyl)-cys are by weight. teine (DANSYL-Cys) was used instead of RGDC SEQ ID NO: 1 (FIGS. 2 and 3). EXAMPLES To address the issue of solvent-induced polymer Swelling, control films of 3 were prepared by soaking in 0.1 mM DAN Example 1 25 SYL-cys solution for 24 hrs. A calibration curve of fluores cence intensity versus concentration was measured for DAN Preparation of RGD-Modified Nylon Substrate SYL-Cys solutions from 0.16 to 21 uMat pH 7.5 and pH 12. Surface complex DANSYL content of 7b and 9b was quan Activated nylon Surface 4 was prepared by first casting tified by immersion in water at pH 12 for 3 hours, which films of 3 (Nylon 6/6; (R-(CH2)CO; R'=(CH)NH) from 30 cleaves the Zr complexes from the surface, precipitates ZrO2. 0.1 mM formic acid solution on glass microscope slides that and releases fluorophore from 7b and 9b into solution. The were rinsed copiously in Millipore R water, and evacuated at amount of DANSYL surface-bound through Zr complexes 7b 10° torr for 3 hours. The coated slides were then placed in a and 9b was measured to be 0.10 nmol/cm and 0.18 nmol/ deposition chamber that was equipped with two stopcocks for cm, respectively. These amounts are consistent with the exposure either to vacuum or to vapor of Zirconium tetra(tert 35 DANSYL:Zr Stoichiometries of 1:1 and 2:1 indicated for 7b butoxide). The chamber was evacuated to 10 torr for 30 and 9b, respectively (FIGS. 2 and 3). Notably, they are at least minutes, and slides of 3 were exposed to vapor of Zirconium an order of magnitude higher than the previously highest tetra(tert-butoxide) (with external evacuation) for 30 seconds followed by 5 minexposure without external evacuation. This reported value of about 10 pmol/cm for polymer surface cycle was repeated twice, then followed by an additional 10 40 derivatization with a peptide. A substantial change in Surface minutes of exposure without external evacuation. The cham hydrophilicity was confirmed by a decrease in water contact ber was then evacuated for 16 hours at 10 torr to ensure angle (75° for 3 compared to 50° for 9a). removal of excess Zirconium tetra(tert-butoxide). The IR The approximate spatial Surface coverage by RGD was spectrum of polymer Surface-bound Zr complex (4) showed calculated from its measured surface loading of 0.2 nmol/ v. 2976 cm, indicative of tert-butoxide groups. 45 cm; assuming an RGD “footprint” of 40 A (determined RGD-modified nylon 7a was prepared by immersing a using Chem3D). This corresponds to coverage of about 0.4 4-coated slide in a 0.1 mM solution of phosphonoundecanol cm per cm of surface, or 40%. Without being bound by (5) in dry THF for 15 minto yield complex 6. Treatment of 6 theory, it is hypothesized that the percent Surface coverage by in a 0.1 mM solution of 3-maleimidopropionic acid N-hy the organic ligand depends upon the relative sizes of the droxysuccinimide ester for 24hrs under dry N, was followed 50 organic ligand and the transition metal complex. by copious rinsing Successively in acetonitrile and Milli pore(R) water and drying in vacuo. As depicted in FIG.2, either Example 4 an RGDCSEQID NO: 1 or DANSYL-cys-tethered surface (7a or 7b) can be obtained via Michael addition, which results Hydrolytic Stability of Modified Nylon Substrates in a complex with a 1:1 ratio of Zrto RGDCSEQID NO: 1 55 or DANSYL-Cys. IR analysis of 6 showed peaks in the ali Nylon films (2 cm) derivatized as 7b and 9b and control phatic region (voirs, 2922 cm; (vcrt2..., 2851 cm) films of 3 were immersed in water at pH 7.5 for 7 days at room characteristic of disordered alkyl chains. Immersion of 6 in a temperature, and the Supernatants were analyzed by fluores 0.1 mMaqueous solution of RGDCSEQID NO: 1) at pH 6.5 cence spectroscopy. The samples were then removed from for 24 hours produced 7a. 60 solution, dried, and immersed in water at pH 12 for 3 hrs, after which the Supernatants were again analyzed by fluorescence Example 2 spectroscopy. Release of DANSYL groups was measured by fluorescence intensities of supernatants from treated 7b and Preparation of RGD-Modified Nylon Substrate 9b which were compared to the control sample (3) over this 65 seven-day period. Unreacted DANSYLating reagent des RGD-derivatized surface 9a (FIG. 3) was prepared by orbed from the Nylon in about 3 hours. No release of surface immersing a 4-coated slide in a 0.1 mM solution of 3-male bound DANSYL material occurred over the next seven days. US 9,056,154 B2 31 32 Thus Zirconium-amide Surface-bound complexes are stable model for polyurethanes, Succinimide as a model for poly to hydrolysis under these conditions. imides, and N-hexyl p-toluene sulfonamide as a model for polysulfonamides. The reaction progress was observed via Example 5 "H NMR and reaction completion was calculated from the reduction in relative integration of the N—H NMR peak in Cell Response to Surface Modification each molecule. All reactions were assumed to proceed via Cell responses to surfaces 3 and 9a were evaluated in vitro. Zirconium complex coordination to "carbonyl oxygen, NIH 3T3 cells were maintained in Dulbecco's Modified N—H proton transfer to ligand, and loss of ligand. Eagle's Medium (DMEM) with 10% calf serum. Cells were The succinimide and N-hexyl p-toluene sulfonamide washed with Phosphate-Buffered Saline (PBS) and released 10 yields were about 90% after 30 minutes. The methyl N-hexyl from tissue culture dishes using 50 mg/mL trypsin and 2 carbamate yield was about 40% after three hours using a mg/mL EDTA in PBS. Trypsinization was stopped by wash Stoichiometric excess of zirconium alkoxide. This is evidence ing cells in Soybean trypsin inhibitor (Sigma). Cells were of the ability of transition metal alkoxides or amides to sur resuspended in serum-free DMEM at 5x10" cells/mL. Two face-modify the polymers. milliliters of the cell suspension were added to wells contain 15 ing the Nylon coated surfaces, which had been pre-blocked Example 10 for 1 hr in 1% Bovine Serum Albumin After 90 minutes, non-adherent cells were removed and replaced with fresh, Polyurea Modification serum-free DMEM. Cells were fixed, permeabilized, and stained for the focal adhesion protein Vinculin at the indicated Examples 7-9 were repeated using dicyclohexylurea as a time points. model for polyurea and tetrakis(diethylamino)Zirconium Fibroblast cell spreading on (a) nylon 3 control at 3 hrs; (b) (IV) as the transition metal alkoxide. The yield was about RGD-modified surface 9a at 3 hrs; and (c) RGD-modified 90% after 30 minutes. This is indicative of the ability of surface 9a at 6hrs is depicted in FIGS. 4a-4c. Staining shows transition metal alkoxides or dialkylamides to surface Vinculin, and scale bars are 50 microns. Compared to control 25 modify this polymer. untreated nylon (3), significant numbers of cells attached to the RGDCSEQID NO: 1-modified surface (9a). Cells were Example 11 well spread on the RGD-modified surface showing many focal adhesions when stained with anti-Vinculin antibodies, Surface Reaction of Collagen with Zirconium while the few cells on untreated nylon remained round. 30 Tetra(tert-butoxide) Surface-bound Zr-amide complexes, which are readily synthesized on the surface of 20 Nylon 6/6, are thus effective Films of type 1 collagen (from bovine achilles tendon, for activation of that surface for further organic chemical Aldrich) were cast from a 0.1M solution in formic acid on transformation. glass microscope slides that were Sonicated in ethanol, rinsed 35 copiously in Millipore water, and evacuated at 10' torr for 3 Example 6 h. The coated slides were then soaked for 24 hours in Milli pore water, and baked at 110° C. for 16 hours to dehydrate Modification of Other Polymer Classes—Polyamide them. These collagen slides were placed in a deposition Control chamber equipped with two stopcocks for exposure either to 40 vacuum or to vapor of zirconium tetra(tert-butoxide). The The ability to modify polyurethanes, polyimides and chamber was evacuated to 10 torr for 30 min, and slides of polysulfonamides with a transition metal alkoxide with Zir collagen were exposed to vapor of Zr tetra(tert-butoxide) conium tetra(tert-butoxide) was evaluated on a small mol (with external evacuation) for 30 sec followed by 5 min ecule model from which the ability to surface-modify the exposure without external evacuation. This cycle was corresponding polymer can be readily predicted. As a control, 45 repeated twice and was then followed by an additional 30 min the small molecule model was first tested for the polyamides exposure without external evacuation. The chamber was then of Examples 1-5 using N-hexylacetamide as the model. evacuated at 10 torr for 1 h to ensure removal of excess of Zr complex 2 (FIG. 1) was prepared by treating N-hexy Zirconium tetra(tert-butoxide), and to give Zr-activated col lacetamide (0.15g, 1.0 mmol) with Zr tetra(tert-butoxide) 1 lagen. (0.40 g, 1.0 mmol) in dry CHCl for 1 hour under nitrogen. 50 Solvent and reaction byproducts were removed in vacuo to Example 12 yield Zr complex 2 in >95% yield as shown by "H NMR (ICDC13: D 0.8 (t, 3H); 1.3 (m, 35H); 1.9 (s, 3H); 3.2 1,12-Diphosphonododecane-Modified Collagen (quartet, 2H)). The reaction proceeded via Zr coordination to the amide carbonyl, followed by N H proton transfer to a 55 Collagen was derivatized with 1,12-diphosphonododecane tert-butoxide ligand, and loss of tert-butanol. The bonding for by immersing a Zr-activated collagen slide in a 0.1 mM solu the amide moiety to Zrin 2 (m°-coordination) is indicated by tion of 1,12-diphosphonododecane in dry THF for 1 h. The the 8 ppm downfield shift of the acylcarbon vs. the free amide surface was rinsed copiously in dry THF to produce 1,12 (C NMR (CDC13: & 170.1 for N-hexyl-acetamide: & 178.1 diphosphonododecane-derivatized collagen. for 2). 60 Example 13 Examples 7-9 11-hydroxyundecylphosphonate-Modified Collagen Modification of Other Polymer Classes 65 Collagen was derivatized with 11-hydroxyundecylphos The N-hexylacetamide example was repeated in tetrahy phonate by immersing a Zr-activated collagen slide in a 0.1 drofuran (THF) at 20 Cusing methyl N-hexylcarbamate as a mM solution of 11-hydroxyundecylphosphonate in dry THF US 9,056,154 B2 33 34 for 1 h. This surface was rinsed copiously in dry THF to to modified and control slides. Collagen coated slides were produce 11-hydroxyundecylphosphonate-derivatized col modified with 1,12-diphosphonododecane, 11-hydroxyunde lagen. cylphosphonate and RGD according to the procedures in Examples 12-14. Bare glass slides and unmodified collagen Example 14 coated glass slides were used as negative controls. Glass slides coated with the cell binding protein fibronectin were RGD-Modified Collagen used as positive controls. Gasket sealed tissue culture cham RGD-derivatized collagen was prepared by immersing a bers were affixed to each slide type and adhesion of human osteoblasts was assayed after 2 and 24 hour incubation peri Zr-activated collagen slide in a 0.1 mM solution of 3-male 10 imidopropionic acid in dry acetonitrile for 1 h to derivatize the ods using a Vybrant Cell Adhesion Assay Kit (invitrogen-V- collagen Surface with maleimido groups. Immersion of this 13181). Representative data is shown in the table below. maleimido-derivatized collagen in a 0.1 mM aqueous solu tion of RGDC SEQ ID NO: 1) at pH 6.5 for 24 h gave Slide Type Percent Adhesion RGD-derivatized collagen. 15 Glass slide (-control) 23.8% Example 15 Collagen coated glass slide (-control) 41.1% Collagen + 11-hydroxyundecylphosphonate 60.9% Collagen + 1,12-diphosphonododecane 82.9% Cell Response to Collagen Surface Modification Collagen + RGD 90.8% Fibronectin coated slide (+control) 94.9% Cellular response to collagen coated glass slides modified with various compounds was assessed by assaying adhesion

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 7

<21 Oc SEO ID NO 1 <211 LENGTH: 4 <212> TYPE PRT <213> ORGANISM: Artificial Sequence <22 Os FEATURE; <223> OTHER INFORMATION: integrin binding sequence

<4 OOs SEQUENCE: 1 Arg Gly Asp Cys 1.

<21 Os SEQ ID NO 2 &211s LENGTH: 7 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: anti-staphylococcal agent 22 Os. FEATURE: <221s NAMEAKEY: misc feature <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs SEQUENCE: 2 Tyr Ser Pro Xaa Thr Asn Phe 1. 5

SEO ID NO 3 LENGTH: 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: anti-staphylococcal agent

<4 OOs SEQUENCE: 3 Tyr Ser Pro Trp Thr Asn Phe 1. 5

<21 Os SEQ ID NO 4 &211s LENGTH: 7 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence US 9,056,154 B2 35 36 - Continued

22 Os. FEATURE: 223 OTHER INFORMATION: anti-staphylococcal agent 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (7) . . (7) 223 OTHER INFORMATION: -NH2

<4 OOs, SEQUENCE: 4 Tyr Ser Pro Trp Thr Asn Phe 1. 5

<210s, SEQ ID NO 5 &211s LENGTH: 504 212. TYPE : DNA <213> ORGANISM: Staphylococcus aureus

<4 OOs, SEQUENCE: 5 atgaagaaac tatata catc. titatggcact tatggattitt tacat cagat aaaaatcaat 6 O aaccc.gaccc at Calactatt CCaattitt Ca gcatcagata citt cagttat ttittgaagaa 12 O actgatggtg agactgttitt aaaat Cacct t caatatatg aagtt attaa agalaattggit 18O gaatticagtg aaCatCattit ctattgttgca a tott cattic catcaiacaga agat catgca 24 O tat caacttg aaaagaaact gattagtgta gacgataatt t cagaaactt tggtggctitt 3OO aaaagctato gtttgttaag acctgctaaa ggtacaiacat acaaaattta titt cqgattit 360 gctgat cac atgcatacga agactittaag caatctgatg c ctittaatga c catttitt ca. aaagacgcat taagt catta ctittggttca agcggacaac attcaagtta ttittgaaaga tatectatacc caataaaaga atag 504

<210s, SEQ ID NO 6 &211s LENGTH: 834 212. TYPE : DNA <213> ORGANISM: Staphylococcus aureus

<4 OOs, SEQUENCE: 6 atggct atta aaaagtataa gcc-aataa.ca aatggtcgtc gtaatatgac titcgittagat 6 O titcgcagaaa t cacgaaaac tacacctgaa aagt cattat taaaaccogct accaaaaaa 12 O

acalaccalagg taaattgact gtaagacacc atggtggtgg acacaaacgt. 18O caat accqtg ttatcgattit caaacgtaac aaagatggta t caatgcaaa agttgattct 24 O attcaatatg atccaaac cq cticagdaaac atcgctittag ttgtatatgc agacggtgaa 3OO aaacgatata t cattgct co taaaggatta gaagtaggto aaatcgttga aagtggtgct 360 gaagctgaca tcaaagttgg taacgcatta CCattacaaa. acatt coagt tgg tacagta gtacacaa.ca tcq agcttaa acctggtaaa ggtggacaaa tcqct cqttic agctggtgca agtgct Caag tacttggtaa agalaggtaaa tacgt attaa t cagattaag atctggtgaa 54 O gttcgt atga tott at citac ttgcc.gtgct acaatcqgtc aagttggtaa CCtacaa.cac gaattagtta acgttggtaa agc.cggacgt. t caagatgga alaggt atcc.g tccaa.cagtt 660 cgtggttctg taatgaac cc taacgat cac ccacacggtg gtggtgaagg tcqtgct cot 72 O atcggtagac Catctocaat gtcac catgg ggtaalaccta cgcttggtaa gaaaact ct cgtggtaaaa aat catcaga caaactitatic gttcgtggac gtaagaaaaa atala 834

<210s, SEQ ID NO 7 &211s LENGTH: 46 212. TYPE : PRT <213> ORGANISM: Staphylococcus aureus US 9,056,154 B2 37 38 - Continued

22 Os. FEATURE: <221> NAME/KEY: mat peptide <222s. LOCATION: (25) ... (33) <223> OTHER INFORMATION: Agr autoinducing peptide

<4 OO > SEQUENCE: 7 Met Asn Thr Lieu. Tyr Lys Ser Phe Phe Asp Phe Ile Thr Gly Val Lieu. -2O - 15 -10 Lys Asn Ile Gly Asn Val Ala Ser Tyr Ser Thr Cys Tyr Phe Ile Met - 5 -1 1 5 Asp Glu Val Glu Ile Pro Llys Glu Lieu. Thr Glin Lieu. His Glu

What is claimed is: 1. A coated Substrate comprising (a) a Substrate comprising (R"), a polymer surface wherein the polymer is selected from the group consisting of polyamides, polyacrylamides, polyim 2O -(RO), -P(O)-(OR), ides, polyurethanes, polyureas, polyamines, polyepoxides, polyesters, polysaccharides, polyethers, polyketones, wherein X is 0 or 1, y is 1, Z is 1 or 2 and x-y--Z is 3: R and polysulfonamides, polysulfides and copolymers of two or R" are each independently radicals having a total of 1 to more thereof; (b) an organometallic coating bonded to said 25 30 carbons; R is H, a metal or lower alkyl having 1-4 polymer Surface comprising a polyalkoxide or a polydialky carbons, and, for at least a portion of the organophos lamide of transition metal atoms selected from the group phorus compounds in the overlayer, R' is H. consisting of atoms of Group 4. Group 5 and Group 6 of the 8. The coated substrate of claim 5, wherein said organo Periodic Chart; and (c) an organophosphorus, organocar phosphorus overlayer comprises an organophosphinic acid boxylic acid or organocarboxylic acid ester compound over 30 compound or mixture of compounds of the structure: layer coated on and covalently bonded to said organometallic layer. 2. The coated substrate of claim 1, wherein the organo (R"), group of the organophosphorus, organocarboxylic acid or organocarboxylic acid ester overlayer compound is a Satu 35 (R"), -P(O)-(OR), rated or unsaturated, Substituted or unsubstituted alkyl group. 3. The coated substrate of claim 2, wherein said alkyl group wherein X is 0, 1 or 2, y is 0, 1 or 2, Z is 1 and x+y+Z is 3: is Substituted in the omega position and the omega-Substitu Rand R" are each independently radicals having a total of 1 to 30 carbons; R is H, a metal or lower alkyland, for ents of the omega-Substituted organophosphorus, organocar 40 at least a portion of the organophosphorus compounds in boxylic acid or organocarboxylic acid ester overlayer com the overlayer, R' is H. pounds are selected from the group consisting of carboxylate, 9. The coated substrate of claim 1, wherein said overlayer carbamate, hydroxyl, keto, ether, oxy, carbonate, amino, comprises an organophosphorus or organocarboxylic acid amide and thiol. compound with an organo group containing a C to C1s 4. The coated Substrate of claim 2, further comprising a 45 hydrocarbon or Substituted hydrocarbon group. second overlayer of a biologically or pharmaceutically active 10. The coated substrate of claim 1, wherein the substrate is compound covalently bonded to said organophosphorus, a molded polymer article. organocarboxylic acid or organocarboxylic acid ester com 11. The coated substrate of claim 1, wherein the polymer is pound overlayer. in the form of a coating. 5. The coated substrate of claim 1, wherein said overlayer 50 12. The coated substrate of claim 11, wherein the polymer comprises an organophosphorus compound selected from the is a coating on an article made from another material selected group consisting of phosphoric acids, phosphonic acids and from the group consisting of glass, silicon dioxide, metal, and phosphinic acids. another polymer. 6. The coated substrate of claim 5, wherein said organo 13. The coated substrate of claim 1, wherein said overlayer phosphorus overlayer comprises an organophosphoric acid 55 comprises a biologically or pharmaceutically active com compound or mixture of compounds of the structure: pound covalently bonded to the organometallic coating layer. 14. The coated substrate of claim 1, wherein said overlayer comprises a biologically active ligand compound selected (RO), P(O)—(OR), from cell attachment mediators or a substance selected from wherein X is 1 or 2, y is 1 or 2 and X-y=3; R is a radical 60 the group consisting of osteoinductive Substances and Sub having a total of 1-30 carbons; where R' is H, a metal or stances that induce cellular growth, proliferation, and/or dif lower alkyl having 1-4 carbons; and, for at least a portion ferentiation. of the organo-phosphorus compounds in the overlayer, 15. The coated substrate of claim 14, wherein said biologi R" is H. cally active ligand compound is selected from the group con 7. The coated substrate of claim 5, wherein said organo 65 sisting of osteoinductive Substances and Substances that phosphorus overlayer comprises an organophosphonic acid induce cellular growth and proliferation and integrin cell compound or mixture of compounds of the structure: attachment mediators. US 9,056,154 B2 39 40 16. The coated substrate of claim 15, wherein said biologi 22. The polymer scaffold of claim 21, wherein said scaf cally active ligand compound is selected from the group con fold is adapted for the re-generation of nervous, musculo sisting of bone morphogenic proteins (BMP), epidermal skeletal, cartilaginous, tendenous, hepatic, pancreatic, ocular, growth factor (EGF), fibroblast growth factor (FGF), platelet integumentary, arterio-venous or urinary tissues or tissues derived growth factor (PDGF), insulin-like growth factor forming solid or hollow organs. (IGF-I and II), TGF-B and vascular endothelial growth factor 23. The polymer scaffold of claim 21, containing cells (VEGF). Selected from the group consisting of cells of the muscular 17. The coated substrate of claim 14, wherein said osteoin and skeletal systems, parenchymal cells, cells of intestinal ductive substances comprise bone morphogenic proteins origin, exocrine cells, bile duct cells, parathyroid cells, thy (BMP), and said substances that induce cellular growth com 10 prise epidermal growth factor (EGF), fibroblast growth factor roid cells, cells of the adrenal-hypothalmic-pituitary axis, (FGF), platelet-derived growth factor (PDGF), insulin-like heart muscle cells, kidney epithelial cells, kidney tubular growth factor (IGF-I and II). TGF-B, and vascular endothelial cells, kidney basement membrane cells, nerve cells, blood growth factor (VEGF). Vessel cells, cells forming bone and cartilage, smooth muscle 18. The coated substrate of claim 1, wherein said overlayer 15 cells, skeletal muscle cells, ocular cells, integumentary cells, comprises a pharmaceutically active compound selected keratinocytes, skin cells and endothelial cells. from the group consisting of anti-neoplastic and anti-prolif 24. The polymer scaffold of claim 21, wherein said cells erative agents. are selected from the group consisting of chondrocytes, fibro 19. The coated substrate of claim 1, wherein said overlayer blasts, osteocytes, osteoblasts, hepatocytes and pancreatic comprises a active agent selected from the group consisting of cells. acyclovir, cephradine, malphalen, tamoxifen, raloxifene, 25. The polymer scaffold of claim 21, containing cells daunomycin, adriamycin, plumbagin, chlorambucil, ephe Selected from the group consisting of cells obtained from drine, atropine, quinine, digoxin, quinidine, biologically donors, embryonic stem cells, non-embryonic stem cells, active peptides, chlorine, cephalothin, proline, proline ana cells from established cell culture lines, cells before genetic logues, penicillin V, aspirin, ibuprofen, steroids and nicotinic 25 engineering and cells after genetic engineering. acid. 26. An implantable medical device characterized by one or more surfaces comprising the coated substrate of claim 1. 20. A polymer scaffold for tissue engineering comprising 27. A method of regulating cellular attachment, migration the coated substrate of claim 1. and proliferation on a polymeric substrate, characterized by 21. A polymer scaffold for tissue engineering comprising contacting living cells, tissues or biological fluids containing the coated substrate of claim 1, wherein said overlayer com 30 prises a biologically active ligand for cellular or tissue living cells with the polymer scaffold of claim 21. ingrowth.