MXPA06009785A - Polymeric compositions and related methods of use - Google Patents

Abstract

Adhesive polymeric compositions which can comprise dihydroxyphenyl moieties and derivatives thereof, and related methods of use.

Description

IM ERIC COMPOSITIONS AND RELATED USE METHODS FIELD OF THE INVENTION The mussel adhesive proteins (MAPs), are remarkable underwater adhesive materials, which form tenacious bonds between mussels and the surfaces on which the mussels reside. During the process of joining to the surfaces, the MAPs are secreted as fluids that are subjected to crosslinking or hardening reaction, which leads to the formation of a solid plate. One of the unique characteristics of MAPs is the presence of L-3, 4-dihydroxyphenylalanine (DOPA), an unusual amino acid, which is believed to be at least partially responsible for adhesion to substrates through mechanisms that are not completely understood. Mussels adhere to a variety of surfaces, including metal, metal oxide, polymers, plastics and wood.

BACKGROUND OF THE INVENTION Control of protein and cell adhesion on surfaces is critical to the performance of biosensors, medical diagnostic products, any instrumentation and used assays that require handling of serum and other human / animal fluids, tissue engineering design , localized in vivo drug delivery, implanted medical devices, surgical incision healing, tissue adhesions such as bone and cartilage for healing, and nanotechnology (therapies based on nanoparticles and diagnostic tools). In many industrial applications, control of cell adhesion and protein to surfaces is also important. Such applications include prevention of the adhesion of mussels to boats and boats, manholes and other structures used in oceans and fresh waters, prevention of bacterial growth and algae in aquatic lines used for industrial and drinking water, and sensors used to measure quantity and quality of the water. In medical sand, the physical or chemical immobilization of poly (alkylene oxides) (PAO), such as polyethylene glycol (PEG), polyethylene oxide (PEO), and PEO-PPO-PEO block copolymers, such as those available under the brand name PLURONICS, and polymers such as PEG / tetraglime, poly (methoxyethyl methacrylate) (PMEMA), and poly (methacryloyl phosphatidylcholine) (PoliMPC) (E. Merril, Ann. NY Acad. Sci., 516, 196 ( 1987), Otsuni et al., Langmuir 2001, 17, 5605-20, which are incorporated herein by reference), on surfaces, have employed as a strategy, to limit the adsorption of proteins and cells on surfaces. The methods currently used to modify surfaces with polymers must be adjusted for each type of material, and therefore require different chemical strategies. For example, noble metal surfaces, such as platinum, silver and gold, can be modified using thiol-containing molecules (-SH), while metal oxides are often modified using silane coupling chemistry. There is no surface modification strategy that can be universally applied to different kinds of materials. However, many of the current methods rely on expensive instrumentation, complex synthetic procedures, or both.

SUMMARY OF THE INVENTION The present invention are compositions which function, for example, as an adhesive, in a substantially aqueous environment. Preferred compositions, generally, comprise an adhesive portion and a polymer portion, the polymer portion having a desired surface active effect (or other desired characteristics). In one aspect, the adhesive portion of a composition of this invention comprises dihydroxyphenyl derivatives including, di (DHPD), wherein the second DHPD is that is, a methylene derivative of dihydroxyphenyl. In yet a further aspect, the polymer portion comprises poly (alkylene oxide). In a highly preferred practice, the adhesive portion comprises DHPD, for example, DOPA (discussed herein), and the polymer portion comprises PEO-PPO-PEO block copolymers (also discussed above). In a more preferred practice, the adhesive portion comprises DHPD including, a pendant chain comprising ethylenic or vinyl unsaturation such as, for example, an alkyl acrylate.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the 1H NMR spectrum of PLURONIC® F127, its carbonate intermediate (SC-PA07) and DME-PA07 in CDC13. Figure 2 provides differential scanning calorimetry thermograms of 30% by weight of DME-PA07, DOPA-PA07, and unmodified aqueous solutions of PLURONIC® F127. The arrows indicate the location of the gelation endotherm. Figure 3 traces cut storage modules, G 'of an aqueous solution of DME-PA07 at 22% by weight as a function of temperature at 0.1 Hz and a strain of 0.45%. Shown in the insertion is the rheological profile of a 22% unmodified aqueous solution of PLURONIC® F127, as a function of temperature. Figure 4 traces cut storage modules, G 'of a 50% aqueous solution of DME-PA08 as a function of temperature at 0.1 Hz and a strain of 0.45%. Shown in the insertion is the rheological profile of a 50% unmodified aqueous solution of PLURONIC® F68, as a function of temperature. Figure 5 traces storage modules of aqueous solutions of DME-PA08 at 45% by weight and 50% by weight, respectively, as a function of temperature at 0.1 Hz and a strain of 0.45%. Figures 6A and 6B show differential scanning calorimetry thermograms of (A) DOPA-PA07 and (B) DME-PA07 at different concentrations after heating. The arrows indicate the location of the gelation endotherm observed only at high polymer concentrations. Figures 7A-C show peaks C (ls) XPS of high resolution for (A) unmodified Au, (B) m-PEG-OH, and (C) m-PEG-DOPA. A dramatic increase in the ether peak at 286.5eV in (C) indicated in the presence of PEG. Figures 8A-C provide positive TOF-SIMS spectra showing peaks representing gold catechol link. The spectrum was normalized to the Au peak (m / z ~ 197). Figure 9 provides TOF-SIMS spectrum showing the secondary positive ion peak at masses m / z ~ 43 for unmodified Au substrate, Au exposed to mPEG-OH, mPEG-DOPA powder, and Au exposed to mPEG-DOPA. Figure 10 shows TOF-SIMS spectrum showing positive secondary ion peaks for Au substrate chemosorbed with mPEG-DOPA. The catecholic link of gold is observed at m / z ~ 225 (AuOC), 254 (AuOCCO), and 309. Less intense peaks are observed AuOaCb at m / z ~ 434, 450, 462, and 478. The periodic triplets seen at m / z that vary from 530-1150 correspond to Au bound to DOPA- (CH2CH20) n, where each subpex is separated by 14 or 16 amu, representing CH2, CH2CH2 and CH2CH20 in the PEG chain. This pattern was observed for n = 1-15. Figure 11 shows the SPR spectrum of protein adsorption (0.1 mg / ml BSA) on modified and unmodified gold surfaces. The modified surfaces of mPEG-DOPA and mPEG-MAPd exhibit adsorption of reduced proteins, compared with gold ingots and modified surfaces of mPEG-OH. Figure 12 shows the dependence of mPEG-DOPA concentration on anti-introgression behavior. Gold surfaces were modified for 24 hours at the indicated mPEG-DOPA concentrations, followed by analysis of the density and area of the adhered cells. (* 0 p < 0.05, ** = p < 0.001; black ingots = total proy area, gray ingots = cell surface density). Figure 13 compares cell attachment and spreading on gold ingots, gold treated with mPEG-OH, and gold modified with mPEG-DOPA 5 K, mPEG-MAPd 2K, and mPEG-MAPd 5K, under optimal conditions (50 mg / ml for 24 hours). (black ingots = total proy area, gray ingots = cell surface density, *** = p <0.001). Figures 14A-C are a series of SEM micrographs indicating the morphology of NIH 3T3 fibroblasts in (a) unmodified Au, (B) Au treated with mPEG-OH, and (C) Au modified with mPEG-DOPA. All treatments were at 50 mg / ml in DCM for 24 hours. Figure 15 shows the UV / vis absorption spectrum of magnetite nanoparticles stabilized with mPEG-DOPA, suspended in various aqueous solutions of NaCl at the concentrations as shown and plotted in this document. The addition of NaCl does not induce nanoparticle precipitation. Figure 16 shows that the addition of salt to untreated Au nanoparticles induces aggregation. UV / vis scans of 10 mm of untreated Au nanoparticles, suspended in aqueous NaCl solutions (concentrations as shown and plotted in this document) are shown. The attenuation and change of the absorption band of 520 nm with increased concentrations of NaCl reflects the aggregation of the nanoparticles. Figure 17 illustrates that the addition of salt to Au nanoparticles stabilized with mPEG-DOPA does not induce aggregation. UV / vis scans of 10 mm of Au nanoparticles stabilized with mPEG-DOPA, suspended in aqueous NaCl solutions (concentrations as shown and plotted herein) are shown. The lack of attenuation and change of the absorption band of 520 nm with increased NaCl concentrations reflects the effective stabilization of the nanoparticles. Figure 18 plots the UV / vis absorption spectrum of CdS nanoparticles stabilized with mPEG-DOPA, suspended in aqueous NaCl solutions (concentrations as shown and plotted in this document).

Figure 19 plots XPS recognition scans of unmodified Ti02 and Ti02 treated with mPEG-DOPAi-s. Figure 20 traces the long term resistance to cell adhesion in Ti02 and Ti02 modified with mPEG-DOPA? _3. The duration of the non-fouling response is proportional to the length of the anchor group of the DOPA peptide. Adherent cells were visualized with calcium AM. Figure 21 plots the high resolution XPS scans of the Cls region of Ti0 substrates modified with mPEG-DOPAl-3. Of interest, it is the increase in the carbon ether peak (286. OeV) with increases in the length of the DOPA peptide anchor. Figure 22 plots the high resolution XPS scans of the Oís region of Ti02 substrates modified with mPEG-DOPAl-3. The peak at 532.9eV represents increases in polymeric oxygen, while the peak Ti-O-H (531.7eV) decreases with increases in the length of the DOPA peptide. Figure 23 plots the results of the Robust Design experiment in 316L stainless steel. Figure 24 traces the cell attachment for 4 hours, on a variety of surfaces modified by mPEG-DOPA? -3, using a modification of 24 hours at 50 ° C at the indicated pH.

Figure 25 traces the% gel conversion against the UV exposure time in minutes. Figure 26 plots the mole fraction of DOPA incorporated against the mole% of 1 or 7 in the precursor solution. Figure 27 plots% gel conversion against% mol 1 or in the precursor solution. Figure 28 is XPS analysis of X-ray Photoelectron Spectroscopy of a silicon nitride surface. Figure 29 is a free monitoring of functionalized silicon nitride overhangs. Figure 30 is an entropic elasticity analysis of poly (ethylene glycol). Figure 31 is a measure of resistance of the modified side chain DOPA. Figure 32 is a proposed model of the DOPA-T? 02 binding mechanism. Figure 33 is an array of atomic force microscopes. Figure 34 is data with respect to force measurement. Figure 35 is an adhesion data. Figure 36 is a synthetic route and data analysis.

DETAILED DESCRIPTION OF THE INVENTION Even more specifically, this invention comprises a dihydroxyphenyl adhesive compound (DHPD) of formula (I), wherein Ri and R2 may be the same or different and are independently selected from the group consisting of hydrogen, C? -4 hidrocar substituted and unsubstituted, branched and unbranched, saturated and unsaturated hydrocarbon; P is separately and independently selected from the group consisting of -NH2, -COOH, -OH, -SH, wherein Rx and R2 are defined above, a single bond, halogen, wherein Ai and A2 are separately and independently selected from the group consisting of H, a single bond; a protecting group, substantially poly (alkylene oxide), D OR where n varies between 1 and approximately 3 and A3 is R is H, lower alkyl C? ~ 6, or poly (alkylene oxide) O R3 R3 is as defined above, and D is indicated in Formula (I). In one aspect, poly (alkylene oxide) has the structure wherein R3 and R4 are independently and independently H, or CH3 and m have a value in the range between 1 and about 250, A4 is NH2, COOH, -OH-, -SH, -H or a protecting group. In a very preferred way, DHPD is Ri R2 and P are defined as above. In a preferred additional form, DHPD is of the structure wherein A2 is -OH and Ai is substantially poly (alkylene oxide) of the structure R3, R and m are defined as in claim 2. Generally speaking, poly (alkylene oxide) is a block copolymer of ethylene oxide and propylene oxide. A method of this invention involves adhering substrates to each other, comprising the steps of providing DHPD of the structure: wherein Ri and R2 are defined as above; applying the DHPD of the previous structure to one or the other or both structures to be adhered; contacting the substrates to be adhered with the DHPD of the previous structure between them, to adhere the substrates to each other, and optionally reposition the relative substrates among themselves, separating the substrates and putting them in contact again with each other with the DHPD of the structure previous among these. In a preferred method, Ra and R2 are hydrogen.

Definition Dihydroxyphenyl derivatives (DHPD) for purposes of this application, must mean dihydroxyphenyl derivatives of the following structure: wherein P, Rx and R2 are defined later, and n varies between 1 and about 5. In a practice, Ri and R2 are hydrogen and P is, the same, dihydroxyphenyl. A DHPD in a practice of the present invention, is 1-3,4, dihydroxyphenylalanine (DOPA), (generically), wherein Ai A2 are as defined above. "Substantially poly (alkylene oxide)", as used herein, must mean predominantly or mainly, alkyl oxide or alkyl ether in composition. This definition contemplates the presence of heteroatoms, for example, N, O, S, P, etc., and of functional groups, for example, -COOH, -NH2, -SH, as well as ethylenic or vinyl unsaturation. It is understood that any such alkylene oxide-free structures will only be present in such relative abundance, as they are not materially reduced, for example, the characteristics of total surfactants, without toxicity, or immune responses, as appropriate, or of this polymer. These adhesives derived from dihydroxyphenyl ("DHPD"), work in an aqueous environment. To form the polymer composition, a DHPD portion, which generally provides adhesive functionality, is coupled to a polymer, which provides the desired active effect of the surface. These components will be described in more detail below. These adhesives and polymer compositions have many uses, including protein prevention and / or cell adhesion to a surface in various medical, industrial and consumer applications. DHPD adhesives can also be used as substitutes for sutures for a wound and as aids in the healing of bone fractures or cartilage-to-bone damage. These and other uses will be described in more detail below. The preferred polymeric compositions of the present invention have the following structure: wherein, for each compound of formula (Ia), Ri and R2 are separately and independently defined as above, Pi and P2 are separately and independently defined as P in formula (I); n and m independently vary from 0 to about 5, provided that at least one of n or m is at least 1; Adhesive Portion The adhesive portion of the present invention is a dihydroxyphenyl derivative ("DHPD") having the following preferred structure: wherein Ri, R2 and P are defined as above and t ranges from 1 to about 10, preferably about 1 to about 5 and more preferably 1 to about 3. The DHPD adhesive can be operated in an aqueous environment. In this context, an aqueous environment is any medium that comprises water. This includes, without limitation, water that includes water and fresh water, cellular and bacterial growth medium solutions, aqueous buffers, other water-based solutions, and body fluids. The DHPD portion can be derivatized. As will be understood by those skilled in the art, such derivatization is limited by the retention of the desired adhesive characteristics.

Polymer Component Various polymeric components provide an active surface effect and other desired characteristics will be well known to those skilled in the art, aware of this invention. The desired surface active effect refers to reduced particle agglomeration and anti-bioincrustation, including resistance to cell and / or protein adhesion. For example, the polymer component can be soluble in water, depending on the end-use application, and / or capable of micelle formation depending on several other end-use applications. Polymers useful in the present invention include, but are not limited to, polyethylene glycol (PEG), polyethylene oxide (PEO), polypropylene oxide (PPO), block copolymers of PEO-PPO-PEO, polyphenylene oxide, PEG / tetraglime, PMEMA, polyMPC and perfluorinated polyethers. The polymer compositions can be synthesized in various forms. For example, the polymer compositions can be synthesized through a general synthetic process for the activation of the polymeric end group. Various polymeric or monomeric components thereof can be activated using carbonate chemistry. In particular, a polymeric component activated with succinimidyl carbonate, which reacts with a portion of DHPD, can provide a stable urethane conjugate. Two of the many possible trajectories (a) and (b) in Reaction Scheme la and Ib below, show coupling with a poly (alkylene oxide) in either aqueous or non-aqueous solvents, without compromising the desired bioadhesion. For example, a DHPD residue can be coupled to a polymer component to provide the desired conjugate composition, through either urethane or amide bond formation. These synthetic counts are shown in Reaction Scheme la and Ib, which is discussed in more detail below.

DME-PA07 m = 100, n = 65 DME-PA08 m = 78, n = 30 REACTION SCHEME 1a D0PA-PA07 m = 100, n = 65 DOPA-PA08 m = 78, n = 30 REACTION SCHEME 1b More particularly, if it is coupled to the polymeric component via urethane linkage formation, a carboxylic acid group of the DHPD component can be esterified or derivatized with several other functional groups. Alternatively, the DHPD component can be coupled to a polymer component (eg, amidation or esterification depending on the final polymer group, -NH2 or -OH), providing a DHPD functionality, which can be derivatized by any of the numerous known protecting groups, including without limitation, Boc, Fmoc, borate, phosphate, and tributyldimethylsilyl. Protecting the N group from a DHPD component can leave the carboxylic acid group available for multi-functional derivatization and / or a higher density of the conjugated polymer components together with these. Accordingly, in part, the present invention is also a method for using urethane synthesis to incorporate a DHPD residue in a polymer system. Such a method includes (1) providing a polymer component terminating in a plurality of monomers, each having a functional end group; (2) preparing a carbonate derivative of the polymer component; and (3) preparing a urethane portion after the reaction of the carbonate derivative and at least one portion of DHPD. As described above, a polymeric component used in conjunction with this method, can include those having reactive terminal monomeric functionality, with a reagent that provides the desired carbonate derivative and, finally, provide a urethane portion coupled to the polymer components and DHPD. Various other coupling reagents and / or hydroxy-terminated polymer components can be used to provide the desired urethane portion. In part, the present invention is also a method for using a carbonate intermediate to maintain the cathcolic functionality of a composition and / or polymer system incorporated into DHPD, or to otherwise improve, the adhesion properties thereof. Such a method includes (1) providing a polymer component terminating in a plurality of monomers, each having a terminal functional group; (2) reacting the polymer component with a reagent, to provide a carbonate intermediate; and (3) reacting the carbonate intermediate with at least a portion of DHPD. Without limitation to any single theory or mode of operation, this inventive method can be considered as a way to improve the reactivity of the final group of the polymer component, via a suitable carbonate intermediate. The subsequent reaction on the amino nitrogen of the DHPD portion provides the corresponding conjugate, while maintaining cathcolic functionality. In accordance with this invention, as demonstrated in Reaction Scheme 1, various synthetic routes can be used to couple DHPD portions to such activated carbonate intermediates. DOPA methyl ester (DME), prepared by the reaction of DOPA with methanol in the presence of thionyl chloride, can be used in organic solvents. The progress of the reaction can be monitored by TLC and NMR, with the coupling reaction virtually complete in one hour (with representative conjugates DME-PA07 (from PAO PLURONIC® F127) and DME-PA08 (from PAO PLURONIC® F68)). The high yields of the product are obtained from the purification of cold methanol. The free DOPA carboxylic acid form can be coupled with the carbonate intermediate in alkaline aqueous solution. It is well known that, the main difficulty in working with DOPA is the ease of oxidation (to DOPA-quinone and other products), which easily occurs in aqueous alkaline solutions. To prevent unwanted oxidation of DOPA catechol side chains during coupling under alkaline conditions, a DOPA protected with borate, can be first formed by adding DOPA to an aqueous solution of borate (Reaction Scheme Ib). The resulting complex is remarkably stable in neutral or alkaline solutions, and can be easily deprotected under acidic conditions. Taking advantage of the complex formation between DOPA and borate, DOPA was coupled to the ends of several commercially available PAOs under alkaline aqueous conditions to provide D0PA-PA07 and D0PA-PA08. Visual inspection of the reaction solution revealed the absence of strongly absorbing DOPA-quinone, an indication that DOPA remains un-oxidized during the reaction. At the completion of the reaction, acidification with HCl resulted in deprotection of the DOPA end groups of the block copolymer. Both the 1 H NMR spectrum and the colorimetric assay confirmed the compositions of the succinimidyl activated reaction intermediates and four DOPA-modified PAOs of Reaction Scheme 1. The spectrum of XH NMR of PAO PLURONIC is shown in Figure 1. ® F127, the intermediate activated with succinimidyl carbonate (SC-PA07), and the PAO modified with corresponding DOPA methyl ester (using PLURONIC® F 127, DME-PA07).

The sharp peaks at ~ 2.8 ppm due to CH protons from the succinimidyl carbonate group and ~ 4.4 ppm due to the -CH20 protons of only the ethylene oxide group adjacent to the activated PAO carbonate group, completely disappear from the spectrum of '' "PAH H NMR containing DOPA, while a series of new peaks appear due to the introduction of DOPA portions in the copolymers.A characterizing factor of the 1H NMR spectrum of the PAO containing DOPA, is the appearance of a singlet and two doublets in the range of 6.5-6.9 ppm, which correspond to the three protons in the phenyl ring of DOPA.Similar characteristics were also observed in the XH NMR spectrum (not shown) of the DOPA-PAO conjugate synthesized from the aqueous solution. Based on the presumption of two groups of succinimidyl carbonate available in the corresponding carbonate intermediates, SC-PA07 and SC-PA08, coupling efficiencies of DO methyl ester were found PA and DOPA to these two PAOs, for being in the range from 76% to 81% as obtained from colorimetric analysis (Table 1). The coupling efficiencies reported are the average values of at least three repeated syntheses performed under the same conditions and were not found to significantly increase when a greater excess of DOPA was used in the reaction. Similar coupling efficiencies were also found for DOPA-PA07 and DOPA-PA08, made from aqueous solutions, suggesting that the hydrolysis of PAO activated with succinimidyl carbonate is slow in the aqueous alkaline solution containing Na2B407. Contrary to the coupling efficiencies, the product yields (shown in Table 1) of PAOS modified with selected DOPA, synthesized in the aqueous solution, were found to be lower than those synthesized in organic solvent. This may be due to the surfactant properties of the starting PAO material, which causes the low efficiency of extraction of PAO modified with DOPA with water dichloromethane. It should be noted that the free carboxylic acid in DOPA-PA07 and DOPA-PA08 can be further functionalized using standard peptide chemistry to adjust the properties of the block copolymers. The four PAOS modified with DOPA of Table 1, could be stored at -20 ° C indefinitely, without discoloration or change in properties.

Table 1 Coupling efficiency and product performance of PLURONIC® modified with DOPA Coupling Performance Efficiency (%) * Product (%) DME-PA07 78.0 + 4.0 7.5 + 5.0 DOPA-PA07 80.0 + 4.0 52.0 + 3.0 DME-PA08 76.0 + 2.0 76.0 + 4.0 DOPA-PA08 81.0 + 2.0 4.9 + 2.0 * Determined by colorimetric analysis as shown by Waite and Benedict (Waite, JH &Benedict, Assay of dihydroxyphenylalanine (dopa) in invertebrate structural proteins, Methods in Enzymology 107, 397-413 (1874), which is incorporated in this document by reference). The control of cell adhesion and protein on surfaces is critical to the performance of biosensors, medical diagnostic products, any instrumentation and used assays that require handling of serum and other human / animal fluids, tissue engineering design, localized delivery of In vivo drug, implanted medical devices, surgical incision healing, adhesions of tissues such as bone and cartilage for healing, and nanotechnology (nanoparticle-based therapies and diagnostic tools). In many industrial applications, the control of cell adhesion and protein to surfaces is also important. Such applications include, without limitation, prevention of the attachment of mussels to boats and boats, manholes and other structures used in oceans and fresh waters, prevention of bacterial growth and algae in aquatic lines used for industrial and drinking water, and sensors used to measure the quantity and quality of water. The polymer compositions of the present invention, can be used as coatings to prevent cell and protein adhesion to devices for research and medical applications. These include, without limitation, such uses as coatings for medical implants, coatings for surgical devices, coatings for devices that handle serum and other materials derived from humans or animals, medical diagnostic devices and biosensors. Alternatively, the polymeric compositions may be polymeric adhesive polymeric hydrogels for medical uses, such as tissue sealants, gels for prevention of surgical adhesion (scar tissue formation), bone and cartilage adhesives, tissue engineering design, and elution of site-specific drug and for research uses, such as immobilization of proteins including, antibodies and small molecule analytes including pharmaceuticals.

In addition, there are several uses of industrial and consumer products of these coatings and hydrogels that include, without limitation, prevention of marine bioinfusion (binding of algae, bacteria and mussels to underwater surfaces), prevention of bacterial contamination of water currents to industrial plants, such as manufacturers of drugs and electronics, prevention of bacterial contamination of drinking water streams, dental and denture adhesives, underwater adhesives to supply indicators, coatings to determine water purity and measurement sensors, paints used for bioinstitution prevention , and use in cosmetics to adhere desired fragrances and dyes to hair, eyelids, lips and skin, to form temporary skin coloration, such as tattoos and the like, and for resealable adhesives for consumer products such as storage bags. The present methods can be used to prepare a variety of polymer modified surfaces for both medical technologies (diagnostics, devices, nanoparticle-based therapies) and non-medical technologies (paints and other particle dispersions, MEMs, quantum dots, non-injected surfaces) . Adhesive hydrogels can also be formed using the current methods. The DHPD adhesive is bonded to polymers capable of forming hydrogels in vivo or in vitro. These hydrogels can be formed by a number of methods including the use of self-assembling polymers that form gels at elevated temperatures such as normal human body temperatures, the use of polymers that can be cross-linked by an enzymatic reaction, the use of polymers that can be subjected to oxidation to form crosslinked hydrogels, and the use of polymers that can be subjected to photoactivation to produce crosslinked hydrogels.

Anti-Incrustation Coatings The anti-inundation coatings of the present invention can be applied to medical devices, such as stent or arterial, pacemakers, heart valves, glucose monitors and other biosensors, vascular casings, defibrillators, orthopedic devices and surgical devices. , which include sutures and catheters. The polymeric compositions of the present invention can be used as coatings to prevent adhesion of protein and / or cell, to a device for medical and research applications. These include without limitation such uses as coatings for medical implants, coatings for surgical devices, coatings for devices that handle serum and other materials derived from animals or humans, medical diagnostic devices, and biosensors. Among the changes in the modification of surfaces of biomaterials, with polymers for resistance to cell adhesion, are to produce a sufficiently high density of polymer able to repel proteins and cells and produce a coating that covers the surface completely. This is particularly a problem with devices that contain multiple components made of different materials. A surface can be modified by the polymer composition of the present invention in any number of ways. For example, the polymer composition can be absorbed onto the surface or a portion of DHPD containing a polymerization initiator can be adsorbed onto the surface and a polymeric growth initiated from the surface. With the latter, a number of polymerization techniques are possible, including without limitation, radical polymerization initiated at the surface, radical polymerization methods, ionic polymerization, ring opening polymerization and photopolymerization. For example, using the unique properties of PEG solubility, the polymer's surface density can be increased by treating surfaces with PEG solutions near the lower critical solution temperature (LCST)., or turbidity point. While not wishing to be bound by theory, Applicants believe that under the conditions of high ionic strength and high temperature used in the present invention, the PEG molecules have a reduced hydrodynamic radius, which in principle, allows a higher density of PEG chains to be packed on a surface that under standard conditions. This method is useful for polymers that show transitions of inverse solubility at high temperature and high ionic strength, such as poly (ethylene glycol), poly (N-isopropylacrylamide) and other N-substituted poly (acrylamides) that show transitions of inverse solubility. By modifying surfaces with various polymeric compositions of the present invention, resistance to cell and protein adhesion is conferred for up to 7 days, 14 days, 21 days, 30 days, 60 days, 90 days and 120 days or more. The number of portions of DHPD in the adhesive component and the pH of the modifier buffer are responsible for most of the variation in the adhesion resistance of cells and / or proteins of the modified materials. For those surfaces that are modified by adsorption, adsorption time and polymer composition, the concentration contributes little to the variation in the cell adhesion and / or protein resistance of the modified materials. The greater the number of monomers of the DHPD portion in the adhesive component, the better the cell and / or protein adhesion resistance. The density of the polymer composition on the surface correlates well with resistance to cell adhesion and / or protein. The thickness of the coating layer can be from about 20 A to about 100 μm, which includes 30 A, depending on the polymer composition used and the pH of the modulation buffer. The concentration of the polymer composition used for modification of a surface can be from about .1 mg / ml to about 75 gm / ml. The pH of the modifier buffer can be from about 3 to about 9. The modification time can be from about 10 minutes to about 72 hours. The temperature of the modification can be from about 25 ° C to about 60 ° C. As shown in Figure 19, the XPS recognition scan of unmodified Ti02 revealed strong peaks at ~ 458 eV (Ti2P) and ~ 530 eV (Ois), characteristic of native oxide, as well as a peak less than 248.7 eV (Cls), as a result of adventitious hydrocarbon contamination. Ti02 substrates treated with mPEG-DOPA? _3, under cloud point conditions, however, showed dramatic increases in surface-bound carbon, as reflected by the Cls peak, suggesting the presence of PEG on the surface. However, the increase in Cls peaks, observed after modification with mPEG-DOPA? _3, was directly proportional to the number of terminal DOPAs present. Additionally, a peak lower than 400 eV (Nls) was observed in the surface spectrum of Ti02 modified with mPEG-DOPA? _3, which represents the amide nitrogen in DOPA. Quantitative analysis of the high resolution XPS data of the substrate surfaces can provide useful information in relative amounts of PEG bound to the surface. Table 2 shows the atomic composition calculations of titanium, oxygen and carbon, for T02 modified with mPEG-DOPA? _3. The oxygen signal is further subdivided into metal oxide (Ti-O-Ti), sodium hydroxide (Ti-O-H), and organic oxygen and coupled water species (C-O, H20).

Table 2 Atomic composition calculations of titanium, oxygen and carbon N trace cliffs were forgotten basumido for being water attached to the surface The ratio of Ti to Ti-O-Ti for all substrates, differs substantially from the theoretical stoichiometry of 2.0; the difference is probably due to a sampling depth that goes beyond the depth of the surface oxide (3-4 mm). This result is expected given by the Fory radius of 5000 Pm PEG (2.8 mm) and a typical XPS sampling depth of 5-10 mm. On surfaces modified with mPEG-DOPA? _3. The atomic ratio Ti to C decreases dramatically with increases in the peptide length of DOPA, which corresponds to increases in the amount of PEG adsorbed. The C observed at the organic oxygen ratio (C-O), exceeds the theoretical value of 2.0 for pure PEG, which suggests that the adventitious contamination of hydrocarbon remains on the modified surfaces. These results are shown in Table 3.

Table 3 Atomic ratio of polymer compositions adsorbed DOPA creates strong, reversible bonds with Ti02. The bond energy is 30.56 kcal / mol and needs approximately 800 pN to be released from Ti02 at the same level of single molecule, which is four times stronger than the interaction between Avidin and Biotin. The DOPA-Ti02 intensity of interaction is approximately half that of Abidine-Biotin, one of the strongest hydrogen-based interactions in biology (0.1-0.2 nN) and a covalent bond (> 2nN). To study the adhesion property of DOPA, the following conditions were used: simple molecule procedure, an aqueous environment and a platform for immobilization of DOPA. Atomic Force Microscope (AFFK) was chosen as a tool for research, which satisfies those three conditions and is sensitive enough to measure the viscoelastic properties of soft materials: protein, DNA and synthetic polymers at a single molecule level. Amine portions were introduced to a cantilever strip (SÍ3N4) and then the mixture of methoxy-poly derivatives (ethylene glycol, mPEG) and PEG terminated in Fmoc (Fmoc-PEG) was conjugated. DOPA (Boc) was coupled to the amine groups generated by unfolding of Fmoc (Figure 33C). An excess of 5 ~ 10 molar of mPEG to DOPA-PEG was used, so that the immobilized DOPA4 PEG could be isolated. This configuration hinders sterically the molecular dynamics of DOPA-PEG, thus providing an explanation for the result of DOPA oxidation experiments afterwards (Figure 36). The chemical reaction stages were monitored by X-ray photoelectron spectroscopy (XPS), showing successful PEG modifications on flat silicon nitride surfaces (1 x 1 x cm2) (Fig. 28). The chemical groups introduced on the surfaces of the silicon nitride tips change the electrostatic properties, which is also a good indicator of surface modifications (Fig. 29-A). It is important to note that the difference in procedural signals was detected between the modified cantilevers and ingots, which represent the strength of resistance due to the molecular layers (Fig. 29-B). The overhangs conjugated to DOPA exhibit significant adhesion exhibited accompanied by entrotropic elasticity of the PEG chain (Figure 34). A histogram of the force distribution shows a uni-modal form that indicates only a single adhesive case, which is different compared to the case of a multivalent protein, Avidin 12. Measurements of Diergence Férza (FD) were collected and performed a statistical analysis (Fig. 34, n = 105). The average force was 785 pN in water at a load ratio of 180 nN / s. More importantly, the length of the widened PEG (36 nm) was consistent with the expected contour length of a PEG molecule (37 nm, Fig. 30). These data are looking at a single molecular case: monovalent DOPA binding, polydispersity of PEG and tip geometry. A single DOPA was conjugated to the end of a polymer chain, to serve as a linkage unit on a Ti02 surface (Fig. 33). This monovalence is different from the metal-Histidinad (Metal- (His) 6) studies, where (His) 6 moored provides three metal chelation sites (3 x metal / (His) 2) 13-15. In addition, a configuration simulating "three mountains" of cantilevered PEG5, can separate two different detached DOPA signals, due to the polydispersity of PEGs (three) and the spherically formed point (mountain) (r = 25 nm). Defined, "d", is the displacement distance z of the piezoelectric device when a single molecule of DOPA-PEG was completely widened during the reaction (Figure 34C). The "d" values appear to be almost constant although many cycles repeated through it vary slightly (Fig. 34A). This small variation may be due to the DOPA link to the surface at different angles. An important feature of our experiment is that the unbound signals are repeats using the "identical" DOPA molecule. This is compared to the traditional single molecule push experiments, where the tips take a molecule randomly. This also shows that the DOPA adhesion chemistry was completely reversible. This reversibility does not lead to the conclusion that the weakest chemical bond of the substrate to the tip (Ti02 ~ Si3N4), is the Ti (surface) -O (DOPA) bond. The result suggests that at ~ 0.8 nN, the DOPA-TÍ02 interactions are mechanically mediated between those of Avidin-Biotin, one of the strongest hydrogen bond-based interactions in biology (0.1 ~ 0.2nN) and covalent bond (>2 nN). The energy formation of the force data changes the charge ratio, the amount of force applied per unit of time. Changes over four orders of magnitude of a load ratio generate four different force distributions to map the power landscape of the DOPA link. The graph of the linear line-log of force versus the load ratio in Figure 34D, provides energy link and a distance after which the association is removed along the applied force direction. The DOPA has an energy barrier of 28.1 kcal / mol, and the distance needed to reach the maximum activation energy was 1.27 A (Fig. 34E). The binding orientation of DOPA is believed to be within the two hydroxyl groups in the aromatic ring indicated descending to the surface. Therefore, the confirmation of the chemical groups responsible for signals of adhesion of single molecule in AFM is important to exclude other binding chemistries, due to the different orientations. Two methods were used. First, the chemical modification of the hydroxyl group covalently by tertabutyldimethylsiloxane (TBDMS), resulted in no link during two hundred cycles of procedures (Fig. 31, first line). However, the deprotection of the TBDMS groups regenerated the binding capacity of DOPA (Fig. 31, lower part, two lines). Second, the protection by formation of ionic complexes with borate, completely suppresses the strong adhesion of DOPA as well (Fig. 31, n = 200). These data clearly confirm the di-hydroxyl group of DOPA as the true structural source for strong and reversible binding. The mussels developed an interesting way to create such a strong bond in water, a post-translational modification of tyrosine by tyrosine hydroxylase. This enzyme catalyzes a reaction of adding a hydroxyl group using tyrosine as a substrate and found a large amount in strands and plates where DOPA also exists. It is surprising to say that the small post-translational modification (-OH) seems to produce an extreme change in adhesive capacity. In this way, experiments were designed to show a correlation between post-translational modification and link capacity. A tyrosine moored overhang was prepared in place of DOPA, and tyrosine adhesion was investigated in Ti02. No detectable force signals were observed, except for some non-specific adhesions with low probability (Figure 35A). To reject the hypothesis that the tip used in this experiment does not have some tyrosine molecules, the surface of Ti02 was replaced with gold. The aromatic ring of tyrosine binds to a surface of gold in an orientation parallel to the surface, through the interaction of the electron p-p, which is a well-known mechanism in the chemistry of surface adsorption 18,19. The same overhang used in Ti02, produces relatively strong adhesions repeatedly on the gold surfaces (Fig. 35B). Statistical analysis of the force distribution showed the binding resistance of the p-p electron of 398 (+98) pN, which was approximately 50% intensity, compared to the DOPA-TIO2 interaction (Figure 35C). The strength signals of the tyrosine-gold bond also showed the same characteristics as previously shown in the interaction of DOPA-TÍ02: elastic widening of PEG with an expected contour length and appearance of repetitive signal with similar "d" (Fig. 33C). In this experiment, it was clearly demonstrated that the post-translational modification mediated by tyrosine hydroxylase, greatly improved the binding capacity of DOPA from almost zero to 800 pN. DOPA biological papers go beyond adhesiveness after oxidation: their crosslinking polypeptide chains result in more rigid materials found through the strands and plates. The crosslinking mechanism has multiple trajectories that start from a chemically unstable structure of DOPAquinone. The coupling of the aryl-aryl ring (di-DOPA), has been found in mussel adhesive proteins20, but the Michael addition products (quinone-alkylamine adducts), have been found in other non-mussel species (Fig. 36A) . Therefore, these structures can occur as a result of oxidation in mussels as well. It is clear, in terms of crosslinking, but it is under debate with respect to the adhesive properties after maturation, i.e., oxidation. It was shown that the structure of DOPAquinone is not a main player for adhesiveness. The chain of DOPAquinone-PEG is spatially and chemically stabilized by con-conjugation in excess of methoxy-PEG molecules (molar equivalent 5 ~ 10), which is an important molecular configuration to prevent further reactions of DOPAquinone. The monitoring force signals resulting with the single DOPAquinone time, activated by pH increase (= 9.7), do not cover interesting things that have been hidden so far. First, the AFM signals measured, showed two clear distributions in terms of force magnitude: high force and low force (Figure 36B). Statistical analysis of the data provided two clear histograms with 178 + 62 pN for low force and 741 + 110 pN for high force (Fig. 36C). The quinone bond can be assigned to the low strength region because it appears only after oxidation has been activated and subsequently becomes more frequent over time (Figure 36D). The slow kinetic characteristic of DOPA oxidation contributes to high initial frequency of DOPA signals. This is the first single molecule experiment about detecting the structural change of a small molecule after the external stimulus. Based on these results, the possibility of the structure of DOPAquinone that is responsible for the high adhesiveness, can be eliminated. Therefore, without being bound by theory, the regeneration of the reduced form ie the di-hydroxyl group of DOPA during oxidation is believed to be a very important requirement to maintain or change the adhesive properties of DOPA-containing materials in an interface In addition, the DOPA anchoring system can be a new platform for studying other extensible biological macromolecules such as polysaccharides, DNA and proteins. In the study carried out, the elastic property of PEG (Pm 3400) was already presented and is believed to be the shortest chain length ever studied until now. This could be achieved simply because two defined anchoring methods were used, at both ends: (1) covalent bonds between PEG and cantilevers and (2) DOPA anchor between PEG and substrate. This method is also highly contrasted with conventional single-molecule experiments, where a tip "looks" different molecules in each unique movement of a cantilever. It has been a great barrier to investigate the molecular responses after the external stimulus, if an external stimulus was not 100 percent effective23. The DOPA-based anchoring system may be an alternative technique to overcome these problems in current single-molecule push experiments. Currently, it is not clear to answer how the DOPA behaves like a reversible glue, similar to a "Post-it". Two molecular bond models, bidentate molecular (Figure 32A, right) and bidentate binuclear (Figure 32A, left), they are available, but both do not consider the reversible link of DOPA because the studies focused on only non-desorption adsorption processes25. Therefore, the nature of the chemical bond was assumed to be primarily covalent independent of factors that result from the removal of water molecules after adsorption. A study using FTIR suggests that the nature of the DOPA-TÍ02 bond can be 60% ionic and 40% covalent. Based on these findings, molecular adsorption models were reviewed to incorporate reversibility where multiple hydrogen bonds were formed in water (Fig. 32B).

Figure 33 Experimental design and a unique molecular DOPA adhesion A photograph describes how the blue mussel (Mytilus Edulis) sticks to metal oxide surfaces. The circle includes a plate where the unusual amino acid, DOPA, was found. (B) Two main protein components not covered in plates in mussels, Mefp-3 and Mefp-5. These mussels have adhesive proteins that have a high DOPA content: 27% n mole of Mefp-5 and 21% of Mefp-3. The bold Y (Y): DOPA, Cursive S (S): phospho-serine, Cursive R (R): hydroxyarginine. (C) AFM tip modifications. The polymerization of 3-aminopropyltrimethoxysilane (APTMS), introduces amine groups on the tip surfaces SÍ3N4 (not drawn). A long chain describes a PEG molecule conjugated with a single (Boc) -DOPA at the end. The mixture of mPEG-NHS (2k) and Fmoc-PEG-NHS (3.4k) at a molar ratio of ~ 10: 1, was used to stabilize the DOPA-PEG molecule (see supplementary section for details).

Figure 34: Single molecule force measurement and landscape determination of DOPA bonding energy on Ti02 surfaces Four unique molecules of representative DOPA AFM, give signals of a cantilever. These four signals were not consecutively regenerated (the signals were suppressed, which did not show some adhesions). Due to the low probability to detect the DOPA adhesion (5 ~ 10%), the detected signals showed similar "d" values (referred to Figure 34C). (B) a histogram that describes the distribution of force. The average is 781 + 151 pN (n = 105) at a load ratio of 180 nN / sec. (C) A definition of distance, "d", the directional z that moves the distance of a piezoelectric device when the DOPA-PEG molecule is fully stretched. (D) A plot of link strength (linear) against the load ratio (log). The load ratio is the product of the spring constant of a cantilever and a thrust speed. Four different load ratios were selected: 1500, 180.7, 28.4 and 2 nN / sec. Average forces were plotted with standard deviation in each given load ratio. The forces were 846.48 + 157 pN (1500 nN / s), 781 + 151 pN (180 nN / s), 744 + 206 pN (28.4 nN / s) and 636.2 + 150 (2 nN / s). (E) A schematic landscape of DOPA link power. The external force titrates the landscape and decreases the energy barrier of the reaction coordinates. The slope (= kBT / xb) of the linear regression graph was 32.31, which results in the distance to the activation barrier (xb) was 12.7 A. The height of the energy barrier was determined by extrapolation when a load ratio it is equal to zero (Eb = 28.1 kcal / mol).

Figure 35. Molecular identification of the DOPA adhesive origin Adhesion of tyrosine single molecule on surfaces Ti02. No clear link signals were detected (representative upper signal, n = 639 of 700 repetitions), except the initial electrostatic interactions, which were not avoidable in some cantilevers. Non-specific adsorption signals (lower representative signal, n = 61 of 700). (B) confirmation of the existence of tyrosine on the surface of the tip. Pi electrons (p) from the phenyl tyrosine group specifically interact with the gold p-electron. (C) distributions of tyrosine binding strength to a gold surface. Tyrosine represents 398 + 98 pN (180 nN / sec) of adhesion strength.

Figure 36. The DOPA adhesiveness change after oxidation (DOPAquinone) A schematic chemistry pathway of DOPA formation and oxidation. DOPA was created by the action of tyrosine hydroxylase and subsequently oxidized to DOPAquinone by pH and enzyme. It is unstable and reactive due to the tendency of radical formation of DOPAquinone. It can crosslink with other molecules DOPA (di-DOPA), also reacts with amine groups of plants. The arrows are the potential reactions found in other species not in mussel adhesive proteins. (B) Signals of representative force (n = 16) with similar "d", defined in Figure 1C (~ 50 nm). They were collected by the AFM experiment of 1 hour (1800 repetitions) in a basic condition (20 mM Tris-Cl, pH 9.7). The progress of time was made from the top to the bottom of the graph. The red signals indicate DOPA-Ti02 and the black signals for DOPAquinone-Ti02. (C) force histograms after the total analysis of 1800 F-D curves. The histogram in a region of slow force, showed 178 + 82 pN (n = 143) and one in a region of high strength, presented 741 + 110 pN (n = 51). (D) A scatter plot of the number of events during a specified time window (10 min). DOPA signals (circle, axis and left), gradually decreased from twenty-two events for the first ten minutes to only three events during the last time window. However, the quinone signals (axis and right triangle), decrease from an event in the first time window to twenty-two events in the last time window (50 ~ 60 min). To summarize, successfully measuring the binding strength of the single molecule of DOPA (~ 0.8 nN), was successfully measured, and reversible link chemistry was shown. This strong adhesion was created by post-translational modification, but was significantly reduced by oxidation of DOPA, to DOPAquinone. The anti-induction coating of the present invention can be either essentially permanent, that is, it lasts 120 days or more, or biodegradable depending on the number of DOPA or portions derived from DOPA in the adhesive component. Figure 20 shows the results of an adhesion of 3T3 fibroblast cells at day 28 and spreading tests on Ti02 treated with mPEG-DOPA1-3. At early time points (ie, less than 7 days), resistance to protein and cell adhesion correlates well with the length of the DOPA peptide anchor group, with resistance increasing in the order of mPEG-DOPA < mPEG-D0PA2 < mPEG-DOPA3. Ti02 substrates treated with mPEG-D0PA2 and mPEG-DOPA3, maintain reduced cell binding, or resistance, through 21 days. The robust design methodology was used to determine the effect of the DOPA peptide anchor length and modification conditions (pH, concentration and time) on the surface density and anti-incrustation performance of metal, metal oxide, semiconductor and polymeric surfaces. The nine experiments used for each substrate are described below in Table 7. For almost all surfaces, the length of the DOPA peptide and the pH of the modulation buffer, lead to the greatest variation in the amount of PEG absorbed, as measured by XPS and adhesion and spreading of 3T3 fibroblasts. The experiments summarized in Table 7 allow the determination of modification conditions that provide resistance to optional cell adhesion by a variety of materials, as measured by the 4-hour cell adhesion assay. After the nine experiments were performed on each substrate, the data were subjected to Robust Design analysis. The presence of large error values when plotting Robust Design data is characteristic of the technique, since a single data point at a factor level contains the variation of the remaining factors averaged over all levels. The polymer compositions of the present invention, they can also be used to coat surfaces of devices and instruments used to manipulate bodily fluids including serum. The coating on the surface of the device or instrument blocks the binding of the protein to the surfaces, thereby reducing or eliminating the need for extensive washing or cleaning of the device or instrument between uses. The device needs to be thoroughly cleaned to prevent contamination between body fluid samples applied to the top of the device. Currently, the cleaning of these instruments and devices between uses requires extensive washing with caustic agents such as 50% bleach and / or elevated temperatures. The coating process could be to circulate a 1 mg / ml aqueous solution of DHPD polymer through the device at room temperature, for a period of a few hours. The coatings of the present invention can be used in medical implants for a wide variety of uses. For example, the coatings can be used to block bacterial adhesion and therefore grow in the implanted device reducing the possibility of infection at the implant site. The coatings can be used to reduce the amount of acute inflammation on the device, reducing the binding of the protein and cell adhesion to the device. The coatings of the present invention can also be used as nanoparticles to prevent the aggregation of particles in the presence of serum.

Hydrogels The polymeric compositions of the present invention can also be as surgical adhesives for medical and dental uses and as vehicles for drug delivery to mucosal surfaces. The polymeric compositions can be used as polymeric adhesive polymeric hydrogels for medical uses, such as tissue sealants, gels for prevention of surgical adhesions (formation of scar tissue), bone and cartilage adhesives, tissue engineering design and elution of site-specific drug and for research uses, such as immobilization of proteins including antibodies and small molecule analytes including pharmaceuticals. The polymer compositions of the present invention can also be used as interfacial bonding agents, wherein the monomers or pure solutions of monomers are applied to a surface as a primer or binding agent between a woven surface or a metal or surface of device / implant of metal oxide and a volume polymer or polymer hydrogel. With an appropriate polymer component, in which one of ordinary skill in the art could be identified, the polymer compositions of the present invention can be injected or delivered in a fluid form and hardened in situ to form a gel network. In situ hardening can occur through light cure, chemical oxidation, enzymatic reaction or through the natural increase in temperature, resulting from the supply in the body. In part, the present invention is also a method for the non-oxidative gelation of a polymeric composition of the present invention. One such method includes (1) providing a polymer composition of the present invention; (2) mixing water and the polymer composition; and (3) increasing the temperature of sufficient mixture to the gel of the composition temperature, such temperature increases without oxidation of the polymer or DOPA or DOPA-derived portion residue incorporated herein. Depending on the choice and identification of the polymer component of such composition, an increase in the concentration of the mixture can reduce the temperature required to effect gelation. Depending on the choice and identity of a particular copolymer component, a larger hydrophilic block thereof, may increase the required gel temperature of the corresponding composition. Various other structural and / or physical parameters may be modified to adjust the gelation, such modifications may be extended to other polymeric compositions and / or systems which are consistent with the broader aspects of this invention. It is widely recognized that commercially available PLURONIC® block copolymers self-assemble in a concentration and temperature dependent manner in icelos consisting of a hydrophobic PPO core and a water-swollen core consisting of PEO segments. At high concentrations, certain PEO-PPO-PEO block copolymers, such as PLURONIC® and PLURONIC® F68, are transformed from a low viscosity solution to a transparent high temperature reversible gel. While it is not desired to bind by theory, it is generally assumed that the interactions between micelles at elevated temperatures lead to the formation of a gel phase, which is stabilized by micellar entanglements. The gelation and micellization processes depend on factors such as molecular weight of block copolymer, relative block sizes, solvent composition, polymer concentration and temperature. For example, increasing the length of the hydrophilic PEO blocks relative to the hydrophobic PPO board results in an increase in micellization and gelation temperature (Tge?). Differential scanning calorimetry (DSC) measurements were made on aqueous solutions of DME-PA07 and D0PA-PA07 at different concentrations to detect the aggregation of block copolymers in micelles. The DSC profiles obtained by PLURONIC® F127, DME-PA07 and D0PA-PA07, were found to be qualitatively similar and were characterized by a large endothermic transition corresponding to micelle formation, followed by a small endotherm to Tgel (Figure 2). The transition temperature of the minor peak was found to correlate strongly with the Tgel determined by the rheometry and the road reversal method (Table 4).

Table 4 Gel temperatures obtained from the road inversion method, rheology or deferential scanning calorimetry by 22% by weight of solutions of DME-PA07, D0PA-PA07, and PLURONIC® F127 Gel temperature (° C) Rheological method DSC inversion vial D E-PA07 (22% in 22.0 + 1.0 20.3 + 0.6 20.9 + 0.1 weight) D0PA-PA07 (22% in 22.0 + 1.0 20.4 + 0.5 21.7 + 0.2 weight) PLURONIC® F127 17.0 + 1.0 15.4 + 0.4 17.5 + 0.4 (22% by weight) Aqueous solutions with concentrations ranging from 10 to 30% (w / w) of DOPA-PA07 copolymers and 35 to 54% (w / w) of DOPA-PA08 copolymers, were prepared by the cold method, in which, the conjugate DOPA was dissolved in distilled water at about 4 ° C with intermittent stirring until a clear solution was obtained. The thermal gelation of concentrated solutions was initially assessed using the road inversion method. In this method, the temperature at which the solution does not flow anymore, is taken as the gelation temperature. The gelation temperature was found to be strongly dependent on the copolymer concentration and the block copolymer composition (ie, PA07 versus PA08). For example, 22 wt% solutions of DOPA-PA07 and DME-PA08 were found to form a clear gel at approximately 22.0 + 1.0 ° C; decreasing the polymer concentration to 18% by weight, resulting in a gelation temperature of approximately 31.0 + 1.0 ° C. However, DOPA-PA07 solutions with concentrations of less than 17% do not form gels when heated to 60 ° C. DOPA-PA07 has a gel temperature slightly higher than that of (17.0 + 1.0 ° C) PLURONIC® F127 unmodified. The gelation behavior of DOPA-PA08 was found to be qualitatively similar, except that many higher polymer concentrations were required to form a gel. Solutions at 54% by weight of DOPA-PA08 and DME-PA08, formed gels at 23.0 + 1.0 ° C, while 50% by weight of gels DOPA-PA08 at 33.0 + 1.0 ° C. However, DOPA-PA08 solutions with concentrations of less than 35% by weight, do not form gels when heated to 60 ° C. DOPA-PA08 has a gel temperature much higher than that (16.0 + 1.0 ° C) of unmodified PLURONIC® F68. These gels were found to be resistant by flowing for long periods of time. From this experiment, it has also been found that both DOPA and DOPA methyl ester derivatives of the same commercially available PAO PLURONIC® have almost the same gel temperature, and the elaborated gel of 54% by weight of either DME-PA08 or DOPA-PA08 at room temperature, it is more rigid than that made of 22% of either DME-PA07 or D0PA-PA07. The viscoelastic behavior of DOPA solutions modified with PLURONIC® was also studied by oscillatory rheometry. Figure 3 shows the elastic storage modulus, G ', of 22 wt% solutions of aqueous solutions of unmodified PLURONIC® F127 and DME-PA07 as a function of temperature. Below the gelation temperature, the storage modulus G 'was negligible, however, the G' increased rapidly to the gel temperature (Tge?), Defined as the beginning of the increase of G 'against the temperature graph. DOPA-PA07 (not shown), presents a similar rheological profile. The Tge? of 22% solutions of DME-PA07 and DOPA-PA07, was found to be identical (20.3 + 0.6 ° C), which is approximately 5 degrees higher than an equivalent concentration of unmodified PLURONIC® F127 (15.4 + 0.4 ° C) ). The G 'of DME-PA07 or DOPA-PA07, approximates a plateau value of 13 kPa, which is comparable with that of unmodified PLURONIC® F127. Shown in Figure 4, there are rheological profiles of 50% solutions of unmodified PLURONIC® F68 and DME-PA08, as a function of temperature. The Tgei of a 50% by weight solution of DME-PAO was found to be 34.1 + 0.6 ° e, while the Tge? of an equivalent concentration of PLURONIC® F68 not modified, was not significantly different, reaching a plateau value as high as 50 kPa. The concentration dependence of the Tge ?, is illustrated in Figure 5, which shows the rheological profile of DME-PA08 at two different concentrations as a function of temperature. The TgI of 45% by weight solution of DME-PA08 was observed to be about 12 ° C higher than that of the 50% by weight solution of DME-PAO8. Since both DOPA and DOPA methyl ester can be considered hydrophilic, the increase in TGEI observed in the DOPA-modified PAO PLURONIC® compared to that of unmodified PAO PLURONIC® is probably due to the increase in the length of the DOPA. the hydrophilic PEO segments resulting from the coupling of DOPA to the final groups. It is clear, from the data shown in Figures 3 and 4, that the coupling of DOPA or methyl ester of DOPA to the final groups of PAO PLURONIC®, has a more significant impact on the Tgel of PLURONIC® F68, compared to PLURONIC® F127. This can be rationalized in terms of the total molecular weights of F68 (approximately 8,600) and F127 (approximately 12,600). The addition of DOPA and DOPA methyl ester to both end groups using the chemistry shown in Reaction Scheme 1, results in an increase in molecular weight of 446 and 474, respectively. This represents a larger% increase in molecular weight for F68, compared to F127, due to the lower base molecular weight of F68. The data presented in this document are in agreement with previous studies of unmodified PAO PLURONIC® calorimetry, which shows that the broad peak at lower temperature is due to micellization, while the lower peak at higher temperature, only observed in concentrated solutions, corresponds to gelation, a closely athermic process. As shown in Table 5, the micellization start temperature, the temperature at maximum heat capacity and the unmodified PLURONIC® F127 Tgei were found to be lower than those of DOPA-PA07, while the specific enthalpies determined from The areas under the transition (Figure 2) are approximately the same. These enthalpies include contributions from both micelization and gelation. However, due to the lower enthalpy of gelation, the observed enthalpy changes can be widely attributed to micellization.

Table 5 Comparisons of solutions at 30% by weight of DME-PA07, DOPA-PA07 and PLURONIC® F127 not modified in temperature from start of micellization, temperature to maximum Heat capacity, enthalpies, and gel temperature from different scanning calorimetry experiments differential Tem. of Temp. a? H Temp. of Gel micellization capacity (J / g) (° C) (° C) max. of heat (° C) DME-PAO7 5.2 + 0.2 8.3 + 0.1 20.3 + 2.4 14.0 + 0.4 (30% by weight) DOPA-PA07 6 + 0.2 .0 + 0.6 19.3 + 1.4 14.0 + 0.2 (30% by weight) PLURONIC® 1.9 + 0.3 6.0 + 0.4 20.6 + 1.6 10.6 + 0.

F127 (30% by weight) The micellization peak was observed extended to temperatures above the beginning of gelation, indicating that additional monomers are added in the micelles to temperatures above the gelation point. The aggregation concentration dependence of DOPA-PA07 and DME-PA07, is shown in Figure 6. The DSC thermograms indicate a Decrease in micelization temperature and Tge? with increases in polymer concentration. The broad endothermic peak corresponding to micellization can also be observed in solutions at concentrations in which gelation does not take place; the temperature characteristics of the broad peak increase linearly with the decrease in polymer concentration, while the lower peak is observed by coinciding with the gel temperature of the concentrated compolymers, but disappears as the copolymer concentration decreases. As can be gathered from the foregoing, various polymeric compositions of this invention can be designed and prepared to provide various micellization and / or thermal gelation properties. Alternatively, or in conjunction therewith, degradation into excretable polymeric components and metabolites can be achieved using, for example, polyethylene glycol and lactic / glycolic acids, respectively. Nonetheless, the polymer compositions of this invention provide improved adhesion by incorporation of one or more DHPD residues, such incorporation results from the coupling of a terminal monomer of the polymer component to such a residue. Another method of non-oxidative gelation of a polymeric composition of the present invention is light curing. A monomer containing a portion of photocurable DHPD is copolymerized with PEG-DA (PEG diacrylate) to form adhesive hydrogels through photopolymerization. Photopolymerization can be achieved at any visible UV wavelength, depending on the monomer used. This is decidedly determined by one of skill in the art. The photocurable monomers consist of an adhesive portion coupled to a monomer polymerizable with a vinyl group, such as a methacrylate group with or without an oligomeric ethylene oxide linker or fluorinated ether linker therebetween. An aqueous mixture of a photopolymerizable monomer containing an adhesive of the present invention and PEG-DA and 1.5 μl / ml of a photoinitiator, such as 2, 2'-dimethoxy-2-phenyl-acetonephenone (DMPA), camphorquinone / acid, is added. - (dimethylamino) -benzoic acid (CQ / DMAB), and sodium salt of fluorescein / ascorbic acid (AA / FNa2), was irradiated using a UV lamp (365 nm), for more than 5 minutes. The presence of the adhesive in the precursor solution was found to affect the polymerization process of the radical. The catechol adhesive decreased the extent of gel formation, reduced the percentage of adhesive incorporation in the gel network and lengthened the gel formation time. As shown in Figure 25, the gel conversion, as determined by measuring the mass of the gel probe, reached more than 75% by weight after 2 minutes of UV irradiation and increased to more than 85% by weight, then of irradiation for more than 5 minutes. The gelation of PEG-DA occurs in 4 minutes or less, when visible light initiators are used (4 minutes for CQ / DMAB and 3 minutes for AA / FNa2). The copolymerization of PEG-DA with 1 or 7 (the synthesis of 1 and 7 is shown in Reaction Schemes 2 and 3), was qualitatively similar to the polymerization of pure PEG-DA, although the addition of 1 or 7 to the PEG-DA precursor solution, resulted in a decrease in the gel conversion that was dependent on the DOPA monomer concentration and initiation system. For example, in UV polymerization initiated with DMPA, the gel conversion was reduced to less than 85% by weight, in the presence of 2.5 mol% or more than 1 or 7. However, the extension of the gel conversion does not It was statistically different between the gels. The concentration-dependent inhibition of DOPA was observed similar to the visible light induced by primers. For initiated mixtures of AA / FNa2 and CQ / DMAB, the addition of 33.3% of mol of 1, increased the gelation time to more than 8 minutes. However, solutions containing 1 and 7 were still able to be cured at a relatively high mol% DOPA.

REACTION SCHEME 2 DOPA REACTION SCHEME 3 DOPA 2 3 For example, 53.8 mol% of either 1 or 7 in the precursor solution, reduces the% by weight of the gel from 88 to 77 and only 85 mol% of the DOPA was incorporated into the hydrogel. A DOPA colorimetric assay developed by Waite and Benedict, made in light-cured hydrogels, revealed the presence of catecholic DOPA in the hydrogel. After photocuring, the gels containing DOPA were dialysed in 0.5 N HCl to extract the unreacted DOPA monomer. To quantify the extent of DOPA incorporation, the dialysates were analyzed in accordance with the DOPA colorimetric assay of Waite and Benedict and the results were used to calculate the amount of DOPA incorporated into the gel network. Figure 26 shows the mole fraction of DOPA incorporated in the gel network, as a function of the% mole of monomer 1 and 7, in the precursor solution. There was no significant difference in the mole fraction of DOPA incorporated between the samples containing 1 and 7. As much as 24.9 μmol / g of DOPA, it was incorporated into the PEG hydrogels. Direct evidence of the presence of DOPA in the gels was obtained by immersing the intact dialyzed hydrogels in nitrite reagent, followed by NaOH. The initially colorless gels changed to bright yellow after the addition of the nitrite reagent and then to red after the addition of excess base. This color transition is typical of catechols, indicating that the non-oxidized form of DOPA was incorporated into the hydrogels through photopolymerization. The intensity of the red color also reflects the concentration of DOPA incorporated in the photocured gels. Mechanical contact tests were carried out on light-cured gels in the form of hemispheres, to obtain information on the mechanical properties of the gels. The elastic modulus (E) was calculated assuming Hertzian mechanics for the specific cases of non-adhesive contact between an elastic hemisphere that can not be compressed and a rigid plane, in any case, the Hertzian relation between the load (P¿¡) and the displacement (dh) becomes: (1) 16R1 / 2 £ s 3/2 Ph = ° h where R is the radius of the curvature of the hemispherical gel. The load against the displacement data was adjusted with Equation (1), which allowed the elastic modulus to be calculated based on the proportionality factor of the adjustment curve. As seen in Table 6, the average Young modulus (E) for gel containing DOPA of about 50 kPa was obtained.

Table 6 Average Young Module for gels containing DOPA + 33 mol% in the precursor solution * determined from the DOPA ** test p < 0.001 in relation to the PEG-DA gels These values of modules are approximately 30% lower than those of the PEG-DA gels, confirming the inhibitory effect of DOPA in the photopolymerization of radicals. Due to the decrease in modules compared to PEG-DA gels, gels containing DOPA still presented suitable modules for many biomedical applications. A suitable module is one greater than 500 Pa. Another use of adhesive hydrogels is for localized drug delivery. For example, adhesive hydrogels can be formed in a mucous membrane in the mouth or oral cavity. The hydrogels can be loaded with a drug such as an antibiotic, and facilitate the slow release of the drug over a period of time. The hydrogel can also be loaded with an analgesic and used to provide pain relief at a localized site. The hydrogel can also be loaded with a chemotherapy drug and inserted into malignant tissue to deliver localized cancer therapy. The hydrogel can also be loaded with a therapeutic proliferation inhibitor drug and used as a stent graft or other vascular device, and used to control cell proliferation at the implant site of the vascular device. A tissue adhesive hydrogel capable of being crosslinked in vivo can be used as a tissue sealant to replace metal or plastic sutures. The adhesives fold into the surrounding tissue at an injury or surgical site and the polymer forms a cohesive bond to close the wound. The hydrogel can also be used to repair bone fractures and cartilage in damaged bone. r uses There are several uses of industrial products of these coatings and hydrogels that include, prevention of marine bioinfusion (adhesion of algae, bacteria and mussels to underwater surfaces), prevention of contamination of bacteria from water streams to industrial plants, such as manufacturers of drugs and electronics, prevention of bacterial contamination of drinking water streams, dental and denture adhesives, underwater adhesives to supply indicators, coatings for water purity and measurement sensors, paints used for bioincruction prevention. There are also a number of consumer products and cosmetic uses of these coatings and hydrogels including, without limitation, use in dental and denture adhesives, use in cosmetics for hair, skin and legs adhesive, use in cosmetics such as eye shadows, lipstick and mascara for eyelashes, use in temporary tattoo application, and use as resealable adhesives for bags and containers. Without limitation to any particular synthetic scheme or method of preparation, suitable compositions of this invention may include, but are not limited to, the urethane portion between each terminal monomer and DOPA residue. As described more fully below, such a portion is a synthetic artifact of the reagent / agent used to couple the DOPA residue with the polymer component. Within the broad aspects of the invention, several r portions are contemplated, as will be understood by those skilled in the art, aware of this invention, depending on the terminal monomer functionality and the choice of the coupling agent.

Examples: General Description The following non-limiting examples and data illustrate various aspects and features that relate to the compositions and / or methods of the present invention, including the production of various polymeric or copolymer compositions having incorporated therein, one or more DHPD components, as they are available through the synthetic methodology described in this document. While the utility of this invention is illustrated through the use of various polymeric or copolymer systems, it will be understood by those skilled in the art, that comparable results are obtained with various r compositions and / or methods of preparation, as is commensurate with the scope of this invention. PEOiooPPOesPEOioo (PLURONIC® F127, average Pm = 12,600), PE078PP03oPE078 (PLURONIC® F68, average Pm = 8,400), PEG (average Pm = 8,000), pentafluorophenol, 1,3-dicyclohexylcarbodiimide (DCC), 4, 7, 10-trioxa -l, 13-tridecandiamine, sodium salt of fluorescein (FNa2), and ascorbic acid (AA), were purchased from Sigma (St. Louis, MO). L-DOPA, thionyl chloride, methacryloyl chloride, t-butyldimethylsilyl chloride (TBDMS-C1), di-t-butyl dicarbonate, methacrylic anhydride, 2, '-dimethoxy-2-phenyl-acetonafenone (DMPA), acryloyl, 1/8-diazabicyclo [5.4.0] undec-7-ene (DBU), tetrabutylammonium fluoride (TBAD), 4- (dimethylamino) -benzoic acid (DMAB), l-vinyl-2-pyrrolidone (VP), N, N-disuccinimidylcarbonate, sodium borate, sodium molybdate dihydrate, sodium nitrite, 4- (dimethylamino) pyridine (DMAP), N-hydroxysuccinimide, N, N-diisopropylethylamine, dimethylformamide, and dichloromethane were purchased from Aldrich (Milwaukee, Wl). Camphorquinone (CQ) was obtained from Polysciences, Inc. (Warrington, PA). The acetone was dried on a 4A molecular sieve and distilled over P205, before use. Triethylamine was recently distilled before use. All r chemical reagents were used as received. L-DOPA methyl ester hydrochloride was prepared according to the procedure of Patel and Price, J. Org. Chem., 1965, 30, 3575, which is incorporated herein by reference. Activated succinimidyl propionate PEG (mPEG-SPA, average Pm = 5000) was obtained from Shearwater Polymers, Inc. (Huntsville, AL). Ethyl acetate saturated with HCl was prepared by bubbling HCl gas through ethyl acetate (50 ml) for approximately 10 minutes. 3,4-Bis (t-butyldimethylsiloxyl) L-phenylalanine (DOPA (TBDMS) 2) and 3,4-bis (t-butyldimethylsiloxyl-N-t-butoxycarbonyl-L-phenylalanine were synthesized.

(Boc-DOPA (TBDMS) 2) in accordance with the method of Sever and Wilker, Tetrahedron, 2001, 57, (29), 6139-6146, which is incorporated herein by reference. Glassware (12 mm day) is used in the following examples, it was cleaned by immersing it in 5% Contrad70 solution, a detergent which is an emulsion of anionic and non-anionic surfactants in an aqueous base of alealtine (Decon Labs, Inc., Bryn Mawr, PA) in an ultrasonic bath for 20 minutes, rinsed with deionized H20 (DI), sonicated in H20 DI for 20 minutes, rinsed in acetone, sonicated in acetone for 20 minutes, rinsed in hexanes, sonicated in hexanes for 20 minutes, rinsed in acetone, sonicated in acetone for 20 minutes, rinsed in H20 DI and sonicated in H20 DI for 20 minutes. The cover was subsequently dried with air in a laminar flow suction hood filtered with HEPA. To create virgin gold substrates, the clean coverslip was sprayed (Cressington 208HR) with 2 nm Cr followed by 10 nm Au (99.9% pure). Titanium oxide surfaces were prepared (Ti02) by physical evaporation of the electron beam in a silicon wafer (Si) and cleaned in a plasma chamber before testing it. The Si pellets (MEMC Electronic Materials, St. Peters, MO, orientation surface 100)) were coated with 100 nm of Ti by an Edwards FL400 electron beam evaporator at < 10"d Torr The Si pad was then cut into 8mm x 8mm pieces which subsequently cleaned by ultrasonication in the following medium: Contrad70 at 5%, ultrapure water (ultra pure water was deionized and purified), acetone and petroleum ether. The substrates were further cleaned in an oxygen plasma chamber (Harrick Scientific, Ossining, NY) to < 200 mTorr and 100W for 3 minutes. The virgin and modified gold surfaces were characterized, as described below, by X-ray photoelectron spectroscopy (XPS). The XPS data were collected in an Omicron ESCALAB (Omicron, Taunusstein, Germany) configured with a monochromatic 300-W AIKOÍ (1486.8eV) X-ray source, size of a circular spot 1.5 mm, a flood shot for loading purposes. counter, and an ultralight vacuum (< 10 ~ 8 Torr). The take-off angle, defined as the angle between the normal substrate and the detector, was set at 45 ° C. The substrates were mounted on standard sample posts by means of double-sided adhesive tapes. All binding energies were calibrated using either the Au gold peak (4f7 2) (84. OeV) or the C carbon peak (Is) (284.6eV). The analyzes consist of extensive study exploration (energy step 50. OeV) and a high resolution scan of 10 minutes (energy step 22. OeV) at 270-300eV for C (ls). The deconvolution of the peak and the calculations of the atomic percentage were made with the EIS analysis software. The spectrum of the secondary ion was collected on a TRIFT III ™ time-of-flight secondary ion mass spectrometer (TOF-SIMS) (Physical Electronics, Eden Prairie, MN) in the mass ratio 0-2000 m / z. A Ga + source was used in an energy beam of 15 leV with a frame size of 100 μm. Both positive and negative spectra were collected and calibrated with a simple series of low mass ions using the Cadenee PHI software. To determine the relative hydrophilic / hydrophobic nature of the surfaces, the contact angle data was collected, as described below, by the sessile drop method. An established contact angle goniometer was used as usual (components from Ramé-Hart, Mountain Lakes, NJ) equipped with a wetted sample chamber to measure both ultrapure water advance and retraction contact angles (18.2MO-cm; Barnstead, Dubuque, IA) on modified and unmodified substrates. For each surface, four measurements were made in different places and the mean and standard deviation were reported. Surface Plasmon Resonance (SPR) measurements were made in a BIACORE 2000 (Biacore International AB; Uppsala, Sweden) using gold sensor cartridges discovered. The resonance response was calculated using NaCl solutions 0-100mg / ml. Diluted solutions (0.1 mM in H20) of mPEG-DOPA, mPEG-MAPd and mPEG-OH were injected into the SPR flow cell for 10 minutes after which the flow was switched back to pure H20 DI. In a separate experiment to measure protein adsorption to modify substrates, sensor surfaces made with PEG films were exposed to 0.1 mg / ml bovine serum albumin solution (BSA) in 10 mM HEPES buffer (0.15 M HaCl , pH = 7.2) and subsequently pure buffer. For use in demonstrating anti-fouling effects, the NIH 3T3-Swiss albino fibroblasts obtained from ATCC (Manassas, VA) at 37 ° C and 100% C02 were maintained in Dulbecco's modified Eagle's medium (DMEM; Cellegro, Herndon, VA) containing 10% (v / v) fetal bovine serum (FBS) and lOOU / ml of both penicillin and streptomycin. The RP-CLAR preparation was performed using a CLAR Waters system (Waters, Milford, MA) on a Vydac 218T reverse phase column with a gradient of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid. It was performed in ESI-MS analysis in one system (Finnigan, Thermoquest, CA). The MALDI-TOF EM analysis was performed on a Voyager DE-Pro mass spectrometer (perspective Biosystem, MA). Α-cyano-4-hydroxycinnamic acid was used as a matrix. The NiTi alloy (10 mm x 10 mm x 1 mm) of Nitinol Devices & Components (Fremont, CA). Yes, SiO2 (thermal oxide 1500A), and GaAs lozenges were purchased from University Wafer (South Boston, MA). With respect to the following cell adhesion tests and / or extension assays, modified and unmodified substrates were pretreated on 12-well TCPS plates with 1.0 ml of DMEM containing 10% FBS for 30 minutes at 37 ° C and C02 at 10% Fibroblasts of 12-16 steps were harvested using 0.25% trypsin-EDTA, resuspended in DMEM with 10% FBS and quantified using a hemocytometer. The cells were seeded at a density of 2.9 x 10 3 cells / cm 2 by diluting the suspension to the appropriate volume and adding 1 ml to each well. Substrates were maintained in DMEM with 10% FBS at 37 ° C and 10% C02 for 4 hours, after which time, the loose cells were aspirated. The cells adhered to the substrates were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently treated with 5 μM of 1,1 '-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (Dil; Molecular Probes, Eugene, OR) in DMSO for 30 minutes at 37 ° C. The dyeing was then aspirated and the substrates were washed (3x) with DMSO for 10 minutes to be mounted on slide using Cytoseal (Stephens Scientific, Kalamazoo, MI) to preserve fluorescence. These experiments were performed in triplicate for statistical purposes. For the electron microscope, some samples were dehydrated with EtOH then fixed, dried to critical point and sprayed with 3 nm Au. To quantify cell binding, substrates were examined with an Olympus BX-40 (? Ex = 549 nm,? Em = 565 nm) and color images were captured with a Coolsnap CCD camera (Roper Scientific, Trenton, NJ). Five images of each of the three replicas of the substrate were taken. The resulting images were quantified using thresholds in MetaMorph (Universal Imaging, Downington, PA). One-way ANOVA and post-hoc Turkey test with 95% confidence intervals (SPSS, Chicago, IL) were used to determine the statistical significance of the data. The mean and the standard deviation of the measurements were reported.

Example 1 Synthesis of Succinimidyl Carbonate PAO, SC-PA07 PLURONIC®F127 (0.60 mmol) was dissolved in 30 ml of dioxane. N, N'-Disuccinimidyl carbonate (6.0 mmol) was added in 10 ml of dry acetone. DMAP (6.0 mmol) was dissolved in 10 ml of dry acetone and added slowly under magnetic stirring. The activation was continued at 6 hours at room temperature, after which SC-PA07 was precipitated in ether. The disappearance of the starting materials during the reaction was followed by CCD in chloroform-methanol solvent system (5: 1). The product was purified in acetone and precipitated with ether four times. The yield of the product was 65%. XH NMR (500 MHz, CDC13): d ppm 0.96-1.68 (br, -OCHCH3CH20-), 2.80 (s, -C00N (C0) 2 (CH2) 2), 3.15-4.01 (br, -OCH2CH20-; -0CHCH3CH20 -), 4.40 (s, -OCH2CH2OCOON (CO) 2CH2CH2-).

Example 2 Synthesis of DME-PA07 A suspension mixture of methyl ester hydrochloride DOPA (1.25 mmols) and triethylamine (2.5 mmols) was mixed with SC-PA07 (0.16 mmols) in 10 ml of chloroform. The disappearance of the materials during the reaction was followed by CCD in a solvent system chloroform-methanol-acetic acid (5: 3: 1). After stirring for 1 hour at room temperature, the solvent was evaporated, and the DME-PA07 was purified by methanol precipitation three times. DME-PA07 gives a positive Arnow test indicating the presence of hydroxyl catechol groups. The yield of the product was 75%. XH NMR (500 MHz, CDC13) d ppm 0.98-1.71 (br, -OCHCH3CH20-), 2.83-3.06 (m, -NHCHCH2C6H3 (OH) 2COOCH3), 3. 15-4.02 (br, -OCH2CH20-; -NHCH (CH2C6H3 (OH) 2COOCH3), 4.05-4.35 (d, -OCH2CH2OCONHCHCH2C6H3 (OH) 2COOCH3), 4.55 (br, NHCHCH2C6H3 (OH) 2COOCH3), 5.30 (d, -NHCHCH2C6H3 (OH) 2COOCH3), 6.45-6.80 (Is, 2d, -NHCHCH2C6H3 (OH) 2COOCH3).

Example 3 Synthesis of DOPA-PA07 L-DOPA (1.56 mmols) was added to 30 ml of 0.1M aqueous Na2B40 solution (pH = 9.32) under Ar atmosphere, followed by stirring at room temperature for 30 minutes. SC-PA07 (0.156 mmol) in 5 ml of acetone was added to the resulting mixture and stirred overnight at room temperature. The pH of the solution was maintained with sodium carbonate during the reaction. The disappearance of the starting materials during the reaction was followed by CCD in chloroform-methanol-acetic acid solvent system (5: 3: 1). The solution was acidified to pH 2 with concentrated hydrochloric acid and then extracted three times with dichloromethane. The combined dichloromethane extracts were dried with anhydrous sodium sulfate and filtered, and dichloromethane was evaporated. The product was further purified by precipitation of cold methanol. D0PA-PA07 gives a positive Arnow test indicating the presence of hydroxyl catechol groups. The yield of the product was 52%. XR NMR (500 MHz, CDC13) d ppm 0.92-1.70 (br, -OCHCH3CH20-), 2.91-3.15 (m, -NHCHCH2C6H3 (OH) 2COOCH), 3.20-4.10 (br, -OCH2CH20-; -OCHCH3CH20-), 4.1-4.35 (d, OCH2CH2OCONHCHCH2C6H3 (OH) 2COOH), 4.56 (m, NHCHCH2C6H3 (OH) 2COOH), 5.41 (d, -NHCHCH2C6H5 (OH) 2COOH), 6.60-6.82 (Is, 2d, -NHCHCH2C6H3 (OH) 2COOH ).

Example 4 Synthesis of Succinimidyl Carbonate PA08, SC-PA08 A similar procedure to that described above was used for the synthesis and purification of SC-PA07 to prepare SC-PA08. The yield of the product was 68%. x NMR (500 MHz, CDC13); d ppm 0.95-1.58 (br, -OCHCH3CH20-), 2.80 (s, -COON (CO) 2 (CH2) 2), 3.10-4.03 (br, -0CH2CH20-; -OCHCH3CH20-), 4.40 (s, -OCH2CH2OCOON (CO) 2CH2CH2).

Example 5 Synthesis of DME-PA08 A procedure similar to that described above was used for the synthesis and purification of conjugate DME-PA07 to make DME-PA08. The yield of the product was 76%. 1 H NMR (500 MHz, CDC13): d ppm 0.98-1.50 (br, -OCHCH3CH20-), 2.85-3.10 (m, -NHCHCH2C5H3 (OH) 2C00CH3), 3.15-4.01 (br, -OCH2CH20 ~; -0CHCH3CH20-; -NHCH (CH2C6H3 (OH) 2C00CH3), 4.03-4.26 (d, -OCH2CH2OCONHCHCH2C6H3 (OH) 2COOCH3), 4.55 (, -NHCHCH2C6H3 (OH) 2COOCH3), 5.30 (d, -NHCHCH2C6H3 (OH) 2COOCH3), 6.45-6.77 (Is, 2d, -NHCHCH2C6H3 (OH) 2COOCH3).

Example 6 Synthesis of DOPA-PA08 A procedure similar to that described above was used for the synthesis of the D0PA-PA07 conjugate to prepare and purify DOPA-PA08. The yield of the product was 49%. XH NMR (500 MHz, CDC13): d ppm 0.92-1.50 (br, -OCHCH3CH20-), 2.91-3.10 (m, -NHCHCH2C6H3 (OH) 2COOH), 3.15-3.95 (br, -OCH2CH20-; -OCHCH3CH20-) , 4.06-4.30 (d, -OCH2CH2OCO NHCHCH2C6H3 (OH) 2COOH), 4.54 (m, -NHCHCH2C6H3 (OH) 2COOH), 5.35 (d, -NHCHCH2C6H5 (OH) 2COOH), 6.50-6.80 (ls, 2d, NHCHCH2C6H3 (OH) 2COOH).

EXAMPLE 7 Colorimetry Assay The coupling efficiency of methyl ester DOPA and DOPA was determined to PLURONICs® F127 and F68 using the colorimetric method of Waite and Benedict. Briefly, the samples were analyzed in triplicate by diluting aliquots of standard and unknown solutions with IN of HCl to a final volume of 0.9 ml. 0.9 ml of the nitrite reagent (1.45 M sodium nitrite and 0.41 M sodium molybdate dihydrate) was added to the DOPA solution, followed immediately by the addition of 1.2 ml of 1N NaOH. Due to the time-dependent changes In absorbance intensity, care was taken to ensure that the time between the addition of NaOH and the recorded absorbance was 3-minutes for all standards and samples. Absorbance was recorded at 500 nm for all standards and samples. DOPA was used as the standard for both DOPA methyl ester and DOPA conjugates.

Example 8 Rheology Rheology measurements of gelation processes were performed using a Bohlin VOR Rheometer (Bohlin) Rheology, Cranbury, NJ). A 30 mm diameter stainless steel cone and a plate geometry with a cone angle of 2.5 degrees were used for all measurements. The temperature was controlled by a circulating water bath. The samples were cooled in the refrigerated one before the transfer of 0.5 ml of liquid solution of the apparatus. The measurements of storage and loss of modules were taken G 'and G "in the oscillatory mode at 0.1 Hz and a strain of 0.45%. The heating rate was 0.5 ° C / min except in the vicinity of the gelation temperature, when it is reduced to 0.1 ° C / min. The amplitude dependence of the strain of the viscoelastic data was verified for several samples, and the measurements were only made in a linear proportion where modules is independent of the amplitude of the strain. Mineral oil was applied to a ring around the outer surfaces of the sample compartment to prevent dehydration during measurements.

Example 9 Differential Scanning Calorimetry (DSC) DSC measurements were performed on a TA calorimeter Instruments DSC-2920 (TA Instruments, New Castle, DE). The spectrum is obtained for three samples of each concentration in heating and cooling cycles. The volumes of 20 μl samples in hermetically sealed aluminum pans were used and explored where they were recorded in a heating and cooling ratio of 3 ° C / min with an empty pan as a reference.

Example 10a Finished amino methoxy-PEG, mPEG-NH2 (2.0 g, 0.40 mmol, Mw = 2000 or 5000, Sun-Bio PEGShop), dicyclohexylammonium salt N-Boc-L-DOPA (0.80 mmol), HOBt were dissolved. (1.3 mmoles), and Et3N (1.3 mmoles) in 20 ml of a 50:50 mixture of dichloromethane (DCM) and DMF. Then DCM was added, and the reaction was carried out under argon at room temperature for 30 minutes. The reaction solution was washed successively with saturated sodium chloride solution, 5% NaHC03, dilute HCl solution and distilled water. The crude product was concentrated under reduced pressure and purified by column chromatography on Sephadex ® LH-20 with methanol as the mobile phase. The product, mPEG-DOPA, was further purified by precipitation in methanol cooled three times, dried under vacuum at room temperature and stored under nitrogen at -20 ° C. "" "H NMR (500 MHz, CDCl 3 / TMS): d 6.81-6.60 (m, 3H, C6H3 (OH) 2-), 6.01 (br, s, ÍH, OH-), 5.32 (br, s, ÍH , OH-), 4.22 (br, s, ÍH, C6H3 (OH) 2-CH2-CH (N-) - C (0) N-), 3.73-3.38 (m, PEO), 3.07 (m, 2H, PEO-CH2-NH-C (0) -), 2.73 (t, 2H, C6H3 (OH) 2-CH2-CH (N-) -C (O) N-), 1.44 (s, 9 H, (CH3 ) 3C-), 1.25 (s, 3 H, CH3CH20-).

H3C 0-f-CH2-CH2 -0-j-CH2-CH2 N c H Example 10b The synthesis and related procedures of the preceding example can be extended, by analogy, using other peptides and oligopeptides containing DOPA, if they are natural or synthetic in origin. Depending on a particular synthetic sequence, the use of an N-terminal protecting group may be optional. As referenced above, various other DOPA-like adhesive components can be used, as will be known to those skilled in the art aware of this invention. For example, B-amino acids and N-substituted glycine DOPA analogs can be used. With respect to particular DHPD adhesive components, a variety of polymeric components can be used according to the synthetic techniques and procedures described above. The polymer component can vary in molecular weight limited only by the corresponding solubility to which it refers. As mentioned above, a variety of other polymers can be used for antifouling and / or particle stabilization surfaces, such polymers include but are not limited to hyaluronic acid, dextrans and the like. Depending on the solubility requirements and desired surface effect, the polymer component may be branched, hyperbranched or dendrimeric, such components available either commercially or by well-known synthetic techniques. While the composition of Example 10a is the amidation product for the referenced starting materials, it will be understood that comparable polymer-DHPD conjugates can be prepared by coupling the N-terminus of a DHPD component to a final group, structure or side chain of a suitably functionalized natural or synthetic polymer, including those described above. For example, and without limitation, as illustrated above, a suitable polymer component terminating with a carbonate functionality can be used to provide the desired conjugate by reaction with N-terminus of the desired DHPD component.

EXAMPLE HI The consensus decapeptide repeat sequence (decapeptide from the mussel adhesive protein, MAPd, NH2-Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-DOPA-Lys-C02H) of foot protein 1 Mytilus was synthesized. mussel edulis (Mefp 1) by synthesis of solid phase peptide in Rink resin (0.6 mMol / g) using protected amino acids Fmoc, BOP, HOBt, and DIEA as activating agents and NMP as solvent. Fmoc deprotection was performed using a 25% piperidine solution in NMP for twenty minutes. The amino acid coupling was performed using two amino acid equivalent BOP-Fmoc: HOBt: DIEA in a 1: 1: 1: 1 ratio for twenty minutes, with an initial ten minute pre-activation step. In performing the decapeptide, the terms free amine of the decapeptide was coupled to activated methoxy-PEG-C02H (mPEG-SPA, Mw = 2k or 5k, Pardela Polymers) using carbodiimide chemistry. The PEG-decapeptide conjugates (mPEG-MAPd, 2k or 5k) were cleaved at 0 ° C for two hours using TMSBr ΔM in TFA, with EDT, thioanisole and m-cresol. The crude mPEG-MAPd products were precipitated in ether at 0 ° C, and purified by preparative HPLC using a Vydac 218TP reverse phase column (220x 22mm x 10 μm). The purity of the products was determined to be > 90% using analytical HPLC, and structures confirmed using MALDI-TOF-MS PerSeptive Biosystem.

Example 11b The synthesis and procedures of Example la can be extended analogously and consistent with the variations illustrated in Example 10b. In addition, other conjugates can be prepared using DOPA-containing polymers prepared by enzymatic conversions of tyrosine residues herein. Other techniques well known in the field of peptide synthesis can be used good effect to provide other desired protein sequences, peptide conjugates and resultant adhesive / anti-fouling effects.

Example 12a Gold surface was modified by adsorption of mPEG-DOPA or mPEG-MAPd (2k, 5k) of the solution in DCM or phosphate buffered saline (PBS, pH = 3, 7.4, and 11) at polymer concentrations varying from 0.1-75 mg / ml. Substrates were placed in a vial and immersed in mPEG-DOPA or mPEG-MAPd solution for up to 24 hours without agitation. In the removal of the solution, the substrates were rinsed with the appropriate solvent (DCM or H20 DI) to remove free polymer, and dried in vacuo. For comparison, identical surface modifications were made using PEG-monomethyl ether (mPEG-OH, avg Mw = 5000). Alternatively, a drop of solution containing mPEG-DOPA or mPEG-MAPd (10 mM in PBS, molecular weight PEG = 2000) was incubated on an Au coated glass cover (Au thickness ~ 10 nm) for 30 minutes at 37 ° C, after which the surface of the object cover was rinsed (3x) with PBS. The analysis of the modification surfaces for forward / backward contact angle, XPS and TOF-SIMS revealed the formation of a chemical layer of mPEG-DOPA or mPEG-MAPd. Figures 7A-C show the XPS spectrum for surfaces modified with mPEG-OH, unmodified and modified with mPEG-DOPA. As expected, the ether peak at 286.5 eV increased only slightly with the mPEG-OH treatment, while a dramatic increase was observed after absorption of mPEG-DOPA, which indicates a large presence of ether carbons. An ether peak of a pure PEG with the same binding energy has been reported in the literature. The smallest peak at 285.0 eV in Figure 7 can be attributed to the aliphatic and aromatic carbons in the main group PEG and DOPA, as well as some hydrocarbon combinations that result from the preparation / evacuation processes. SIMS flight time data corroborated the XIPS results. The TOF-SIMS analysis was carried out on Au substrate modified with mPEG-DOPA and unmodified, as well as mPEG-DOPA powder and a gold substrate exposed to mPEG-OH. The data was collected from each substrate for approximately 4 minutes. The positive ion spectrum of unmodified Au exhibits peaks (CnH2n +?) + And (CnH2n _?) +, Typical for hydrocarbon contamination (data not shown). Additional minor contaminants are present, including NH4 +, Na + and relatively small amounts of CaHbOc + species. Because the process used to deposit the Au film, a peak was observed for Cr a m / z ~ 52, in addition to the Au peak at m / z ~ 196.9. That exposes the gold surfaces to mPEG-OH results in only modest increment in the peaks representing PEG CaHbOc + fragments, which are probably attributable to non-specific contamination or absorption of mPEG-OH. This is evidenced by the peaks at m / z -225 (AuOC +) and 254 (AuOCCO +) which do not show dramatic increases when compared to substrates modified with mPEG-DOPA (Figures 8A-C). The positive ion spectrum of the Au surface modified with mPEG-DOPA was dominated by the presence of CaHbOc + peaks representing the adsorbed molecule. As illustrated in Figure 9, the relative abundance of C2H30 + and C2H50 + increased with respect to surfaces modified by mPEG-OH and unmodified. There is also a dramatic increase in the relative abundance of C3H + (m / z ~ 43) and CH50 + (m / z ~ 53), as well as, which can probably be attributed to hydrocarbon contamination or the fragmentation of t-butyl in the protection group Boc. Perhaps the most notable feature of the positive ion spectrum of the PEGylated Au substrate are the triplet repeats printed in the high mass ratio (Figure 10). Each of these triplet clusters corresponds to an Au-DOPA- (CH2CH20) n fragment. When further resolved, each subset within the triplet represents the addition of CH2, 'CH2CH2 or CH2CH20, as each of these peaks is approximately 14-16 amu apart. These repetition patterns are identified from n = 0-15, beyond which the signal is below detectable limits. In a negative ion spectrum for the virgin Au surface, little note was observed apart from sharply defined peaks for O ", HO" and Aun_ for n = 1-3 (data not shown). There is a small amount of hydrocarbon contamination present at m / z -13 (CH "), 24 (C2H2"), and 37 (C3H "). The negative ion spectrum of the PEGylated Au surface was dominated by the peak for C7Hn02 + am / z ~ 126 893. The presence of this peak at modest intensity in the Au spectrum modified with mPEG-OH suggests that it represents a large de-ethylene glycol fragment.The most interesting peaks remain in the high mass ratio (>200 m / z) and represent the catecholic oxygen coupling to Au. The spectrum suggests that an Au atom can be attached to six oxygen atoms, which correspond to three DOPAs. The contact angle data demonstrates a signature dependence on the character of the adsorption solvent used when modifying the gold films with mPEG-DOPA (data not shown). The modified surface in DCM showed a significantly lower? Than the unmodified surface (p <0.001) and the modified surfaces in all aqueous solutions (p < 0.05). Generally expressed, as the pH of the aqueous solutions increased, the hydrophilicity of the treated surfaces decreased, indicating a decreased ability to PEGylate the surfaces, perhaps due to the tendency of DOPA to be oxidized to its lower quinone adhesive form at pH elevated, an interpretation that is supported by previous studies that show the unopposed catechol form of DOPA is primarily responsible for adherence.

Example 12b The protein uptake and the binding / separation of the cells on treated and untreated coverslips were evaluated as follows. Surface plasmon resonance (SPR) experiments demonstrated that the DOPA-containing polymers bind rapidly to the surface and the resulting modified surfaces possess improved resistance to protein adsorption (Figure 11). The protein adsorption in gold modified with mPEG-MAPd (5k) is approximately 70% less than the unmodified gold surface. Analysis of fibroblasts grown on modified substrates showed a strong dependence on cell binding at concentrations of mPEG-DOPA (Figure 12), adsorption solvent and modification time used during the preparation of PEG-modified substrates. The modified surfaces for 24 hours with > 25 mg / ml of mPEG-DOPA or mPEG-MAPd exhibit a statistically significant reduction in cell attachment and separation (Figures 12-14). The gold surface modified with mPEG-MAPd (5k) exhibits a reduction of 97% in the total projected cell area and a 91% reduction in the density of cells bound to the surface.

Example 12c The modification illustrated in Example 12a, optionally varies as referenced in Examples 10b and llb, may be extended to other noble metals, including without limitation, silver and platinum surfaces. Such applications may be extended, as described herein, to include surface modifications of any bulk metal or metal alloy having a passive surface and rust. For example, volume metal oxide and related ceramic surfaces can be modified, as described herein. Such techniques can also be extended to semiconductor surfaces, such as those in the manufacture of integrated circuits and MEMS devices, as also illustrated below in the context of nanoparticle stabilization.

Example 13 Silicate crystal surfaces (cover glassware) were modified by absorption of mPEG-MAPd (2k) from a 10 mM solution in water, using the method described in Example 12a. The cell density of NIH 3T3 cells bound to glass surfaces, modified and unmodified, was evaluated as described above. The glass surface modified by 24 hours with mPEG-MAPd exhibits a 43% reduction in cell density compared to unmodified glass surfaces (Cell Density (cells / mm2): 75.5 +/- 6.5 in unmodified glass; / - 9.8 in glass modified with mPEG-MAPd).

Example 14a To illustrate the stabilization of metal oxides and, in particular, metal oxide nanoparticles, 50 mg of mPEG-DOPA (5k) was dissolved in water (18 MO-cm, Millipore) and combined with 1 mg of powder of magnetite (Fe304). Similar preparations were also prepared using an mPEG-NH2 (5k) Fluka) and an mPEG-OH (2k) (Sigma) as controls. Each of these aqueous solutions was sonicated using a Branson Ultrasonics 450 Sonicator for one hour while immersing in a 25 ° C bath. The probe has a frequency of 20 kHz, length of 160 mm, and an end diameter of 4.5 mm. The samples were then removed and allowed to stand at room temperature overnight to allow any unmodified magnetite to precipitate out of the solution. The solutions prepared using the control polymers (mPEG-NH2 and mPEG-OH) rapidly precipitated to give a brown solid and clear, colorless supernatant. The samples prepared using nanoparticles stabilized with PEG-DOPA, the sample was clear and brown. The light brown swirling envelope was isolated and dialyzed for three days in water using Spectro / Por® membrane tubing (MWCO: 15,000). After dialysis, the sample was lyophilized and stored under vacuum at room temperature until used.

Example 14b Nanoparticles stabilized with mPEG-DOPA were characterized by electron transmission microscope (TEM), thermogravimetric analysis (TGA) Fourier transform infrared spectroscopy (FTIR) and spectroscope UV / vis. The TEM results showed that most of the nanoparticles are of diameter of 5-20 nm (data not shown). TGA analysis of 0.4 mg magnetite stabilized with mPEG-DOPA indicated that the particles contain 17% by weight of mPEG-DOPA (data not shown). FTIR was performed on untreated magnetite showed relatively little absorbance with the wavelength ratio of 4000-400 cm "1, while the nanoparticles treated with mPEG-DOPA exhibit absorption bands at 800-1600 cm" 1 and 2600-3200 cm "1, confirming the presence of mPEG-DOPA.

Example 14c The magnetite nanoparticles stabilized with mPEG-DOPA are already dispersed in polar and aqueous organic solvents (eg, dichloromethane) to provide clear brown suspensions that are stable for months without the formation of noticeable precipitates. The suspensions of nanoparticles stabilized with mPEG-DOPA in several solvents were prepared by dispersing 1 mg of magnetite treated with mPEG-DOTA in 1 ml of water (18 MO-cm filtered using a Millex® AP 0.22 μm filter), DCM or Toluene. The suspensions were placed in a bath sonicator for ten minutes to disperse the nanoparticles. All three solutions are stable at room temperature for at least six months, while the magnetite control suspensions are modified and magnetite stabilized by mPEG-OH or mPEG-NH2 precipitated in less than 24 hours in each solvent.

Example 14d Suspensions of nanoparticles stabilized with mPEG-DOTA were also found to be stable under physiological salt concentrations. To determine if mPEG-DOPA can inhibit salt-induced nanoparticle aggregation, 0.3 mg of magnetite treated with mPEG-DOPA was placed in a quartz cuvette and combined with 0.7 ml of water (18 MO-cm filtered using a 0.25 filter). μ). Aliquots of NaCl solution (5 μl, 10 μl, 20 μl, 50 μl, 100 μl) were added sequentially to the cuvette and allowed to stand for ten minutes before taking the UV-VIS spectrum (Figure 15). The absorbance spectrum of nanoparticles stabilized with mPEG-DOPA suspended in solutions containing increased NaCl concentrations are almost identical, demonstrating that PEG-DOPA is effective in the stabilization of nanoparticles and prevents aggregation. The peak centered at 280 nm is indicative of the catechol side chain of DOPA.

Example 14e The methods and techniques illustrated in Examples 14a-14d can be extended to other metal oxides and ceramic nanoparticles, as will be understood by one skilled in the art, aware of this invention. Likewise, such applications of the present invention may further include use of a broad proportion of polymer-DHPD conjugates analogous to and consistent with those compositions and variants of that described in examples 10b and llb. As illustrated below in the preparation of semiconductor compositions, metal oxide and ceramic nanoparticles can be stabilized in situ in the presence of a polymer-DHPD conjugate of this invention.

Example 15a The demonstrated stabilization of metal nanoparticles, a commercial colloidal gold suspension is placed (Sigma, particle size 5 or 10 nm) inside of shielded dialysis (cut Mw of 8000 by 5 nm and 1500 by 10 nm) and dialyzed in ultrapure water for 2-3 days to remove sodium azide present in the commercial preparation . The dialyzed suspensions were then placed in small glass vials and mPEG-DOPA (10 mg / ml) was added. The samples were allowed to stand at room temperature for approximately 2 days, then the samples were dialyzed again to remove excess mPEG-DOPA. Untreated Au nanoparticle of 10 nm are unstable in the presence of NaCl and aggregate (Figure 16) while the treated Au nanoparticles remain stably suspended in the presence of aqueous NaCl (Figure 17).

Example 15b Various other metal nanoparticles, including but not limited to silver, platinum and the like, can be stabilized as described in the preceding example. While stabilization was demonstrated using a representative conjugate composition of this invention, several other compositions can be prepared analogous to and consistent with the alternate embodiments described in Examples 10b and llb. Comparable results can be obtained by in situ formation of the stabilized nanoparticles synthesized from the corresponding metal precursor in the presence of an appropriate adhesive conjugate polymer of this invention.

Example 16a The data of this example demonstrate the stabilization of semiconductor nanoparticles. CdS nanoparticles (quantum dots) were prepared by a standard method based on the slow mixing of diluted Cd (N03) 2 and NaS solutions. Fresh base solutions (2 mM) of Cd (C03) 2 and Na2S were prepared in nanopure water. The Na2S solution was injected slowly into 50 ml of Cd (N03) 2 solution using a gas permeable syringe at a rate of 20 μl s "1. The solution turned yellow with the addition of Na2S, and then 2 ml was injected. of Na2S, resembled a yellow precipitate due to the aggregation of CdS nanoparticles.The CdS precipitate was isolated and dried for further use.Using the method described above for magnetite, the dry CdS powder was dispersed in a mPEG-DOPA solution by sonication To provide a light yellow solution The yellow aqueous suspension was stored in the dark for several months at room temperature without visible formation of precipitate Control experiments were performed in the absence of polymer and in the presence of mPEG-OH or mPEG-NH2 providing a yellow precipitate and a clear, colorless supernatant The nanoparticles stabilized with mPEG-DOPA remain stably suspended in the presence of charged NaCl (Fig. ra 18).

Example 16b The results of this example illustrates the in situ formation of stabilized semiconductor nanoparticles. The CdS nanoparticles (quantum points) were formed in the presence of mPEG-DOPA by slowly mixing diluted methanolic solutions of Cd (N03) 2 and Na2S. Freshly prepared base solutions (2 mM) of Cd (N03) 2 and Na2S were prepared in methanol. 25 mg of mPEG-DOPA (molecular weight PEG = 2000) were dissolved in 5 ml of 2 mM Cd (N03) 2 in methanol, then 5 ml of a 2MM Na2S solution was added slowly with a syringe at a ratio of 20 μl s-1. The solution gradually turned yellow during the addition. No yellow precipitates were observed, and the dynamic light scattering revealed particles with an average diameter of 2.5 nm. Control experiments were performed in the absence of polymer or in the presence of mPEG-OH providing yellow precipitate and a clear, colorless supernatant. Various other substrates of inorganic particles can be prepared, as will be understood by those skilled in the art, depending on the material selected and corresponding to ionic substitution or exchange reaction, as carried out in the presence of an adhesive composition by the class described in this document.

Example 16c The polymer conjugate compositions of this invention can also be used to stabilize a variety of other semiconductor materials. For example, central skeleton nanoparticles can be surface stabilized in accordance with this document.

Example 17 The optimization of experiments of the Examples 17-20 was performed with mPEG-DOPA-5K. Several parameters were examined to optimize the adsorption of mPEG-DOPA in solution gold, which includes type and pH of solvent, adsorption time and solution concentration of mPEG-DOPA. The bound and separated cells do not vary widely with used adsorption solvent. The number of cells in the substrates and their total projected area is not significantly different between DCM and three different aqueous solutions. Substrates adsorbed on neutral, basic and organic mPEG-DOPA solutions all have significantly improved antifouling properties when compared to the unmodified substrate (p <; 0.01). Although no differences in cell binding and separation between solutions were observed, contact angle data should support the use of an organic solvent in an optimal modification protocol as a means of reducing catechol oxidation. Additionally, only the DCM modified surface demonstrated significantly fewer cells on the surface and lower total projected cell area.

Example 18 The bound and separated cells show a strong dependence on solution concentration of mPEG-DOPA (Figure 12). Previously 25 mg / ml of mPEG-DOPA, significantly fewer cells bound and separated on the modified substrate than on the surface of virgin gold (p <0.001) and the modified surface in a 10 mg / ml solution (p < 0.05). Below 10 mg / ml, there are no differences in cell attachment and separation compared to the unmodified substrate. There is no difference in the binding and cell separation observed between the modified surfaces in mPEG-DOPA solutions ranging from 25-75 mg / ml when compared to some other.

Example 19 Few bound and separated fibroblasts were observed with increased duration of adsorption of mPEG-DOPA, too. Although cell attachment and separation seemed to decrease as little as 5 minutes of substrate modification, and adsorption time of 24 hours resulting in significantly fewer cells bound and separated in the PEGylated substrate than in the unmodified substrate (p <0.001) and substrates treated for short periods (p < 0.05).

Example 20 The morphology of fibroblasts grown on both unmodified and modified PEG surfaces was examined via electron microscope (Hitachi 3500 SEM). Fibroblasts in unmodified Au and Au modified with mPEG-OH are generally flat and well separated, while those cultured in modified Au mPEG-DOPA are so far less separated (Figures 14A-C). It should also be noted that in the MPEG-DOPA surface, a lower number of cellular processes was observed in the other structures which contribute to cell adhesion via integrins and focal adhesion. Figure 13 illustrates the differences in binding and separation of fibroblasts in uncovered Au, Au treated with mPEG-OH and Au modified with mPEG-DOPA 5K, mPEG-MAPd 2K or mPEG-MAPd 5K under optimal conditions (50 mg / ml per 24 hours) . The surfaces modified with DOPA-containing conjugates have significantly less cell adhesion and separation than either of the other two surfaces. The mPEG-MAP 5K modification, although, considered by a reduction of 97% in the total projected cell area and a 91% reduction in density of cells on the surface, a reduction so far greater than that achieved by mPEG-DOPA 2K. The differences in adhesion and cell separation between surfaces modified with DOPA and PEG conjugated with MAPd in Figure 13 can probably be attributed to the physical characteristics of the associated attached PEG layer. The SPR results analyzes indicate that MAPd-PEGs form thickness, plus robust thin layers with a high concentration of PEG per unit area that makes the PEGs fixed to DOPA of equivalent molecular weight. The thin layers of thickness resulting from PEGylation mediated by MAPd are more successful in inhibiting protein inhibition and, in turn, cell adhesion.

Example 21 Synthesis of Boc-DOPA (TBDMS) 2-0Su N-Hydroxysuccinimide (NHS) (0.110 g, 0.95 mmol) was added to a solution of Boc-DOPA (ATBDMS) 2 (0.500 g, 0.95 mmol) in dry dichloromethane ( DCM) (DCM) (8.0 ml). The solution was stirred in a cold bath, and 1,3-dicyclohexylcarbodiimide (DCC) (0.197 mmol) was added under a nitrogen atmosphere. The reaction was stirred for 20 minutes at 0 ° C and then warmed to room temperature and stirred for an additional 4 hours. The reaction mixture was filtered to remove the urea derivative and subsequently vaporized to 1/5 of its original volume. The solution was cooled to 4 ° C, allowed to stir for 2 hours to precipitate the remaining urea derivative, filtered and evaporated to give Boc-DOPA (TBDMS) 2-OSu as a white foam (0.567 g, 96% yield). %).

Example 22 Synthesis of Boc-DOPA2 (TBDMS) 4 Boc-DOPA (TBDMS) 2-OSu (0.567 g, 0.91 mmol) was dissolved in dimethylformamide (DMF) (2.5 ml) and DOPA (TBDMS) 2 (0.405 G, 0.95 mmol ) was added once under a nitrogen atmosphere. The mixture was stirred in an ice bath, and diisopropylethylamine (DIEA) (158 μl, 0.91 mmol) was added via drip via a syringe. After 20 minutes, the reaction was warmed to room temperature, stirred for an additional 17 hours, filtered (if necessary), diluted with ethyl acetate (EtOAc) (40 ml), transferred to a separate funnel and filtered. washed with 5% aqueous HCl. The aqueous layer was extracted again with EtOAc. The organic layers were combined and washed with 5% aqueous HCl (3x), H20 (lx), dried with MgSO4, and evaporated to give Boc-DOPA2 (TBDMS) as a white foam (0.83 g, 98% yield). %).

Example 23 Synthesis of Boc-DOPA2 (TBDMS) 4 ~ OSu The procedure of Example 21 was repeated using Boc-DOPA2 (TBDMS) 4 to obtain Boc-DOPA2 (TBDMS) -OSu.

Example 24 Synthesis of Boc-DOPA3 (TBDMS) 6 The procedure of Example 22 was repeated using Boc-DOPA2 (TBDMS) 4-OSu to obtain Boc-DOPA3 (TBDMS) 6.

Example 25 Synthesis of D0PA2 Boc-DOPA2 (TBDMS) (0.5 g, 0.54 mmol) was dissolved in HCl / saturated EtOAc (3 mL), and the solution was stirred under nitrogen. After 5 hours, additional HCl gas was bubbled gently through the solution for 25 minutes. The reaction was allowed to stand overnight, and subsequently concentrated to the original volume. The resulting precipitate was collected by centrifugation, washed with cold EtOAc (3x), and dried to provide DOPA2 as a white powder (0.15 g, 74% yield). The product was further purified by RP-HPLC, and characterized by ESI-MS.

Example 26 Synthesis of D0PA3 Boc-DOPA3 (TBDMS) 6 (1.06 g, 0.79 mmol) was dissolved in HCl / saturated EtOAc (3 mL) and the solution was stirred under nitrogen. After 12 hours, additional HCl gas was bubbled gently through the solution for 30 minutes, and the reaction was allowed to continue for 40 hours. More HCl gas was bubbled through the solution for another 30 minutes, and the stirring was stopped. The resulting precipitate was collected by centrifugation, washed with cold EtOAc (3x) and dried to give DOPA3 as a white powder (0.424 g, 96% yield). The product was further purified by preparative RP-HPLC, and characterized by ESI-MS.

Example 27 Synthesis of mPEG-DOPAl -3 A solution of 0. ÍM of borate buffer (50 ml, pH 8.5) was degassed with argon for 20 minutes, and L-DOPA (0.197 g, 1.0 mmol) was added. After the solution was stirred for 15 minutes, PEG-SPA terminated with methoxy (mPEG-SPA) 5K (0.5 g, 0.1 mmol) was added in portions, and the reaction was allowed to stir for 3 hours. The resulting clear solution was acidified to pH 1-2 with aqueous HCl, and extracted with DCM (3x). The combined organic layers were washed with 0.1M HCl, dried over MgSO4 and concentrated. The remaining residue was dissolved in DCM and precipitated with ethyl ether three times to provide mPEG-DOPA as a white powder (0.420 g, 84% yield). The product was characterized by MALDI-MS and XE NMR spectroscopy.

Example 28 Surface Modification Solid metal substrates (Al, 316 L, stainless steel and NiTi) were milled and polished, latterly with 0.04 m colloidal silica (Syton, DuPont). Si pellets were evaporated with either 20 nm Ti02 or 10 nm TiO2 / 40 nm Au using an Edwards FL400 electron beam evaporator at < 10 ~ 6 Torr and subsequently it is given in pieces of 8 mm x 8 mm. All substrates were ultrasonically cleaned for 20 minutes in each of the following: Contrad70 at 5% (Fisher Scientific), H20 ultrapure, acetone, and petroleum ether. Subsequently, the surfaces were further cleaned by exposure to 02 plasma (Harrick Scientific) at 150 mTorr and 100 W for 5 minutes. To prevent the formation of a gold oxide layer (Au203), some substrates were not exposed to 02 plasma. To generate an analogous biopolymer surface, the glass object cover (Fischer Scientific) was cleaned as described above and immersed in a 0.01% solution of poly-L-lysine (Sigma) for 5 minutes, rinsed with ultra pure H20 and dried under nitrogen. To explore a variety of modification conditions with a minimum number of samples, a Robust Desing procedure of new elements was used. Substrates were modified under direct coding conditions by immersion in mPEG-DOPAi-3 solutions in 0.6 M K2S04 buffered with 0.1 M MOPS at 50 ° C, modification time and concentration of mPEG-DOPA were modified as shown in Table 1. The modified substrates were subsequently rinsed with ultra pure H20 and dried under a stream of nitrogen.

Table 1 Example 28a Substrates Ti02 Substrates Ti0 were modified under direct coding point conditions by immersion in mPEG-DOPA? _3 solutions in 0.6 K K2SO buffered with 0.1 m N-morpholinpropanesulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28b 316L stainless steel (Goodfellow, Devon PA) was modified under direct coding point conditions by immersion in mPEG-DOPA solutions;? -3 in 0.6 M K2SO4 buffered with 0.1 M N-morpholinepropanesulfonic acid (MOPS) a 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28c Al203 (Goodfellow, Devon PA) was modified under direct coding point conditions by immersion in mPEG-DOPA? _3 solutions in 0.6 M K2SO4 buffered with 0.1 M N-morpholinepropensulfonic acid (MOPS) at 50 ° C per 24 ° C. hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28d Si02 (thermal oxide 1500A, University was modified Wafer, South Boston, MA) under direct coding point conditions by immersion in mPEG-DOPA1-3 solutions in 0.6 M K2SO4 buffered with 0.1 M N-morpholinepropensulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28e NiTi alloy (10 mm x 10 mm x 1 mm) was obtained from Nitinol Devices & Components (Fremont, CA) and modified under direct coding point conditions by immersion in mPEG-DOPA? _3 solutions in 0.6 M K2S04 buffered with 0.1 M N-morpholinepropensulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28f Au was modified (electron beam evaporated in Si pill from University Wafer) under conditions of direct coding point by immersion in solutions mPEG-DOPA1-3 in 0. 6 M of K2S04 buffered with 0.1 M of N-morpholinepropensulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28g A 2? 3 was modified (Au samples as described in Example 28f were exposed in an oxygen plasma to form Au203) under direct coding point conditions by immersion in solutions mPEG-D0PA? _3 in 0.6 M K2S04 buffered with 0.1 M of N-morpholinepropensulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28h GaAs (University .Wafer, South Boston, MA) was modified under direct coding point conditions by immersion in mPEG-D0PA? _3 solutions in 0.6 M K2S0 buffered with 0.1 M N-morpholinepropensulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

Example 28i p-L-Lys surfaces were prepared by immersing the glassware cover (Fisher Scientific) in a 0.01% solution of poly-L-lysine (p-L-Lys, Sigma) for 5 minutes, rinsing with ultra pure H20 under N2. They were then modified under direct coding point conditions by immersion in mPEG-DOPA? _3 solutions in 0.6 M K2SO4 buffered with 0.1 M N-morpholinepropensulfonic acid (MOPS) at 50 ° C for 24 hours. The modified substrates were rinsed with ultra pure H20 and dried under a stream of nitrogen.

EXAMPLE 29 Cell Adhesion 3T3 Swiss fibroblasts (ATCC, Manassas, VA) were grown from inlet 12-16 normally at 37 ° C and 5% C02 in Dulbecco's modified Eagle's medium (DMEM) (Cellgro, Herdon, VA) supplemented with 10% fetal bovine serum (FBS) (Cellgro, Herndon, VA), 100 g / ml penicillin, and 100 U / ml streptomycin. Before the cell adhesion assays, the fibroblasts were harvested using 0.25% trypsin-EDTA -, they were resuspended in growth medium and counted with a hemacytometer.

General Procedures for Four-Hour Trials. Test substrates were prepared in polystyrene plates of the 12-well tissue culture with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1, 1-dioctadecyl-3, 3,3 ', 3'-tetramethylindolcarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on the size of the substrate) from random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT digital camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using a threshold in MetaMorph. (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and the standard deviation of the measurements were reported.

Example 29a Substrate Ti02 (4 hour test) Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3 ', 3'-perchlorate tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29b TiO substrate? (long term studies) For long term studies on Si02 substrates they were reseeded twice a week at the same density as for the 4 hour trial. At periodic intervals, the non-adherent cells were removed by aspirating the medium into each cavity.

Example 29c 316L stainless steel substrate (4 hour test) Test substrates were prepared on 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in peraformaldehyde at 3.7% for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3 ', 3'-perchlorate tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29d Substrate A1203 (4 hour test) Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3,3'-perchlorate. tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29e Substrate Si02 (4 hour test) Test substrates were prepared on 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3,3'-perchlorate. tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29f NiTi Substrate (4 hour test) Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3,3'-perchlorate. tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29g Au Substrate (4 hour test) Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assayAdherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethyldocarbocyanine (Dil) perchlorate (Molecular Probes, Eugene , OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) from random locations on each substrate using a Leifca epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29h Au2 substrate 3 (4 hour test) Test substrates were prepared on 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3 ', 3'-perchlorate tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29i GaAs Substrate (4 hour test) Test substrates were prepared on 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3,3'-perchlorate. tetramethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements were reported.

Example 29j p-L-Lys substrate (4-hour test) Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 ml of DMEM with FBS for 30 minutes at 37 ° C and 5% C02. The cells were seeded in the substrates at a density of 2.9 x 10 3 cells / cm 2 and maintained in DMEM with 10% FBS at 37 ° C and 5% C02 for 4 hours. For the 4 hour cell adhesion assay, the adherent cells were fixed in 3.7% peraformaldehyde for 5 minutes and subsequently stained with 5 μM of 1,1'-dioctadecyl-3,3,3,3'-perchlorate. tetra ethylindocarbocyanine (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37 ° C. Quantitative cell attachment data were obtained by acquiring 9-16 images (depending on substrate size) of random locations on each substrate using a Leica epifluorescent microscope equipped with a SPORT RT camera (Diagnostic Instruments, Steriing Heights, MI). The resulting images were quantified in terms of total projected cell area using threshold in MetaMorph (Universal Imaging Corporation ™, a subsidiary of Molecular Devices Corporation, Dowmington, PA). The mean and standard deviation of the measurements are reported.

EXAMPLE 30 Twenty four hour modification of substrates and four test hours Each surface was modified for 24 hours in a 1.0 mg / mL solution of mPEG-D0PA3 (or mPEG-OH as a control) at 50 ° C at the pH values shown in Figure 24. A four-hour cell spreading and adhesion test was conducted as described above, in Example 9. The results are shown in Figure 24. The cell adhesion resistance was conferred for all treated substrates with mPEG-DOPA3. Cell adhesion and spreading on substrates treated with mPEG-OH, does not differ from unmodified surfaces (data not shown).

Example 31 Surfaces and Surface Preparation Silicon pellets (WaferNet GmbH, Germany) were coated with Ti02 (20 nm) by physical vapor deposition using reactive magnetron ion spray (PSI, Villigen, Switzerland). The metal oxide coated pellets were subsequently cut into 1 cm x 1 cm pieces for ex situ epsilometry measurements. The optical waveguide chips for OWLS measurements were purchased from Microvacuum Ltd. (Budapest, Hungary) and consist of an AF45 glass substrate (8 x 12 x 0.5 mm) and a waveguide waveguide surface layer. Sio.25Tio. S02 200 mm thick. An 8 nm Ti02 layer was deposited on top of the waveguide layer under the same conditions described above for the silicon wafers. Prior to the polymer modification, Ti02-coated silicon wafers and waveguide chips were sonicated in 2-propanol for 10 minutes, rinsed with ultrapure water, and dried under a stream of nitrogen, followed by an exposure of 3 minutes to plasma 02 (Harrick Scientific, Ossining, USA), to remove all the organic components from the surface. After the OWLS measurements, the waveguides were regenerated to be reused by sonication (10 minutes), in cleaning solution (300 mM HCl, 1% detergent, Roche Diagnostics, Switzerland) and subsequently rinsed with ultrapure water to remove the adsorbates.

Surface Modification Surfaces were modified by mPEG-DOPA1-3, produced according to Example 27, for 24 hours at a polymer concentration of 1.0 mg / ml using a turbidity point absorber (PT buffer: 0.6M of buffered K2SO4) at pH = 6.0 with 0. MO of MOPS) at temperatures ranging from 25 ° C to 50 ° C. After modification, the substrates were rinsed with water, dried in N2 stream, and immediately analyzed as described below.

X-ray Photoelectron Spectroscopy (XPS) Measurements High resolution and recognition spectra were collected in a SAGE 100 (SPECS, Berlin, Germany), using a standard AlKa (non-monochromatized) X-ray source, operating at 325 W (13kV, 25mA) and an angle taken from 0o, defined as the angle between the photoelectron detector and the normal surface. The energy passage of 50 eV and 14 eV was used for the high resolution and recognition spectrum, respectively. The pressure of the analytical chamber remains below 2 x 10"8 Pa during the acquisition of data All the XPS spectra were referenced to the aliphatic hydrocarbon component of the Cls signal at 284.7 eV. CasaXPS software, using Shirley background subtraction and the sum of a Lorentzian function at 10% and Gaussian at 90% The measured intensities (peak areas) were converted to normalized intensities by atomic sensitivity factors, of which, they were calculated the atomic compositions of the surfaces.

The average values obtained from three substrate replicates are reported in Tables 7-8. The standard deviations were typically < 10% of the medium and are omitted for clarity.

Table 7 Quantitative analysis of XPS data for atomic concentrations of Ti02 surfaces modified with mPEG-DOPA Table 8 Calculated atomic ratios of XPS data for atomic ratios of Ti02 surfaces modified with mPEG-DOPA ^ Contributions A, B and C, are defined in Table 7 Spectroscopic Elipsometry For ELM measurements, substrates dispersed from Ti02, were modified ex situ, as described above, with the temperature of the modification solution varying from 25 ° C to 50 ° C. After modification, the substrates were rinsed with H20, incubated at room temperature in 10 mM HEPES buffer (pH = 7.4) for 48 hours, rinsed again with H20 and dried with N2. To examine protein resistance, modified and unmodified substrates were exposed to pure human serum for 15 minutes, rinsed with water, and dried in a stream of N2. ELM measurements were made on a M-2000D spectroscopic eliopsometer (JA Woollam Co., Inc., Lincoln, USA) at 65 °, 70 ° and 75 °, using wavelengths from 193-1000 nm, before and immediately after of the modification, after incubation with HEPES and after exposure to serum. The ELM spectrum was adjusted with multiple layer models in the WVASE32 analysis software, using the optical properties of a generalized rubber polymer layer (An = 1.45, Bn = 0.01, Cn = 0), to obtain the "dry" thickness of the adsorbed PEG and thin layers. (The "dried" or dehydrated thickness is that measured under ambient conditions after drying with N2). The average thickness measured of the three replicates is reported in Tables 9-10.

Table 9 Effect of Adsorption of temperature on the thickness of thin layers of PEG in Ti02a Thickness temperature (A) adsorption (° C) 25 10.6 + 1.8 28 13.9 + 0.3 31 16.4 + 1.1 34 19.4 + 1.9 37 19.6 + 3.5 40 22.5 + 2.0 45 24.4 + 2.0 50 33.8 + 4.6 Ti02 surfaces were exposed to mPEG- DOPA3 (1 mg / ml) for 2 hours, after which each surface was rinsed for 48 hours in HEPES.

Table 10 Apparent thickness (Á) of organic thin layers in Ti02, measured by spectroscopic ellipsometry a Ti02 surfaces were exposed to mPEG-D0PA3 / l mg / ml) for 0-1080 minutes, rinsed with water, incubated for 48 hours in HEPES and then exposed to serum for 15 minutes. bel pure increase in the thickness of the thin layer after exposure to serum was 0.5, the approximate resolution of the ELM technique.

Light Wave Spectroscopy Optical Waveguide (WLS) Waveguides coated with Ti02 in 2-propanol and 02 plasma were cleaned as described above. The clean guides were mounted on the measuring head of an OWLSllO (Microvacuum Ltd.) and stabilized for at least 48 hours at room temperature in turbidity point buffer (PT buffer: 0.6 M K2S04 buffered at pH = 6.0 with 0.1M MOPS). The stabilization period allowed the exchange of ions on the surface of Ti02, reach equilibrium and obtain a stable baseline. To monitor polymer adsorption, mPEG-DOPA was injected into PT buffer in flow stop mode, followed by CP buffer to remove unbound PEG, after which, the signal was allowed to stabilize. The coupling angles aTM and aTE were recorded and converted to refractive indexes (NTM, NTe), for the software provided by the manufacturer.

Real-time changes in the effective refractive index of the sensor were converted to adsorbed mass using the Feijter formula (de Feijter, 1978 # 14). The increase of the refractive index, dn / dc, for each mPEG-DOPA polymer, was calculated by linear interpolation between 0.13 cm3 / g for pure PEG and 0.18 cm3 / g for pure poly (amino acid). For protein adsorption experiments, the temperature of the measuring head was equilibrated at 37 ° C, until the signal stabilized, after which the serum was injected for 15 minutes, followed by injection of buffer. The substantial differences in the adsorbed mass were not observed with increases in the time of exposure to the serum.

Example 32 Synthesis of N-methacryloyl 3,4-dihydroxyl-L-phenylalanine 1.15 g (5.69 mmol) of Na 2 B 407 was dissolved in 30 ml of water. The solution was degassed with Ar for 30 minutes, after which 0.592 g (3.0 mmol) of L-DOPA was added and stirred for 15 minutes. Then 0.317 g (3.0 mmol) of Na 2 CO 3 was added, the solution was cooled to 0 ° C, and 0.3 ml (3.0 mmol) of methacryloyl chloride was added slowly with stirring. The pH of the solution was maintained above 9 with Na 2 CO 3 during the reaction. After stirring for 1 hour at room temperature, the solution was acidified to a pH of 2 with concentrated HCl. The mixture was extracted with ethyl acetate three times. After washing with 0.1N HCl, and drying over anhydrous MgSO, the solvent was removed in vacuo to give a crude light brown solid. The product was further purified by elution of a column of silica gel with dichloromethane (DCM) and methanol (95: 5). After evaporation of the solvent, a sticky solid with a product yield of 35% was obtained. X H NMR (500 MHz, acetone-d 6): and 7.1 d (H H, -NH-); 6.6-6.8 (3H, C6H3 (OH) 2-); 5.68 s (ÍH, CHH =); 5,632 s (unknown peak); 5.33 s (ÍH, CHH =); 4.67 m (ÍH, -CH-); 2.93-3.1 m (2H, CH2-); 1877 s (3 H, -CH3).

Example 33 Synthesis of 3,4-bis (t-butyldimethylsilyloxy) -L-phenylalanine 3. 60 g (24.0 mmol) of TBDMS-C1 was dissolved in 18 ml of anhydrous acetonitrile. 1.60 g (8.0 mmol) of L-DOPA was added to the solution, the suspension was stirred and cooled to 0 ° C, and 3.6 ml of DBU (24.0 mmol) was added. The reaction mixture was then stirred for 24 hours at room temperature. The addition of cold acetonitrile to the reaction solution resulted in a colorless precipitate. The precipitate was filtered and washed with cold acetonitrile several times, followed by drying in vacuo. White powder was obtained with a yield of 78%. 1 H NMR (500 MHz, methanol-d); and 6.7-6.9 (e H, C6H3 (0-Si-) 2-); 3.72 (m, 1 H, -CH-); 2.82-3.2 (m, 2 H, -CH2-); 1.0 (d, 18 H, -C (CH3)); 0.2 (d, 12 H, Si-CH3).

Example 34 Synthesis of 3,4-bis (t-butyldimethylsilyloxy) -N-butyloxycarbonyl-L-phenylalanine 1.60 g (3.77) of 3,4-bis (t-butyldimethylsilyloxy) -L-phenylalanine was added to 10 ml of water containing 0.34 g (4.05 mmol) of NaHCO 3. 0.96 g was added (4.30 mmol) of di-t-butyl dicarbonate in 10 ml of tetrahydrofuran and the reaction mixture was stirred for 24 hours at room temperature. After evaporation of tetrahydrofuran, 10 ml of water was added to the residue. The solution was acidified with diluted HCl to pH 5 and extracted three times with ethyl acetate. After drying over anhydrous MgSO, the solvent was removed in vacuo. The crude product was purified by column chromatography (silica gel, eluent, 10% methanol in DCM). A white solid was obtained with a 70% yield after evaporation of the elution solvent. 1 H NMR (500 MHz, methanol-d); and 6.68-6.81 (3 H, C6H3 (O-Si-) 2-); 4.28 (m, 1 H, -CH-); 2.78-3.08 (m, 2 H, -CH3-); 1.4 (s, 9 H, -0-C (CH3) 3); 1.0 (d, 18 H, -Si-C (CH 3) 3); 0.2 (d, 12 H, Si- (CH3) 2).

Example 35 Synthesis of pentaf luoro phenyl ester of 3,4-bis (t-butyldimethylsilyloxy) -Nt-butyloxycarbonyl-L-phenylalanine 1 g (1.90 mmol) of 3,4-bis (t-butyldimethylsilyloxy) -N- was dissolved. butyloxycarbonyl-L-phenylalanine and 0.351 g (1.90 mmol) of pentafluorophenol, in a solvent mixture of 24 ml of dioxane and 1 ml of DMF, and 0.432 g (2.10 mmol) of DCC was added at 0 ° C. The solution was stirred for 1 hour at 0 ° C and for 1 hour at room temperature, after which the solution was filtered to remove the dicyclohexylurea and evaporated in vacuo. Product 4 was purified by column chromatography (silica gel, eluent, hexane / ethyl acetate = 11.2). After removing the eluent, a sticky, pure white solid was obtained, with a yield of 55%. - "? NMR (500 MHz, CDC13): and 6.65-6.81 (3 H, C6H3 (O-Si-) 2-); 4.85 (m, 1 H, -CH-); 3.05-3.2 (m, 2 H , -CH3-), 1.41 (s, 9 H, -0-C (CH3) 3), 1-0 (d, 18 H, -Si-C (CH3) 3), 0.2 (d, 12 H, Si - (CH3) 2).

Example 36 Synthesis of N- (13'-amino-4 ', 1", 10'-trioxatridecanyl) -t-butyloxycarbonyl-3', 4'-bis (t-butyldimethylsilyloxy) -L-phenylalanamide 0.869 g ( 1.26 mmol) of 3,4-bis (t-butyldimethylsilyloxy) -Nt-butyloxycarbonyl-L-phenylalanine pentafluorophenyl ester in 10 ml of DMC, dropwise to a mixture of 2.08 ml (9.44 mmol) of 4, 7, 10- trioxa-1, 13-tridecandiamine and 1.32 ml (9.44 mmol) of Et 3 N in 1 ml of DMF for 30 minutes at 0 ° C. The solution was stirred at room temperature for another 2 hours, and then the solvent was removed under vacuum. The crude product was loaded onto silica gel and eluted with DCM, 5% methanol in DCM, 10% methanol in DCM and 15% methanol in DCM. The solvent was removed under vacuum to provide 5 as a white solid. The yield was 63%. 1 H NMR (500 MHz, acetone-de); and 7.38 (m, 1 H, -CONH-); 6.60-6.80 (3 H, C6H3 (0-Si-) 2-); 5.26 (m, 1 H, -CONH-); 4.30 (m, 1 H, -CH-); 3.4-3.8 (m, 12 H, -CH20-, 3.03-3.4 (m, 4 H, -CH2-NH-, -CH-NH2); 2.78-3.02 (m, 2 H, -CH2-); 2.0 ( m, 2 H, -CH2-), 1.7 (m, 2 H, -CH2-), 1.39 (s, 9 H, -0-C (CH3) 3), 1.0 (d, 18 H, Si-C ( CH3) 3) / 0.2 (d, 12 H, Si-C (CH3) 2).

Example 37 Synthesis of N- (13- (N'-t-Butyloxycarbonyl-L-amino-3 ', 4'-bis (t-butyldimethylsilyloxy) -4,7,7-trioxa tridecanyl) -methacrylamide 0.57 g ( 0.79 mmol) of N- (13'-Amino-4 ', 7', lO'-trioxatridecanyl) -t-butyloxycarbonyl-3 ', 4'-bis (t-butyldimethylsilyloxy) -L-phenylalanamide and 0.166 ml (1.18 mmol) of Et3N, in 5 ml of anhydrous chloroform, to which 0.176 ml (1.18 mmol) of methacrylic anhydride was added. The solution was stirred at room temperature for 3 hours, then the solvent was removed in vacuo. The pure 6 was obtained by column chromatography (silica gel, eluent: ethyl acetate), as a white, sticky solid, with a yield of 61%. 1 H NMR (500 MHz, CDC13) and 6.60-6.80 (3 H, C6H3 (O-Si-) 2-); 6.40 (m, 1 H, -CONH-); 5.71 s (1H, CHH =); 5.30 s (ÍH, CHH =); 5,096 (m, 1 H, -CONH-); 4.21 (m, 1 H, -C H-); 3.2-3.65 (m, 16 H, -CH20, -CH2-NH-CH2-NH2); 2.80-2.99 (, 2 H, -CH2-); 1.96 s (3 H, -CH3); 1.81 (m, 2 H, -CH2-); 1.68 (m, 2 H, -CH2-); 1.40 (s, 9 H, -0-C (CH3) 3); 1.0 (d, 18 H, Si-C (CH 3) 3); 0.2 (d, 12 H, Si-C (CH3) 2).

Example 38 Synthesis of N- (13- (N'-t-Boc-L-3 ', 4'-dihydroxy-phenylalaninamido) -4,7,10-trioxatridecanyl) -methacrylamide To a 10 ml beaker, 0.344 was added. g (0.433 mmol) of N- (13- (N '-t-butyloxycarbonyl-L-Amino-3', 4'-bis (t-butyldimethylsilyloxy) -4,7, 10-trioxatridecanyl) -methacrylate, 3 ml of THF and 0.137 g (0.433 mmol) of TBAF The solution was stirred at room temperature for 5 minutes, then 3 ml of 0.1 N HCl was added The solution was extracted three times with DCM, after which the solvent was evaporated in vacuo The 7 was obtained as a white solid by column chromatography (silica gel, eluent: 7% methanol in DCM), with a yield of 63% XE NMR (500 MHz, acetone-de), and 7.90 (m, 1 H, -CONH-); 7.23-7.40 (d 2 H, C6HH2 (OH) 2-); (3 H, C6HH2 (OH) 2-; 5.930 (m, 1 H, -CONH-); 5.71 s (ÍH, CHH =), 5.30 s (ÍH, CHH =), 4.20 (m, 1 H, -CH-), 3.1-3.60 (m, 16 H, -CH20, -CH2-NH-, -CH2 -NH2), 2.70-2.95 (m, 2 H, -CH2-), 1.96 s (3 H, -CH3), 1.78 (m, 2 H, -CH2-), 1.65 (m, 2 H, -CH2- ); 1.39 (s, 9 H, -0-C (CH3) 3).

Example 39 Synthesis of PEG-Diacrylate (PEG-DA) 40 g (5 mmol) of PEG were dried by azeotropic evaporation in benzene and then dissolved in 150 ml of DCM. 4.18 ml (30 mmol) of Et3N and 3.6 ml (40 mmol) of acryloyl chloride were added to the polymer solution. The mixture was refluxed with stirring for 5 hours and allowed to cool to room temperature overnight. Ether was added to the mixture to form a weak yellow precipitate. The crude product was then dissolved in saturated NaCl solution, which was heated to 60 ° C to form two layers. DCM was added to the top layer and MgSO was added to remove the moisture. After filtration of MgSO, the volume of the solvent was reduced in vacuo and the sample was precipitated in ether. The final product was then vacuum dried and stored at -15 ° C. The yield was 75%. 1H NMR (400 MHz, D20): d 6.47 (d, 1 H, CHH = C-); 6.23 (m, 1 H, C = CH-C (= 0) -0-); 6.02 (d, 1 H, CHH = C-); 4.35 (m, 2'H, -CH2-0-C (= 0) -C = C); 3.23- 3.86 (PEG CH2).

Example 40 Polymerization of PEG-DA PEG-DA, 1.7 and photoinitiator precursor solutions were prepared and mixed immediately prior to photopolymerization. The base solutions of PEG-DA (20 mg / ml) and 1 (40 mg / ml) were dissolved in phosphate-buffered saline purged with N2 (PBS, pH 7.4), where 7 (60 mg / ml) was dissolved in 50:50 of PBS / 95% ethanol, previously purged with N2. To prepare a final polymerization mixture, solutions of 1 or 7 were combined with PEG-DA to achieve a final concentration of PEG-DA and DHPD derivatives of 150 mg / ml. 100 μl was then added to a disk-shaped mold (100 μl, diameter 9 mm, depth = 2.3 mmol, Secure Seal® SA8R-2.0, Grace Bio Lab, Inc., OR) and irradiated for up to 20 minutes with either a UV lamp (Black Ray® lamp, 365 nm, Model UVL-56, UVP, CA) or a blue light lamp (VIP, 400-500 nm, BISCO, Inc., IL). For UV-initiated photocuring, DMPA (600 mg / ml in VP) was added to the polymer solution, to make a final concentration of 34 nM. Healing induced by visible light was performed using either CQ (100 mg / ml in VP, final concentration = 150 mM) with DMAB (30 mg / ml in VP, final concentration = 151 mM), or FNa2 (188 mg / ml in PBS, final concentration = 2 mM), with AA (100 mg / ml in PBS, final concentration = 17 mM) as the photoinitiator The final VP concentration was adjusted to be between 135 and 300 mM. After irradiation, the gels were stained with filter paper to remove the liquid surface layer and weighed. The gel conversion percentage was then determined by dividing the weight of the gel, by the weight of 100 μL of precursor solution.

Example 41 Determination of DOPA incorporation The amount of DOPA incorporated into the photopolymerized gel was determined using a modification of the colorimetric DOPA assay developed by Waite and Benedict. The photoreticulated gels were shaken in 3 mL of 0.5 N HCl to extract DOPA monomers that were not incorporated into the gel network. 0.9 ml of nitrite reagent (1.45 M sodium nitrite and 0.41 M sodium molybdate dihydrate) and 1.2 ml of 1M NaOH, were added to 0.9 mL of the extraction solution, and the absorbance (500 nm) of the mixture was recorded using a Hitachi U-2010 UV-Vis spectrophotometer with 2 to 4 minutes of addition of NaOH. The standard curves were constructed using known concentrations of 1 to 7.

Example 42 Mechanical Test Hydrogels were formed in the form of a hemisphere by charging 25 μl of the polymer mixture onto a glass slide treated with 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane. The gels were irradiated for 10 minutes, dialyzed in 0.15M HCl for at least 24 hours, to extract the unincorporated DOPA monomers, and then equilibrated in PBS for more than 15 minutes before being tested. To determine the gel modulus, the semi-spherical gel shells were attached to one end of a steel cylinder (diameter = 6 mm, length = 30 mm) using super-glue. The other side of the cylinder was attached to a piezoelectric stepper motor (IW-701-00, Burleigh Instruments, NY), aligned in series with a 50 g load transducer (FTD-G-50, Schaevitz Sensors, VA) , with a resolution of approximately 0.1 N. A fiber optic displacement sensor (RC100-GM20V, Philtec, Inc., MD), measured the axial movement of the steel rod. An Si pellet coated with Ti02 was positioned below the hydrogel, and the surface of Ti02 was flooded with PBS to maintain hydration of the gel. The nicker was advanced to 5 μm / s until a maximum compression load of 4 mN was measured. The elastic modules were calculated assuming Hertzian mechanics for the specific case of non-adhesive contact between an elastic hemisphere that can not be compressed and a rigid plane, in such case, the Hertzian relation between the charge Ph) and the displacement (dh) reaches be: 16R1,2E -3/2 (1) Ph =! dh wherein R and E are the radius of the curvature and the elastic modulus of the hemispherical gel, respectively. The radius of curvature of the gels was determined from the measurements of height and amplitude obtained from a photograph of the gel.

Example 43 Chemical Oxidation of PEG-DOPA in a Hydrogel 4-branched PEG-amine (PEG- (NH2), Pm = 10,000) was purchased from SunBio, Inc. (Walnut Creek, CAv), while the PEG-bis-amine linear (PEG- (NH 2) 2, Pm = 3,400) and methoxy-PEG-amine (mPEG-NH 2, Pm = 5,000), were purchased from Shearwater Polymers, Inc. (Huntsville, AL). Sephadex® LH-20 was obtained from Fluka (Milwaukee, Wl). The dicyclohexylammonium salt of N-Boc-L-DOPA, sodium periodate (NaI04), mussel tyrosinase (MT, EC 1.14.18.1) and horseradish peroxidase (HRP, EC 1.11.1.17), were purchased from Sigma Chemical Company (St. Louis, MO). Triethylamine (Et3), hydrogen peroxide (30% by weight of H202), sodium molybdate dihydrate, and sodium nitrite were purchased from Aldrich Chemical Company (Milwaukee, Wl). L-DOPA was purchased from Lancaster (Windham, NH). 1-Hydroxybenzotriazole (HBOt) was obtained from Novabiochem Corp. (La Jolla, CA) and O- (Benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU), were purchased from Advanced ChemTech (Lousiville, KY).

Synthesis of DOPA Modified PEG Modified linear and branched DOPA PEG, containing up to four DOPA end groups, was synthesized using standard carbodiimide coupling chemistry as described below. The structure of the four PEG modified with DOPA, is shown in Figure 1.

Synthesis of PEG- (N -Boc-DOPA) 4, I. PEG- (NH2) 4 (6.0 g, 0.60 mmol) was reacted with dicyclohexylammonium salt of N-Boc-L-DOPA (4.8 mmol), HOBt ( 8.0 mmoles), and Et3N (8.0 mmoles), in 60 ml of a 50:50 mixture of dichloromethane (DCM) and dimethylformamide (DMF). HBTU (4.8 mmol) was then added in 30 ml of DCM and the coupling reaction was carried out under argon at room temperature for one hour. The solution was washed successively with saturated sodium chloride solution, 5% NaOH, dilute HCl solution and distilled water. The crude product was concentrated under reduced pressure and purified by column chromatography on Sephadex® LH-20 with methanol as the mobile phase. The product was further purified by cold methanol precipitation three times, dried under vacuum at room temperature and stored under nitrogen at -20 ° C. 1 H NMR (500 MHz, CDCl 3 / TMS) d: 6.81-6.77 (m, 2 H, C 6 H H 2 (OH) 2-), 6.6 (d, ÍH, C 6 H 2 H (OH) 2-), 6.05 (br, s, ÍH) , 5.33 (br, s, ÍH), 4.22 (br, s, 1H, C6H3 (OH) 2-CH2-CH (N -) - C (0) N-), 3.73-3.41 (m, PEO), 3.06 (m, 2H, PEO-CH2-NC (O) -), 2.73 (t, 2H, C6H3 (OH) 2-CH2-CH-), 1.44 (s, 9 H, (CH3) 3C-). GPC-MALLS: Pm = 11,000, Pm / Pn = 1.01.

Synthesis of PEG- () 4, II- 3.0 g of I (0.25 mmoles) were dissolved in 15 ml of DCM at room temperature. 15 ml of TFA was added to the mixture to react for 30 minutes under argon. After evaporation of the solvent in a rotary evaporator, the product was precipitated with cold methanol three times, dried under vacuum at room temperature, and stored under nitrogen at -20 ° C. XR NMR (500 MHz, D20) d: 6.79 (d, 1H, C6H2H (OH) 2-), 6.66 (s, 1H, C6H2H (OH) 2-), 6.59 (d, 1H, C6H2H (OH) 2- ), 4.00 (t, 1H, C6H3 (OH) 2-CH2-CH (N-) -C (O) N-), 3.70-3.34 (M, PEO), 3.24 (m, 2H, PEG-CH2-NC (0) ~), 3.01-2.88 (m, 2H, C6H3 (OH) 2-CH2-CH (N-) -C (O) N-). GPC-MALLS: Pm = 11,400, Pm / Pn = 1.02 Synthesis of PEG- (¥ ¡-Boc-) 2, III PEG- (NH2) 2 (5.0 g, 1.5 mmoles), dicyclohexylammonium salt of N-Boc-L- (5.9 mmol), HOBt (9.8) was dissolved mmoles) and Et3N (9.8 mmoles), in 50 ml of a 50:50 mixture of DCM and DMF. HBTU (5.9 mmol) was then added in 25 ml of DCM, and the reaction was carried out under argon at room temperature for 30 minutes. Recovery and purification of the product is carried out as described above for I. aH NMR (500 MHz, CDC13 / TMS): d 6.81-6.77 (m, 2H, C6HH2 (OH) 2 ~), 6.59 (d, ÍH, C6H2H (OH) 2-), 6.05 (br, s, ÍH), 5.33 (br, s, ÍH), 4.22 (br, s, ÍH, C6H3 (OH) 2-CH2-CH (N -) - C (0 ) N-), 3.73-3.42 (M, PEO), 3.06 (m, 2H, PEO-CH2-NC (0) -), 2.74 (t, 2H, C6H3 (OH) 2-CH2-CH (N-) -C (O) N-), 1.44 (s, 9 H, (CH 3) 3 CO-). GPC-MALLS: Pm = 6,100 P / p7? = 1.02.

Synthesis of methoxy-PEG- (N-Boc-), IV. MPEG-NH2 (2.0 g, 0.40 mmol), dicyclohexylammonium salt of N-Boc-L- (0.80 mmol), HOBt (1.3 mmol) and Et3N (1.3 mmol) were dissolved in 20 ml of a 50:50 mixture. of DCM and DMF. HBTU (0.80 mmol) was then added in 10 ml of DCM, and the reaction was carried out under argon at room temperature for 30 minutes. Recovery and purification of the product was performed as described above for I. XH NMR (500 MHz, CDC13 / TMS): d 6.79 (d, 1H, C6H2H (OH) 2-), 6.66 (s, ÍH, C6H2H (OH) 2-), 6.59 (d, ÍH, C6H2H (OH) 2-), 4.00 (t, 1H, C6H3 (OH) 2-CH2-CH (N-) -C (O) N-), 3. 70-3.34 (M, PEO), 3.24 (, 2H, PEG-CH2-N-C (O) -), 3.01-2. 88 (, 2H, C6H3 (OH) 2-CH2-CH (N-) -C (O) N-). GPC-MALLS: Determination of Content The content of the modified PEG was determined by integration of the relevant peaks in the 1 H NMR spectrum and by a colorimetric assay. In the NMR method, the content was measured by comparing the integral value of Boc methyl protons at d = 1.44 to the methylene protons of PEG at d = 3.73-3.38. The trial is based on the previously described method of Waite and Benedict. Briefly, aqueous solutions of PEG- were treated with nitrite reagent (1.45 M sodium nitrite and 0.41 M sodium molybdate dihydrate), followed by the addition of excess NaOH solution. The absorbance (500 nm) of the mixture was recorded using a Hitachi U-2010 Uv / vis spectrophotometer, within 3 to 4 minutes of the addition of NaOH. A standard curve was constructed using solutions of known concentrations.

Formation of PEG- hydrogels To form PEG- hydrogels, sodium periodate (NaI04), horseradish peroxidase and hydrogen peroxide (HRP / H202), or mussel and oxygen tyrosinase (MT / 02), were added to solutions of PEG- (200 mg / ml) in phosphate buffered saline (PBS, pH 7.4). For MT-induced gelation, the PBS was dispersed with air for 20 minutes before the addition of MT. The gelation time was qualitatively determined to be when the flow of the mixture has stopped, measured by inversion of a vial containing the fluid.

Oscillatory reometry Oscillatory rheometry was used to monitor the gelation process and to determine the mechanical properties of the hydrogels. Crosslinking agent was added to the aqueous solution of PEG- and the well-mixed solution was loaded onto a Bohlin VOR rheometer. The analysis was carried out at a frequency of 0.1 Hz, a strain of 1% and a diameter of 30 mm cone and plate fitting with a cone angle of 2.5 ° C.

Spectroscopic oxidation evaluation of DOPA PEG modified with DOPA, was dissolved in 10 mM of PBS solution (bubbled with argon by HRP / H202 and NaI04 or air for MT experiments). After adding the oxidation reagent, the time-dependent UV / vis spectrum of the solution was monitored at wavelengths from 200 to 700 nm at a scanning speed of 800 nm / min. All samples were initially subjected to blanks against PBS buffer and recorded at room temperature using a UV / vis Hitachi U-2010 spectrophotometer.

Molecular weight analysis Molecular weights were determined by GPC-MALLS in a DAWN EOS (Wyatt Technology), using Shodex-OH Pack columns in mobile aqueous phase (50 mM PBS, 0.1 M NaCl, 0.05% aN3, pH = 6.0) and an Optilab DSP refractive index detector (Wyatt Technology). For molecular weight calculations, he used the experimentally determined dn / dc value of IV (0.136). ip IV Example 44 Materials and Methods Tip Modification Prior to surface modification of silicon nitride tips (SÍ3N4), cleaning procedures were carried out using plasma 02 (name of a machine) for 3 minutes and subsequently, they were transferred to a solution of piranha (sulfuric acid: H202 = 8.2) for 30 minutes. They were moved to 20% (v / v) 3-aminopropyltrimethoxysilane in toluene, to be functionalized with amines for 30 to 60 minutes after rinsing with H20. Two derivatives of polyethylene glycol (PEG) were chosen for PEGylation at the AMF tips: mPEG-N-hydroxysuccinimide (NHS) (Pm 2000) and Fmoc-PEG-NHS (PM 3400) (Nektar Inc.). The mixture (Fmoc-PEG-NHS: mPEGNHS = 1: 5-10.5 mM) of PEG was prepared in 50 mM sodium phosphate buffer, 0.6 M K2SO4, pH 7.8 and chloroform. The PEGylation reactions were sequentially conducted first in sodium phosphate buffer at 40 ° C and subsequently in chloroform for 3 hours in each stage. The reasons for using the PEG mixture are, to prevent multiple DOPA binding in Ti02. The Fmoc-PEG-NHS provides an amine for the Boc-DOPA conjugation after unfolding of Fmoc. Piperidine (20% v / v in NMP) was used, to deprotect the Fmoc for 5 minutes, and subsequently transferred, overhangs to BOP / HOBt / DOPA solutions (a molar ratio of 1: 1: 1, final 8 mM in NMP), with 10 μL of DIPEA. The same procedure was used for tyrosine modification.

AFM experiment All data was collected in an instrument AFM (Asylum Research, Santa Barbara, CA), on top of an inverted Nikon microscope. The spring constant (approximately 45, 100 and 300 pN / per manufacturer's information), of an individual cantilever, was calibrated by applying the equipartition theorem in the thermal noise (SI) spectrum. A drop of water was applied to a surface of Ti02 pre-cleaned (sonication in organic solvent and plasma of 02). The force distance curves containing PEG elasticity and contour length were selected for further statistical analysis. For the DOPAquinone experiments, all experiments were done in 20 mM Tris, at pH 9.8.

Dynamic force experiments The force measurement dependent on the load ratio revealed the energy landscape of the DOPA link (17). An inclination (= kBT / xb) of the linear graph (force against In (charge ratio)), determines the distance of the energy barrier xb along the applied force axis. The link energy barrier is calculated by the logarithmic intercept force at zero load ratio, from the force transition occurred by the change in thrust ratio and xb of the tilt. The AFM overhangs of silicon nitride (Bio-Levers, Olympus, Japan) were used due to their lower spring constants (~ 5 pN / nm and ~ 28 pN / nm). The lower load ratio of 2 nN / sec in this study could be achieved using the thrust ratio of 400 nm / sec and the cantilever (~ 5 pN / nm). The highest load ratio (1500 nN / sec) was produced by operation of 5 μm / sec of piezoelectric device and the use of rigid cantilever (300 pN / nm, Veeco).

Surface characterization The surfaces were analyzed by photoelectron microscope by X-ray (XPS), (Omicron, Taunusstein Germany), equipped with an X-ray source of 300 W Al Ka (1486.8 eV) Al non-monochromatic Ka and an electron shot to eliminate the accumulated load. The silicon nitride surfaces (0.7 x 0.7 cm2), prepared in the chamber at elevated temperature (ask Keun Ho), were cleaned and modified as the same procedures described in AFM tip modification. The photoelectron signal from the carbon orbital Is, was the main indicator for surface modification, considering all the abundant Si, O and N species in surfaces of SÍ3N4. While the principles of this invention have been described in conjunction with the specific embodiments, it should be clearly understood that these descriptions are aggregated only by way of examples and are not intended to limit in any way the scope of the invention. For example, the present invention can improve the adhesive properties of a wide variety of polymeric compositions, whether or not capable of hydrogenation. Likewise, the present invention can be used with various other synthetic techniques well known to those skilled in the art, to functionally modify a particular polymer component for subsequent coupling and preparation of the corresponding DOPA conjugate. Other advantages, features and benefits will become apparent from the scope of the claims of this document below, within the scope thereof, as determined by their reasonable equivalents and as would be understood by those skilled in the art.

Claims (25)

NOVELTY OF THE INVENTION CLAIMS

1. A dihydroxyphenyl adhesive compound (DHPD) of formula (I), characterized in that Ri and R2 may be the same or different and are independently selected from the group consisting of hydrogen, C?-Substituted and unsubstituted, branched and unbranched, saturated and unsaturated hydrocarbon; P is separately and independently selected from the group consisting of -NH2, -COOH, -OH, -SH, where Ri and R2 are defined above, a single bond, halogen, wherein Ai and A2 are separately and independently selected from the group consisting of H, a single bond; a protecting group, substantially poly (alkylene oxide), D where n varies between 1 and approximately 3 and A3 is R is H, lower alkyl C? -6, or poly (alkylene oxide) O R3 R3 is as defined above, and D is indicated in Formula (I).

2. A compound according to claim 1, characterized in that the poly (alkylene oxide) has the structure wherein R3 and R4 are independently and independently H, or CH3 and m have a value in the range between 1 and about 250, A is NH2, COOH, -OH-, -SH, -H or a protecting group.

3. A compound according to claim 1, characterized in that DHPD is of the structure:

4. A compound according to claim 1, characterized in that DHPD is of the structure:

5. A compound according to claim 1, characterized in that DHPD is of the structure: wherein A2 is -OH and Ai is substantially poly (alkylene oxide) of the structure

R3, R4 and m are defined as in claim 2. 6. A DHPD according to claim 5, characterized in that the poly (alkylene oxide) is a block copolymer of ethylene oxide and propylene oxide.

7. A method according to this invention that involves adhering substrates to others, characterized in that it comprises the steps of providing DHPD of the structure: where Rj. and R2. they are defined as above; apply the DHPD of the previous structure to one or the other or both structures to be adhered to; contact the substrates to be adhered with the DHPD of the previous structure between them, to adhere the substrates to each other, and optionally reposition the substrates relative to each other, separating the substrates and putting them in contact again with each other with the DHPD of the structure previous among these.

8. A method according to claim 7, characterized in that Ri and R2 are hydrogen.

9. A method according to claim 7, characterized in that one or the other portions of DHPD are of the structure:

10. A method according to claim 7, characterized in that both portions of DHPD are of the structure:

11. An adhesive characterized in that it comprises a compound of formula (II): wherein Rl r R2 and P are as defined in claim 1.

12. The adhesive according to claim 11, characterized in that the adhesive is bonded in an aqueous medium.

13. The adhesive according to claim 11, characterized in that a compound of formula (II) is DOPA.

14. A composition comprising a compound of formula (III): characterized in that, for each compound of formula (Ia), Ri and R2 are separately and independently defined as defined in claim 1; wherein for each compound of formula (Ia), Pi and P2 are separately and independently defined as P according to claim 1, n and ra independently vary from 0 to about 5, provided that at least one of nom is at least 1 .

15. A composition according to claim 14, characterized in that Rx and R2 are all hydrogen.

16. The composition according to claim 14, characterized in that at least one of Pi or P2 substantially comprises poly (alkylene oxide). The composition according to claim 14, characterized in that at least one of Pi or P2 comprises at least one site of ethylenic unsaturation. 18. The composition according to claim 14, characterized in that at least one of Pi or P2 comprises PEG. 19. The composition according to claim 14, characterized in that at least one of (la) is tri-DOPA, and P is PEG. 20. A composition according to claim 14, characterized in that Pi and P2 are coupled together. 21. A coated medical device, characterized in that it comprises a device, and a coating, the coating comprises the composition according to claim 14. 22. The medical device coated according to claim 20, characterized in that the device is selected from the group consisting of stents and pacemakers. 23. The medical device according to claim 14, characterized in that the coating is biodegradable. 24. A method for preventing cells or proteins from adhering to a surgical incision site comprising the steps of coating the site with the composition according to claim 14. 25. An interaction, which is intermediate in resistance between a bond of hydrogen and a covalent bond, the interaction is substantially reversibly formed, breakable and reformable.