WO2005118831A2 - Compositions poylmeres et procedes associes d'utilisation - Google Patents

Compositions poylmeres et procedes associes d'utilisation Download PDF

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WO2005118831A2
WO2005118831A2 PCT/US2005/006418 US2005006418W WO2005118831A2 WO 2005118831 A2 WO2005118831 A2 WO 2005118831A2 US 2005006418 W US2005006418 W US 2005006418W WO 2005118831 A2 WO2005118831 A2 WO 2005118831A2
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Prior art keywords
dopa
mpeg
dhpd
peg
substrates
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PCT/US2005/006418
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WO2005118831A3 (fr
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Philip B. Messersmith
Jeffrey Dalsin
Lijun Lin
Bruce P. Lee
Kui Huang
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Northwestern University
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Priority to EP05804747A priority Critical patent/EP1735456A4/fr
Priority to JP2007500804A priority patent/JP5133048B2/ja
Priority to AU2005250314A priority patent/AU2005250314A1/en
Priority to CA002557330A priority patent/CA2557330A1/fr
Publication of WO2005118831A2 publication Critical patent/WO2005118831A2/fr
Publication of WO2005118831A3 publication Critical patent/WO2005118831A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J4/00—Adhesives based on organic non-macromolecular compounds having at least one polymerisable carbon-to-carbon unsaturated bond ; adhesives, based on monomers of macromolecular compounds of groups C09J183/00 - C09J183/16

Definitions

  • MAPs Mussel adhesive proteins
  • DOPA L-3,4- dihydroxyphenylalanine
  • Control of cell and protein adhesion on surfaces is critical to the performance of biosensors, medical diagnostic products, any instrumentation and assays used requiring handling serum and other human/animal fluids, tissue engineering, localized in vivo drug delivery, implanted medical devices, healing of surgical incisions, adhesion of tissues such as bone and cartilage for healing, and nanotechnology (nanoparticle-based therapies and diagnostic tools).
  • control of cellular and protein adhesion to surfaces is also important. Such applications include prevention of mussel attachment to boats and ships, piers, and other structures used in oceans and fresh water, prevention of algal and bacterial growth on water lines used for industrial and drinking water, and sensors used to measure water quality and purity.
  • PAO poly(alkylene oxides)
  • PEG polyethylene glycol
  • PEO polyethylene oxide
  • PEO-PPO-PEO block copolymers such as those available under the PLURONICS brand name
  • PEG/tetraglyme poly(methoxyethyl methacrylate)
  • PMEMA poly(methoxyethyl methacrylate)
  • polyMPC Poly(methacryloyl phosphatidylcholine)
  • compositions which function e.g., as an adhesive, in a substantially aqueous environment.
  • the preferred compositions generally comprise an adhesive moiety and a polymer moiety, the polymer moiety having a desired surface active effect (or other desired characteristics).
  • the adhesive moiety of a composition of this invention comprises dihydroxyphenyl derivatives including, di (DHPD) wherein the second DHPD is
  • the polymer moiety comprises poly (alkyleneoxide).
  • the adhesive moiety comprises DHPD, e.g., DOPA (discussed herein), and the polymer moiety comprises PEO-PPO-PEO block polymers (also discussed above).
  • the adhesive moiety comprises DHPD including a pendent chain comprising ethylenic or vinylic unsaturation such as, for example, an alkyl acrylate.
  • this invention comprises dihydroxyphenyl (DHPD) adhesivie compound of formula (I) wherein
  • Ri and R 2 may be the same or different and are independently selected from the group consisting of hydrogen, saturated and unsaturated, branched and unbranched, substituted and unsubstituted C 1-4 hydrocarbon;
  • P is separately and independently selected from the group consisting of -NH 2 , - COOH, -OH, -SH,
  • Ri and R 2 are defined above. a single bond, halogen,
  • Ai and A 2 are separately and independently selected from the group consisting of H, a single bond; a protecting group, substantially poly(alkyleneoxide), D
  • R 4 R 4 is H, C
  • _ 6 lower alkyl, or poly(alkylene oxide)-C-C CH 2 ,
  • R 3 is defined as above, and D is indicated in Formula (i). 1]
  • the poly(alkylene oxide) has the structure
  • R 3 and R 4 are separately and independently H, or CH 3 and m has a value in the range between 1 and about 250, A 4 is NH 2 , COOH, OH, -SH, -H or a protecting group.
  • DHPD is
  • DHPD is of the structure:
  • a 2 is -OH and Ai is substantially poly(alkylene oxide) of the structure
  • R 3 , i and m being defined as in claim 2.
  • the poly(alklene oxide) is a block copolymer of ethylene oxide and propylene oxide.
  • a method of this invention involves adhering substrates to one another comprising the steps of providing DHPD of the structure:
  • Ri and R 2 are defined as above; applying the DHPD of the above structure to one or the other or both of the substrates to be adhered; contacting the substrates to be adhered with the DHPD of the above structure therebetween to adhere the substrates to each other, and optionally repositioning the substrates relative to each other by separating the substrates and recontacting them to each other with the DHPD of the above structure therebetween.
  • DHPD Dihydroxyphenyl derivatives
  • Ri and R 2 are defined below, and n ranges between 1 and about 5.
  • Ri and R 2 are hydrogen and P is, itself, dihydroxy phenyl.
  • a preferred DHPD in a practice of the present invention is 1-3,4, dihydroxy phenyl alanine (DOPA), (generically),
  • Subtantially poly(alkylene oxide) shall mean predominantly or mostly alkyloxide or alkyl ether in composition. This definition contemplates the presence of heteroatoms e.g., N, O, S, P, etc. and of functional groups e.g., -COOH, - NH 2 ,-SH, as well as ethylenic or vinylic unstaturation. It is to be understood any such non-alkyleneoxide structures will only be present in such relative abundance as not to materially reduce, for example, the overall surfactant, non-toxicity, or immune response characteristics, as appropriate, or of this polymer.
  • Figure 1 shows 1H NMR spectra of PLURONIC® F127, its carbonate intermediate (SC-PAO7) and DME-PAO7 in CDC1 3 .
  • Figure 2 provides differential scanning calorimetry thermograms of 30 wt
  • FIG. 3 plots shear storage modulus, G', of a 22 wt % DME-PAO7 aqueous solution as a function of temperature at 0.1 Hz and a strain of 0.45%. Shown in the inset is the rheological profile of a 22 wt % unmodified-PLURONIC® F127 aqueous solution as a function of temperature.
  • FIG. 4 plots shear storage modulus, G', of a 50 wt % DME-PAO8 aqueous solution as a function of temperature at 0.1 Hz and a strain of 0.45%. Shown in the inset is the rheological profile of a 50 wt % unmodified PLURONIC® F68 aqueous solution as a function of temperature.
  • Figure 5 plots storage moduli of DME-PAO8 aqueous solutions at 45 wt
  • Figures 6A and 6B show differential scanning calorimetry thermograms of (A) DOPA-PAO7 and (B) DME-PAO7 at different concentrations upon heating. Arrows indicate the location of gelation endotherm observed only at higher polymer concentrations.
  • Figures 7A-C show high-resolution C(ls) XPS peaks for (A) un-modified
  • Figures 8A-C provide TOF-SIMS positive spectrum showing peaks representing catechol binding of gold. Spectra were normalized to Au peak (m/z ⁇ 197).
  • Figure 9 provides TOF-SIMS spectra showing the positive secondary ion peak at mass 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 spectra showing the positive secondary ion peaks for Au substrate chemisorbed with mPEG-DOPA.
  • Catecholic binding of gold is observed at m/z ⁇ 225 (AuOC), 254 (AuOCCO), and 309. Less intense AuO a C b peaks are seen at m/z ⁇ 434, 450, 462, and 478.
  • Figure 11 shows SPR spectra of protein (0.1 mg/ml BSA) adsorption onto modified and unmodified gold surfaces.
  • mPEG-DOPA and mPEG-MAPd modified surfaces exhibited reduced protein adsorption compared to bare gold and mPEG-OH modified surfaces.
  • Figures 14 A-C are a series of SEM micrographs indicating the morphology of NIH 3T3 fibroblasts on (A) unmodified Au, (B) Au treated with mPEG-OH, and (C) mPEG-DOPA-modified Au. All treatments were at 50mg/ml in DCM for 24 h.
  • Figure 15 shows the UV/vis absorption spectrum of mPEG-DOPA stabilized magnetite nanoparticles suspended in several aqueous NaCl solutions at the concentrations as shown and plotted therein. Addition of NaCl did not induce nanoparticle precipitation.
  • Figure 16 shows addition of salt to untreated Au nanoparticles induces aggregation. Shown are UV/vis scans of lOnm untreated Au nanoparticles suspended in aqueous NaCl solutions (concentrations as shown and plotted therein). The attenuation and shift of the 520 nm absorption band with increasing NaCl concentration reflects aggregation of the nanoparticles.
  • Figure 17 illustrates addition of salt to mPEG-DOPA stabilized Au nanoparticles does not induce aggregation. Shown are UV/vis scans of lOnm mPEG- DOPA stabilized Au nanoparticles suspended in aqueous NaCl solutions (concentrations as shown and plotted therein). The lack of attenuation and shift of the 520nm absorption band with increasing NaCl concentration reflects effective stabilization of the nanoparticles.
  • Figure 18 plots the UV/vis absorption spectrum of mPEG-DOPA stabilized CdS nanoparticles suspended in aqueous NaCl solutions (concentrations as shown and plotted therein.
  • Figure 19 plots XPS survey scans of unmodified TiO 2 and TiO 2 treated with mPEG-DOPA ⁇ -3 .
  • Figure 20 plots the long-term resistance to cell adhesion on TiO 2 and TiO 2 modified with mPEG-DOPA ⁇ -3 .
  • the duration of the non-fouling response is proportional to the length of the DOPA peptide anchoring group. Adherent cells were visualized with calcium AM.
  • Figure 21 plots the high-resolution XPS scans of the Cis region of TiO 2 substrates modified with mPEG-DOPA 1-3 . Of note is the increase in the ether carbon peak (286. OeV) with increasing length of the DOPA peptide anchor.
  • Figure 22 plots the high-resolution XPS scans of the Ols region of TiO 2 substrates modified with mPEG-DOPA ⁇ -3 .
  • the peak at 532.9eV representing polymeric oxygen increases while the Ti-O-H peak (531.7eV) decreases with increasing DOPA peptide length.
  • Figure 23 plots the results of the Robust Design experiment on 316L stainless steel
  • Figure 24 plots the 4-hour cell attachment to a variety of surfaces modified by mPEG-DOPA ⁇ -3 using a 24-hour modification at 50°C at the indicated pHs.
  • Figure 25 plots the % gel conversion versus the UV exposure time in minutes.
  • Figure 26 plots the mole fraction of DOPA incorporated versus the mol % of 1 or 7 in the precursor solution.
  • Figure 27 plots the % gel conversion versus mol % 1 or in the precursor solution.
  • Figure 28 X-ray Photoelectron Spectroscopy XPS analysis of a silicon nitride surface.
  • Figure 29 is a free monitoring of functionalized silicon nitride cantilevers.
  • Figure 30 is an analysis of entropic elasticity of poly(ethylene glycol).
  • Figure 31 is a force measure of side chain modified DOPA.
  • Figure 32 is a proposed model of DOPA-T 1 O 2 binding mechanism.
  • Figure 33 is an atomic force microscopy arrangement.
  • Figure 34 is data regarding force measurement.
  • Figure 35 is adhesion data.
  • Figure 36 is synthetic route and data analysis.
  • DHPD dihydroxyphenyl derivative
  • DHPD adhesives and polymeric compositions have many uses, including prevention of protein and/or cell adhesion to a surface in various medical, industrial and consumer applications.
  • the DHPD adhesives can also be used as substitutes for sutures for a wound and as aids in healing bone fractures or cartilage-to-bone damage. These and other uses will be described in more detail below.
  • compositions of the present invention have the following structure:
  • Pi and P 2 are separately and independently defined as P in formula (I);. n and m independently range from 0 to about 5, provided that at least one of n or m is at least 1;
  • Adhesive Moiety is a dihydroxyphenyl derivative (“DHPD”) having the following preferred structure:
  • the DHPD adhesive can function in an aqueous environment.
  • an aqueous environment is any medium comprising water. This includes without limitation water, including salt water and fresh water, cell and bacterial growth media solutions, aqueous buffers, other water-based solutions, and body fluids.
  • the DHPD moiety can be derivatized. As would be understood by those skilled in the art, such derivatization is limited by the retention of the desired adhesive characteristic.
  • polymeric components providing a surface active effect and other desired characteristics will be well-known to those skilled in the art made aware of this invention.
  • the desired surface active effect relates to reduced particulate agglomeration and anti-biofouling, including resistance to cell and/or protein adhesion.
  • the polymer component can be water soluble, depending upon end-use application, and/or capable of micelle formation depending upon various 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), PEO-PPO-PEO block copolymers, polyphenylene oxide, PEG/tetraglyme, PMEMA, polyMPC, and perfluorunated polyethers.
  • the polymeric compositions can be synthesized in several ways.
  • the polymeric compositions may be synthesized through a general synthetic procedure for polymer end-group activation.
  • Various polymers or monomeric components thereof can be activated using carbonate chemistry.
  • a succinimidyl carbonate- activated polymeric component reacted with DHPD moiety can provide a stable urethane conjugate.
  • Two of the many possible pathways (a) and (b) in Scheme la and lb, below, show coupling with a poly(alkylene oxide) in either aqueous or non-aqueous solvents, without compromising desired bioadhesion.
  • a DHPD residue can be coupled to a polymeric component to provide the desired conjugate composition, through either urethane or amide bond formation.
  • a carboxylic acid group of the DHPD component can be esterified or derivatized with various other functional groups.
  • the DHPD component can be coupled to a polymeric component (e.g., amidation or esterification depending on polymer end group, -NH 2 or -OH) providing a DHPD functionality which can be derivatized by any of numerous known protecting groups, including without limitation Boc, Fmoc, borate, phosphate, and tributyldimethylsilyl.
  • the present invention is also a method of using urethane synthesis to incorporate a DHPD residue into a polymeric system.
  • a method includes (1) providing a polymeric component terminating in a plurality of monomers, each having a functional end group; (2) preparing a carbonate derivative of the polymeric component; and (3) preparing a urethane moiety upon reaction of the carbonate derivative and at least one DHPD moiety.
  • a polymeric component utilized in conjunction with this method can include those having terminal monomeric functionality reactive with a reagent providing the desired carbonate derivative and, ultimately, providing a urethane moiety coupling the polymeric and DHPD components.
  • a reagent providing the desired carbonate derivative and, ultimately, providing a urethane moiety coupling the polymeric and DHPD components.
  • Various other coupling reagents and/or hydroxy-terminating polymeric components can be used to provide the desired urethane moiety.
  • the present invention is also a method of using a carbonate intermediate to maintain catecholic functionality of a DHPD-incorporated polymeric composition and/or system, or to otherwise enhance the adhesion properties thereof.
  • a method includes (1) providing a polymeric component terminating in a plurality of monomers each having a functional end group; (2) reacting the polymeric component with a reagent to provide a carbonate intermediate; and (3) reacting the carbonate intermediate with at least one DHPD moiety.
  • this inventive method can be considered a way of enhancing the reactivity of the polymeric component end group, via a suitable carbonate intermediate. Subsequent reaction at the amino-nitrogen of DHPD moiety provides the corresponding conjugate while maintaining catecholic functionality.
  • DOPA methyl ester prepared by the reaction of DOPA with methanol in the presence of thionyl chloride, can be used in organic solvents. Reaction progress can be monitored by TLC and NMR, with the coupling reaction virtually complete in one hour (with representative conjugates DME-PAO7 (from PAO PLURONIC® F127) and DME-PAO8 (from PAO PLURONIC® F68)). High product yields were obtained upon purification from cold methanol.
  • the free carboxylic form of DOPA can be coupled with the carbonate intermediate in alkaline aqueous solution. It is well known that the chief difficulty in working with DOPA is its ease of oxidation (to DOPA-quinone and other products), which readily occurs in alkaline aqueous solutions.
  • a borate-protected DOPA can be first formed by adding DOPA to aqueous sodium borate (Scheme lb). The resulting complex is remarkably stable in neutral or alkaline solutions, and can be readily deprotected under acidic conditions.
  • DOPA was coupled to the ends of several commercially-available PAOs under alkaline aqueous conditions to yield DOPA-PAO7 and DOPA-PAO8.
  • Visual inspection of the reaction solution revealed the absence of strongly absorbing DOPA-quinone, an indication that DOPA remains unoxidized during the reaction.
  • acidification with HCI resulted in deprotection of the DOPA endgroups of the block copolymer.
  • the product yields (shown in Table 1) of selected DOPA-modified PAOs synthesized in 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, causing the low efficiency of extraction of DOPA-modified PAO with dichloromethane from water. It should be noted that the free carboxylic acid in DOPA-PAO7 and DOPA-PAO8 can be further functionalized using standard peptide chemistry to tailor the properties of the block copolymers.
  • the four DOPA-modified PAOs of Table 1 could be stored at -20°C indefinitely with no discoloration or change in properties.
  • Control of cell and protein adhesion on surfaces is critical to the performance of biosensors, medical diagnostic products, any instrumentation and assays used requiring handling serum and other human/animal fluids, tissue engineering, localized in vivo drug delivery, implanted medical devices, healing of surgical incisions, adhesion of tissues such as bone and cartilage for healing, and nanotechnology (nanoparticle-based therapies and diagnostic tools).
  • the polymeric compositions of the present invention can be used as coatings to prevent protein and cellular adhesion to devices 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 animal or human derived materials, medical diagnostic devices, and biosensors.
  • the polymeric compositions can be tissue 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, and site specific drug elution and for research uses such as immobilization of proteins including antibodies and small molecule analytes including pharmaceuticals.
  • tissue 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, and site specific drug elution and for research uses such as immobilization of proteins including antibodies and small molecule analytes including pharmaceuticals.
  • these coatings and hydrogels including without limitation prevention of marine biofouling (attachment of algae, bacteria, and mussels to surfaces underwater), prevention of bacteria contamination of water streams to industrial plants such as electronic and drug manufacturers, prevention of bacterial contamination of drinking water streams, dental and denture adhesives, underwater adhesives to deliver indicators, coatings for water purity and measurement sensors, paints used for prevention of biofouling, and use in cosmetics to adhere desired fragrances and colorants to hair, eyelids, lips, and skin, to form temporarily skin coloring such as tattoos and the like, and for resealable adhesives for consumer products such as storage bags.
  • Adhesive hydrogels can be also formed using the present methods.
  • the DHPD adhesive is attached to polymers capable of forming a hydrogels in vivo or in vitro.
  • hydrogels can be formed by a number of methods including the use of self-assembling polymers that form gels at higher 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 cross-linked hydrogels, and the use of polymers that can be subjected to photoactivation to produce cross-linked hydrogels.
  • the anti-biofouling coatings of the present invention may be applied to medical devices, such as vascular or arterial stents, pacemakers, heart valves, glucose monitors and other biosensors, vascular wraps, defibrillators, orthopedics devices, and surgical devices, including sutures and catheters.
  • medical devices such as vascular or arterial stents, pacemakers, heart valves, glucose monitors and other biosensors, vascular wraps, defibrillators, orthopedics devices, and surgical devices, including sutures and catheters.
  • the polymeric compositions of the present invention can be used as coatings to prevent protein and/or cellular adhesion 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 animal or human derived materials, medical diagnostic devices, and biosensor.
  • a surface may be modified by the polymeric composition of the present invention in any number of ways.
  • the polymeric composition may be absorbed onto the surface or a DHPD moiety containing a polymerization initiator may be adsorbed onto the surface and polymer growth initiated from the surface.
  • a number of polymerization techniques are possible, including without limitation surface initiated radical polymerization, radical polymerization methods, ionic polymerization, ring-opening polymerization, and photopolymerization.
  • the surface density of polymer can be increased by treating surfaces with PEG solutions near the lower critical solution temperature (LCST), or cloud point.
  • LCST critical solution temperature
  • This approach is useful for polymers that show inverse solubility transitions at high temperature and high ionic strength, such as poly(ethylene glycol), poly(N-isopropylacrylamide) and other N-substituted poly(acrylamides) that show inverse solubility transitions.
  • DHPD moieties in the adhesive component and the pH of the modification buffer are responsible for most of the variation in the cell and/or protein adhesion resistance of the modified materials.
  • adsorption time and polymeric composition concentration contributes little to the variation in cell and/or protein adhesion resistance of the modified materials.
  • the greater the number of DHPD moiety monomers in the adhesive component the better the cell and/or protein adhesion resistance.
  • the density of the polymeric composition on the surface correlates well with resistance to cell and/or protein adhesion.
  • the thickness of the coating layer can be from about 20 A to about 100 ⁇ m, including 30 A, depending on the polymer composition used and the pH of the modification buffer.
  • the concentration of the polymer composition used for modification of a surface can be from about .1 mg/ml to about 75 mg/ml.
  • the pH of the modification 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.
  • XPS survey scans of unmodified TiO 2 revealed strong peaks at -458 eV (Ti2p) and -530 eV (Ols) characteristic of native oxide, as well as a small peak at 248.7 eV (Cis) as a result of adventitious hydrocarbon contamination.
  • TiO 2 substrates treated with mPEG-DOPA 1-3 under cloud point conditions demonstrated dramatic increases in surface-bound carbon as reflected by the Cis peak, suggesting the presence of PEG on the surface.
  • the increases in the Cis peaks observed after modification with mPEG-DOPA ⁇ -3 were directly proportional to the number of terminal DOP As present.
  • a small peak at 400 eV ( ⁇ ls) was seen in the spectrum of the TiO 2 surface modified with mPEG-DOPA ⁇ -3 , representing the amide nitrogen in DOPA.
  • Table 2 shows the titanium, oxygen, and carbon atomic composition calculations for TiO 2 - modified with mPEG-DOPA ⁇ -3 .
  • the oxygen signal is further subdivided into metal oxide (Ti-O-Ti), surface hydroxide (Ti-O-H), and organic oxygen and coupled water (C- O, H 2 O) species.
  • Table 2 Titanium, oxygen, and carbon atomic composition calculations
  • DOPA creates strong, reversible bonds with TiO 2 .
  • the energy of the bond is 30.56 kcal/mol and needs about 800 pN to be detached from TiO 2 at the single molecule level, which is four times stronger than the interaction between Avidin and Biotin.
  • the DOPA-TiO 2 strength of interaction is about midway between that of Avidin-Biotin, one of the strongest hydrogen bond based interactions in biology (0.1-0.2 nN) and a covalent bond (>2nN).
  • DOPA conjugated cantilevers displayed significant adhesion accompanied by entropic elasticity of the PEG chain (Fig 34).
  • a histogram of the force distribution shows a uni-modal shape indicating only single adhesive event, which is different compared to the case of a multivalent protein, Avidinl2.
  • the length of stretched PEG (36 nm) was consistent with the expected contour length of a PEG molecule (37 nm, Fig 30).
  • d is the z-displaced distance of piezoelectric device when a single DOPA-PEG molecule was fully stretched during retraction (Fig 34C).
  • the 'd' values appeared to be almost constant throughout many repeated cycles although it did vary slightly (Fig 34A). This small variation might be due to DOPA binding to the surface at different angles.
  • An important feature of our experiment is that the unbinding signals are epetitions using the 'identical' DOPA molecule. This is compared to the traditional approach of single molecule pulling experiments where tips picked up one molecule randomly. This also demonstrates that the DOPA adhesion chemistry was completely reversible.
  • Mussels developed an interesting way to create such a strong binding in water, a post-translational modification of tyrosine by tyrosine hydroxylase.
  • This enzyme catalyzes a reaction of adding one hydroxyl group using tyrosine as a substrate and a large amount is found in threads and plaques where DOPA exists as well. It is surprising to say that the small post-translational modification (-OH) seems to produce a huge change of adhesive ability. Thus, experiments were designed to show a correlation between the posttranslational modification and binding ability.
  • a tyrosine tethered cantilever was prepared instead of DOPA, and tyrosine adhesion on TiO2 was investigated. No detectable force signals were observed except some non-specific adhesions with low probability (Fig 35A).
  • the TiO2 surface was replaced with gold.
  • the aromatic ring of tyrosine binds to a gold surface in a parallel orientation to the surface through ⁇ - ⁇ electron interaction, which is a well-known mechanism in surface adsorption chemistryl8,19.
  • the same cantilever used in TiO2 produced relatively strong adhesions repeatedly on gold surfaces (Fig 35B).
  • DOPA Biological roles of DOPA go beyond adhesiveness upon oxidation: it crosslinks polypeptide chains resulting in stiffer materials found throughout threads and pads.
  • the crosslinking mechanism has multiple pathways starting from a chemically unstable DOPAquinone structure.
  • Aryl-aryl ring coupling (di-DOPA) has been found in mussel adhesive proteins 20 but Michael addition (quinone-alkylamine adducts) products have been found in other species not mussels (Fig 36A). Therefore, these structures may occur as results of oxidation in mussels as well. It is clear in terms of crosslinking but is under debate with respect to adhesive properties after maturation i.e. oxidation. It was demonstrated that the DOPAquinone structure is not a major player for adhesiveness.
  • DOPAquinone-PEG chain is spatially and chemically stabilized by excess co- conjugation of methoxy-PEG molecules (5-10 molar equivalent) which is an important molecular configuration for preventing further reactions of DOPAquinone.
  • measured AFM signals showed two clear distributions in terms of force magnitude: high force and low force (Fig 36B).
  • Statistical analysis of the data yielded two clear histograms with 178 ⁇ 62 pN for low and 741 ⁇ 110 pN for high force (Fig 36C).
  • the quinone binding can be assigned to the low force region because it appeared only after the oxidation was triggered and subsequently became more frequent over time (Fig 36D).
  • the slow kinetic feature of DOPA oxidation contributed to initial high frequency of DOPA signals. This is the first single molecule experiment about detecting the structural change of a small molecule upon external stimulus. Based on these results, the possibility of the DOPAquinone structure being responsible for the high adhesiveness can be ruled out. Therefore, without being bound to any theory, the regeneration of reduced form i.e. di-hydroxyl group of DOPA during oxidation, is believed to be a very important requirement for maintaining or changing adhesive properties of DOPA containing materials at an interface.
  • the DOPA anchoring system can be a new platform to study other extensible biological macromolecules such as polysaccharides, DNA, and proteins.
  • This method is also highly contrasted with the conventional single molecule experiments where a tip 'sees' different molecules at every single movement of a cantilever. It has been a big barrier to investigate molecular responses upon external stimuli if a given stimulus was not hundred percent effective23,.
  • the DOPA-based anchorage system can be an alternative technique to overcome these problems in current single molecule pulling experiments.
  • a picture describes how the blue mussel (Myt ⁇ lus Edulis) sticks to metal oxide surfaces.
  • the circle included one plaque where the unusual amino acid, DOPA, was found.
  • (B) Two major protein components uncovered in plaques in mussels, Mefp-3 and Mefp-5. These mussel adhesive proteins have high content of DOPA: 27 mole % of Mefp-5 and 21 % of Mefp-3.
  • (C) AFM tip modifications Polymerization of 3- aminopropyltrimethoxysilane (APTMS) introduced amine groups on Si3N4 tip surfaces (not drawn). A long chain describes a PEG molecule conjugated with single (Boc)-DOPA at the end. Mixture of mPEG-NHS(2k) and Fmoc-PEG-NHS(3.4k) at a molar ratio of 5-10:1 was used to stabilize DOPA-PEG molecule (see supplemental section for details).
  • APIMS 3- aminopropyltrimethoxysilane
  • (D) A plot of bonding strength (linear) vs. loading rate (log). Loading rate was the product of spring constant of a cantilever and a pulling speed. Four different loading rates were selected: 1500, 180.7, 28.4 and 2 nN/sec. Averaged forces with standard deviation were plotted at each given loading rate. 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). [0099] (E) A schematic energy landscape of DOPA binding.
  • DOPA was created by the action of tyrosine hydrxylase and subsequently oxidized to DOPAquinone by pH and the enzyme. It is unstable and reactive due to tendency of radical formation of DOPAquinone. It can crosslink with other DOPA molecules (di- DOPA) as well as reacts with amine groups from lysines. The arrows are the potential reactions found in other species not in mussel adhesive proteins.
  • DOPA signals (circle, left y-axis) gradually decreased from twenty-two events for the first ten minutes to only three events during the last time window.
  • quinone signals (triangle, right y-axis) increased from one event at the first time window to forty-two events at the last time window (50-60 min).
  • the antifouling coating of the present invention can either be essentially permanent i.e., lasting 120 days or more, or biodegradeable depending on the number of DOPA or DOPA-derived moieties in the adhesive component.
  • Figure 20 shows the results of a 28-day 3T3 fibroblast cell adhesion and spreading assay on TiO 2 treated with mPEG-DOPA 1-3 . At early time points (i.e.
  • the protein and cell attachment resistance correlates well with the length of the DOPA peptide anchoring group, with resistance increasing in the order mPEG-DOPA ⁇ mPEG-DOPA 2 ⁇ mPEG- DOPA 3 .
  • TiO 2 substrates treated with mPEG-DOPA 2 and mPEG-DOPA 3 maintain reduced cell attachment, or resistance, through 21 days.
  • Robust design methodology was used to determine the effect of DOPA peptide anchor length and modification conditions (pH, concentration and time) on the PEG surface density and antifouling performance of metal, metal oxide, semiconductor, and polymer surfaces. The nine experiments utilized for each substrate are described below in Table 7.
  • the polymeric compositions of the present invention can be also used to coat the surfaces of devices and instrumentation used for handling body fluids including sera.
  • the coating on the surface of the device or instrument blocks protein binding to the surfaces thus reducing or eliminating the need for extensive washing or cleaning of the device or instrument between uses.
  • the devices need to be thoroughly cleaned to prevent cross contamination between samples of bodily fluids applied top the device.
  • caustic agents such as 50% bleach and/or elevated temperatures.
  • the coating process would be to circulate an aqueous solution of 1 mg/ml 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 on medical implants for a wide variety of uses.
  • the coatings can be used to block bacterial adhesion and therefore growth on the implanted device reducing the possibility of infection at the site of implant.
  • the coatings can be used to reduce the amount of acute inflammation on the device by reducing protein binding and cell adhesion to the device.
  • the coatings of the present invention can also be used as nanoparticles to prevent aggregation of these particle 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 tissue adhesive polymeric hydrogels for medical uses such as tissue sealants, gels for prevention of surgical adhesions (scar tissue formation), bone and cartilage adhesives, tissue engineering, and site specific drug elution and for research uses such as immobilization of proteins including antibodies and small molecule analytes including pharmaceuticals.
  • the polymeric compositions of the present invention may also be used as interfacial bonding agents, wherein the neat monomers or solution of monomers are applied to a surface as a primer or bonding agent between a tissue surface or a metal or metal oxide implant/device surface and a bulk polymer or polymer hydro gel.
  • the polymeric compositions of the present invention can be injected or delivered in a fluid form and harden in situ to form a gel network.
  • the in situ hardening can occur through photocuring, chemical oxidation, enzymatic reaction or through the natural increase in temperature resulting from delivery into the body.
  • 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 polymeric composition of the present invention; (2) admixing water and the polymeric composition; and (3) increasing admixture temperature sufficient to gel the polymeric composition, such temperature increase without oxidation of the polymer or DOPA or DOPA-derived moiety residue incorporated therein.
  • an increase in admixture concentration can reduce the temperature required to effect gelation.
  • a larger hydrophilic block thereof can increase the temperature required to gel the corresponding composition.
  • Various other structural and/or physical parameters can be modified to tailor gelation, such modifications as can be extended to other polymeric compositions and/or systems which are consistent with the broader aspects of this invention.
  • PLURONIC® block copolymers self-assemble in a concentration- and temperature-dependent manner into micelles consisting of a hydrophobic PPO core and a water-swollen corona consisting of PEO segments.
  • certain PEO-PPO-PEO block copolymers such as PLURONIC® F127 and PLURONIC® F68, transform from a low viscosity solution to a clear thermoreversible gel at elevated temperature. While not wanting to be bound by theory, it is generally assumed that the interactions between micelles at elevated temperature lead to the formation of a gel phase, which is stabilized by micelle entanglements.
  • micellization and gelation processes depend on factors such as block copolymer molecular weight, relative block sizes, solvent composition, polymer concentration, and temperature. For example, increasing the length of the hydrophilic PEO blocks relative to the hydrophobic PPO block results in an increase in micellization and gelation temperature (E ge ⁇ ).
  • DSC Differential scanning calorimetry
  • PLURONIC® F127, DME-PAO7 and DOPA-PAO7 were found to be qualitatively similar and were characterized by a large endothermic transition corresponding to micelle formation followed by a small endotherm at T gs ⁇ ( Figure 2).
  • the transition temperature of the small peak was found to correlate strongly with ge ⁇ determined by rheometry and the vial inversion method (Table 4).
  • Table 4 Gel temperatures obtained from vial inversion method, rheology or differential scanning calorimetry for 22 wt % DME-PAO7, DOPA-PAO7 and PLURONIC® F127 solutions.
  • DOPA-PAO7 copolymers and 35 to 54 % (w/w) of DOPA-PAO8 copolymers were prepared by the cold method, in which DOPA conjugate was dissolved in distilled water at about 4°C with intermittent agitation until a clear solution was obtained. Thermal gelation of concentrated solutions was initially assessed using the vial inversion method. In this method, the temperature at which the solution no longer flows is taken as the gelation temperature.
  • the gelation temperature was found to be strongly dependent on copolymer concentration and block copolymer composition (i.e., PAO7 versus PAO8).
  • PAO7 block copolymer composition
  • 22 wt % solutions of DOPA-P AO7 and DME-PAO8 were found to form a transparent gel at approximately 22.0 ⁇ 1.0°C; decreasing the polymer concentration to 18 wt % resulted in a gelation temperature of approximately 31.0 ⁇ 1.0°C.
  • DOPA-PAO7 solutions with concentrations less than 17 wt % did not form gels when heated to 60°C.
  • DOPA-PAO7 exhibits a slightly higher gel temperature than that (17.0 ⁇ 1.0°C) of unmodified PLURONIC® F127.
  • DOPA- PAO8 The gelation behavior of DOPA- PAO8 was found to be qualitatively similar, except that much higher polymer concentrations were required to form a gel. 54 wt % solutions of DOPA-PAO8 and DME-PAO8 formed gels at 23.0 ⁇ 1.0°C, while 50 wt % of DOPA-PAO8 gels at 33.0 ⁇ 1.0°C. However, DOPA-PAO8 solutions with concentrations less than 35 wt % did not form gels when heated to 60°C. DOPA-PAO8 exhibits a much higher gel temperature than that (16.0 ⁇ 1.0°C) of unmodified PLURONIC® F68. These gels were found to be resistant to flow over long periods of time.
  • FIG. 4 Shown in Figure 4 are the rheological profiles of 50 wt % solutions of unmodified PLURONIC® F68 and DME-PAO8 as a function of temperature.
  • the E ge ⁇ of a 50 wt % DME-PAO8 solution was found to be 34.1 ⁇ 0.6°e, whereas the r ge ⁇ of an equivalent concentration of unmodified PLURONIC® F68 was approximately 18°C lower (16.2 ⁇ 0.8°C).
  • the plateau storage moduli of 50 wt % solutions of DME-PAO8 and unmodified PLURONIC® F68 were not significantly different, approaching a plateau value as high as 50 kPa.
  • T ge ⁇ The concentration dependence of T ge ⁇ is illustrated in Figure 5, which shows the rheological profile of DME-PAO8 at two different concentrations as a function of temperature. Jgei of 45 wt % solution of DME-PAO8 was observed to be approximately 12°C higher than that of 50 wt % solution of DME-PAO8.
  • both DOPA and DOPA methyl ester can be considered hydrophilic, the increase of r ge ⁇ observed in the DOPA-modified PLURONIC® PAOs, compared with that of unmodified PLURONIC® PAOs, is likely due to the increase in length of the hydrophilic PEO segments resulting from coupling of DOPA to the endgroups.
  • micellization peak was seen to extend to temperatures above the onset of gelation, indicating that additional monomers aggregate into micelles at temperatures above the gelation point.
  • concentration dependence of DOPA-PAO7 and DME- PAO7 aggregation is shown in Figure 6.
  • DSC thermograms indicate a decrease in micellization temperature and ge ⁇ with increasing polymer concentration.
  • the broad endothermic peak corresponding to micellization can also be observed in solutions at concentrations at which no gelation takes place; the characteristic temperature of the broad peak increases linearly with decreasing copolymer concentration, whereas the small peak was observed to coincide to the gel temperature of the concentrate copolymers but disappears as copolymer concentration decreases.
  • various polymeric compositions of this invention can be designed and prepared to provide various micellization and/or thermal gelation properties.
  • degradation into excretable polymer components and metabolites can be achieved using, for instance, polyethylene glycol and lactic/glycolic acids, respectively.
  • the polymeric compositions of this invention provide improved adhesion by incorporation of one or more DHPD residues, such incorporation resulting from the coupling of a terminal monomer of the polymeric component to such a residue.
  • a photocurable DHPD-moiety-containing monomer is copolymerized with PEG-DA (PEG-diacrylate) to form adhesive hydrogels through photopolymerization.
  • Photopolymerization can be achieved at any visible of UV wavelength depending on the monomer used. This is decidedly determined by one skilled in the art.
  • the photocurable monomers consist of an adhesive moiety coupled to a polymerizable monomer with a vinyl group, such as a methacrylate group with or without an oligomeric ethylene oxide linker or fluorinated ether linker in between.
  • a photoinitiator such as 2,2'-dimethoxy-2-phneyl-acetonephenone (DMPA), camphorquinone/4-
  • gel conversion as determined by measuring the mass of the sondgel reached more than 75 wt % after 2 minutes of UV irradiation and increased to greater than 85 wt % upon irradiation for more than 5 minutes.
  • Gelation of PEG-DA occurred in 4 minutes or less when visible light initiators were used (4 minutes for CQ/DMAB and 3 minutes for AA/FNa 2 ).
  • Copolymerization of PEG-DA with 1 or 7 was qualitatively similar to polymerization of pure PEG-DA, although addition of 1 or 7 to the PEG-DA precursor solution resulted in a decrease in gel conversion that was dependent on DOPA monomer concentration and initiating system.
  • gel conversion was reduced to less than 85 wt % in the presence of 2.5 mol % or more of 1 or 7.
  • the extent of gel conversion was not statistically different between the gels. Similar DOPA concentration dependent inhibition was observed for the visible light induced initiators.
  • Figure 26 shows the mole fraction of DOPA incorporated into the gel network, as a function of mol % monomer 1 and 7 in the precursor solution. There was no significant difference in the mole fraction of DOPA incorporated between samples containing 1 and 7. As much as 24.9 ⁇ mol/g of DOPA was incorporated into the PEG hydrogels.
  • Direct evidence for 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 turned bright yellow after the addition of the nitrite reagent and then red following the addition of excess base. This color transition is typical of catechols, indicating that the unoxidized form of DOPA was incorporated into the hydrogels through photopolymerization. The intensity of the red color also reflects the concentration of DOPA incorporated into the photocured gels.
  • Equation (1) where R is the radius of curvature of the hemispherical gel. Load versus displacement data was fitted with Equation (1), which allowed the elastic moduli to be calculated based on the proportionality factor of the curve fit. As seen in Table 6, average Young's moduli (E) for DOP A-containing gel of around 50 kPa was obtained. Table 6 Average Young's Moduli for DOPA containing gels
  • modulus values are about 30% lower than that of PEG-DA gels, confirming the inhibitory effect of DOPA on radical photopolymerization.
  • DOP A-containing gels still exhibited moduli suitable for many biomedical applications.
  • a suitable modulus is one greater than 500 Pa.
  • Another use of the adhesive hydrogels is for localized drug delivery.
  • adhesive hydrogels can be formed on a mucous membrane in the mouth or oral cavity.
  • the hydrogels can be loaded with a drug such as an antibiotic and facilitate slow release of the drug over a period of time. They hydrogel can also be loaded with an analgesic and used to deliver pain relief at a localized site.
  • 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 cell proliferation inhibitor therapeutic drug and used as a coating stent or other vascular device and used to control cell proliferation at the site of an implant of the vascular device.
  • a tissue adhesive hydrogel capable of being cross-linked in vivo can be used as a tissue sealant for replacing metal or plastic sutures.
  • the adhesive bends to the surrounding tissue at a surgical or injury site and the polymer forms a cohesive link to close the wound.
  • the hydrogel can also be used for repair of bone fractures and cartilage to bone damage.
  • compositions of this invention can include but are not limited to a urethane moiety between each such terminal monomer and DOPA residue.
  • a moiety is a synthetic artifact of the agent/reagent utilized to couple the DOPA residue with the polymeric component.
  • various other moieties are contemplated, as would be understood by those skilled in the art made aware of this invention, depending upon terminal monomer functionality and choice of coupling agent.
  • L-DOPA thionyl chloride, methacroyloyl chloride, /-butyldimethylsilyl chloride (TBDMS-C1), di-t-butyl dicarbonate, methacrylic anhydride, 2,2'-dimethoxy-2-phenyl-acetonephenone (DMPA), acryloyl chloride, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), tetrabutylammonium fluoride (TBAF), 4- (dimethylamino)-benzoic acid (DMAB), l-vinyl-2 -pyrrolidone (VP), N,N-disuccinimidyl carbonate, sodium borate, sodium molybdate dihydrate, sodium nitrite, 4- (dimethylamino)pyridine (DMAP), ⁇ -hydroxysuccinimide, ⁇ , ⁇ -diisopropylethylamine, dimethylformamide, and dichlorome
  • Camphorquinone (CQ) was obtained from Polysciences, Inc. (Warrington, PA). Acetone was dried over 4A molecular sieve and distilled over P 2 O 5 prior to use. Triethylamine was freshly distilled prior to use. All other 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.
  • Shearwater Polymers, Inc. Hauntsville, AL
  • Ethyl acetate saturated with HCl was prepared by bubbling HCl gas through ethyl acetate (50 mL) for approximately 10 minutes.
  • Glass coverslips (12mm dia.) used in the following examples were cleaned by immersing in 5% Contrad70 solution, a detergent which is an emulsion of anionic and nonionic surfactants in an allealtine aqueous base (Decon Labs, Inc., Bryn Mawr, PA) in an ultrasonic bath for 20 minutes, rinsed with deionized (DI) H 2 O, sonicated in DI H 2 O 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 DI H 2 O, and sonicated in DI H 2 O for 20 minutes.
  • DI deionized
  • coverslips were subsequently air-dried in a HEPA-filtered laminar flow hood.
  • clean coverslips were sputtered (Cressington 208HR) with 2 nm Cr followed by 10 mn Au (99.9% pure).
  • Titanium oxide (TiO 2 ) surfaces were prepared by electron beam physical evaporation onto silicon (Si) wafer and cleaned in a plasma chamber prior to testing.
  • Si wafers MEMC Electronic Materials, St. Peters, MO, surface orientation (100)
  • the Si wafer was then cut into 8mm x 8mm pieces which were subsequently cleaned by ultrasonication in the following media: 5% Contrad70, ultrapure water (ultrapure water is deionized and distilled), acetone, and petroleum ether.
  • the substrates were further cleaned in an oxygen plasma chamber (Harrick Scientific, Ossining, NY) at ⁇ 200 mTorr and 100W for 3 minutes.
  • XPS X-ray photoelectron spectroscopy
  • BIACORE 2000 (Biacore International AB; Uppsala, Sweden) using bare gold sensor cartridges.
  • the resonance response was calibrated using 0-lOOmg/ml NaCl solutions.
  • Dilute solutions (0.1 mM in H 2 O) of mPEG-DOPA, mPEG-MAPd, and mPEG-OH were injected into the SPR flow cell for 10 minutes after which flow was switched back to pure DI H O.
  • BSA bovine serum albumin
  • NIH 3T3-Swiss albino fibroblasts obtained from ATCC (Manassas, VA) were maintained at 37°C and 10% CO in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Herndon, VA) containing 10% (v/v) fetal bovine serum (FBS) and lOOU/ml of both penicillin and streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • lOOU/ml fetal bovine serum
  • modified and unmodified substrates were pretreated in 12-well TCPS plates with 1.0 ml of DMEM containing 10% FBS for 30 minutes at 37°C and 10% CO 2 .
  • Fibroblasts of passage 12-16 were harvested using 0.25% trypsin-EDTA, resuspended in DMEM with 10% FBS, and counted using a hemocytometer.
  • Cells were seeded at a density of 2.9 x 10 3 cell/cm 2 by diluting the suspension to the appropriate volume and adding 1 ml to each well.
  • the substrates were maintained in DMEM with 10% FBS at 37°C and 10% CO 2 for 4 hours, after which time unattached cells were aspirated.
  • Adherent cells on the substrates were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently treated with 5 ⁇ M l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil; Molecular Probes, Eugene, OR) in DMSO for 30 minutes at 37°C.
  • the stain was then aspirated and substrates were washed (3x) with DMSO for 10 minutes and mounted on glass slides using Cytoseal (Stephens Scientific, Kalamazoo, MI) to preserve fluorescence.
  • PLURONIC® F127 (0.60 mmols) was dissolved in 30 mL of dry dioxane.
  • N,N J -Disuccinimidyl carbonate (6.0 mmols) in 10 mL dry acetone was added.
  • DMAP (6.0 mmols) was dissolved in 10 mL dry acetone and added slowly under magnetic stirring. Activation proceeded 6 hours at room temperature, after which SC-PAO7 was precipitated into ether. The disappearance of the starting materials during the reaction was followed by TLC in chi oroform-m ethanol (5:1) solvent system. The product was purified by dissolution in acetone and precipitation with ether, four times. The product yield was 65%.
  • PAO7 conjugate was used to prepare and purify DOPA-PAO8.
  • the product yield was
  • PLURONICs® F127 and F68 were determined using the colorimetric method of Waite and Benedict. Briefly, samples were analyzed in triplicate by diluting aliquots of standards or unknown solutions with 1 N HCl to a final volume of 0.9 mL. 0.9 mL of 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 1 N NaOH. Due to time-dependent changes in absorbance intensity, care was taken to ensure that the time between the addition of NaOH and recording of the absorbance was 3 minutes for all standards and samples. The absorbance was recorded at 500 nm for all standards and samples. DOPA was used as the standard for both the DOPA methyl ester and DOPA conjugates. Example 8 Rheology
  • Bohlin VOR Rheometer Bohlin Rheologi, Cranbury, NJ. A 30 mm diameter stainless steel cone and plate geometry with a cone angle of 2.5 degrees was used for all measurements. The temperature was controlled by a circulating water bath. Samples were cooled in the refrigerator prior to transfer of 0.5 mL of liquid solution to the apparatus. Measurements of storage and loss moduli, G' and G", were taken 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 was reduced to 0.1°C/min. The strain amplitude dependence of the viscoelastic data was checked for several samples, and measurements were only performed in the linear range where moduli were independent of strain amplitude. Mineral oil was applied to a ring surrounding the outer surfaces of the sample compartment to prevent dehydration during measurements.
  • DSC Differential Scanning Calorimetry
  • 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 cold methanol three times, dried in vacuum at room temperature, and stored under nitrogen at -20°C.
  • DHPD adhesive component Regardless of a particular DHPD adhesive component, a variety of polymeric components can be used in accordance with the synthetic techniques and procedures described above.
  • the polymeric component can vary in molecular weight limited only by corresponding solubility concerns.
  • a variety of other polymers can be used for surface anti-fouling and/or particle stabilization, such polymers including but not limited to hyaluronic acid, dextrans and the like.
  • the polymeric component can be branched, hyperbranched or dendrimeric, such components available either commercially or by well-known synthetic techniques.
  • Example 10a is the amidation product of the referenced starting materials
  • comparable polymer-DHPD conjugates can be prepared coupling the N-terminus of a DHPD component to an end group, back bone or side chain of a suitably functionalized natural or synthetic polymer, including those described above.
  • a suitable polymeric component terminating with a carbonate functionality can be used to provide the desired conjugate by reaction with the N-terminus of the desired DHPD component.
  • Example 11a The consensus decapeptide repeat sequence (mussel adhesive protein decapeptide, MAPd, NH 2 -Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-DOPA-Lys-CO 2 H) of the blue mussel Mytilus edulis foot protein 1 (Mefp 1) was synthesized by solid phase peptide synthesis on Rink resin (0.6 mMol/g) using Fmoc protected amino acids, 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 PEG-decapeptide conjugates (mPEG-MAPd, 2k or 5k) were cleaved at 0°C for two hours using 1 M TMSBr 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 lO ⁇ m). The purity of the products was determined to be >90% using analytical HPLC, and the structures confirmed using a PerSeptive Biosystem MALDI-TOF-MS.
  • Example lib [00163] The synthesis and procedures of Example 11a can be extended analogous to and consistent with the variations illustrated in Example 10b.
  • other conjugates can be prepared using DOP A-containing polymers prepared by enzymatic conversion of tyrosine residues therein.
  • Other techniques well-known in the field of peptide synthesis can be used with good effect to provide other desired protein sequences, peptide conjugates and resulting adhesive/anti-fouling effects.
  • Example 12a [00164] Gold surfaces were modified by adsorption of mPEG-DOPA or mPEG-
  • MAPd (2k, 5k) from solution in DCM or phosphate-buffered saline (PBS; pH 3, 7.4, and 11) at polymer concentrations ranging from 0.1-75 mg/ml.
  • Time-of-flight SIMS data corroborated the XPS findings.
  • TOF-SIMS analysis was carried out on unmodified and mPEG-DOPA-modified Au substrates, as well as mPEG-DOPA powder and a gold substrate exposed to mPEG-OH. Data was collected from each substrate for about 4 minutes.
  • Example 12c [00173]
  • the modification illustrated in Example 12a can be extended to other noble metals, including without limitation, silver and platinum surfaces.
  • Such application can also be extended, as described herein, to include surface modification of any bulk metal or metal alloy having a passivating or oxide surface.
  • bulk metal oxide and related ceramic surfaces can be modified, as described herein.
  • Example 13 Silicate glass surfaces (glass coverslips) were modified by adsorption 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 attached to modified and unmodified glass surfaces were evaluated as described, above.
  • mPEG-DOPA 50 mg was dissolved in water (18 M ⁇ -cm, Millipore) and combined with 1 mg of magnetite (Fe 3 O 4 ) powder. Similar preparations were also prepared using a mPEG-NH 2 (5k) (Fluka) and a mPEG-OH (2k) (Sigma) as controls. Each of these aqueous solutions was sonicated using a Branson Ultrasonics 450 Probe Sonicator for one hour while being immersed in a 25°C bath. The probe had a frequency of 20 kHz, length of 160 mm, and tip diameter of 4.5 mm.
  • Example 14b mPEG-DOPA stabilized nanoparticles were characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), fourier transform infrared spectroscopy (FTIR), and UV/vis spectroscopy. TEM results demonstrated that the majority of nanoparticles were of diameter of 5-20 nm (data not shown). TGA analysis of 0.4 mg of mPEG-DOPA stabilized magnetite indicated that the particles contain 17% by weight mPEG-DOPA (data not shown).
  • Example 14c The dry PEG-DOPA stabilized magnetite nanoparticles readily dispersed in aqueous and polar organic solvents (e.g., dichloromethane) to yield clear brown suspensions that were stable for months without the formation of noticeable precipitates.
  • aqueous and polar organic solvents e.g., dichloromethane
  • Suspensions of mPEG-DOPA stabilized nanoparticles in various solvents were prepared by dispersing 1 mg of mPEG-DOPA treated magnetite in 1 ml of water (18 M ⁇ -cm filtered using a Millex® AP 0.22 ⁇ m filter (Millipore)), DCM or Toluene. Suspensions were placed in a bath sonicator for ten minutes to disperse the nanoparticles. All three solutions were stable at room temperature for at least six months, whereas control suspensions of unmodified magnetite and magnetite stabilized by mPEG-OH or mPEG- NH 2 precipitated out in less than 24 hours in each solvent.
  • Example 14d Suspensions of mPEG-DOPA stabilized nanoparticles were also found to be stable under physiologic concentrations of salt.
  • mPEG-DOPA treated magnetite was placed in a quartz cuvette and combined with 0.7 ml of water (18 M ⁇ -cm filtered using a 0.25 ⁇ filter).
  • Aliquots of saturated NaCl solution (5 ⁇ l,10 ⁇ l, 20 ⁇ l, 50 ⁇ l, 100 ⁇ l) were sequentially added to the cuvette and allowed to stand for ten minutes before UV-VIS spectra were taken (Figure 15).
  • Example 15a Demonstrating stabilization of metal nanoparticles, commercial gold colloid suspension (Sigma, particle size 5 or 10 nm) was placed inside dialysis tubing (M w cutoff of 8000 for 5nm and 15000 for 10 nm) and dialyzed in ultrapure water for 2-3 days to remove the sodium azide present in the commercial preparation. The dialyzed suspensions were then placed into small glass vials and mPEG-DOPA added (10 mg/ml).
  • Example 15b [00181] 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, various other compositions can be prepared analogous to and consistent with the alternate embodiments described in Examples 10b and l ib.
  • Example 16a The data of this example demonstrates stabilization of semiconductor nanoparticles.
  • CdS nanoparticles Quantum dots
  • Fresh stock solutions (2 mM) of Cd(NO 3 ) 2 and Na 2 S were prepared in nanopure water.
  • the Na 2 S solution was injected slowly into 50 ml of Cd(NO 3 ) 2 solution using a gastight syringe at a rate of 20 ⁇ l s "1 .
  • the solution turned yellow with the addition of Na 2 S, and after 2 mL of Na 2 S was injected, a yellow precipitate appeared 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 yield a clear yellow solution. The yellow aqueous suspension was stored in the dark for several months at room temperature without visible formation of precipitate. Control experiments performed in the absence of polymer and in the presence of mPEG-OH or mPEG-NH 2 yielded yellow precipitate and a clear, colorless supernatant.
  • Example 16b [00183] The results of this example illustrate the in situ formation of stabilized semiconductor nanoparticles.
  • CdS nanoparticles (quantum dots) were formed in the presence of mPEG-DOPA by slowly mixing dilute methanolic solutions of Cd(NO 3 ) 2 and Na 2 S. Freshly prepared stock solutions (2 mM) of Cd(NO 3 ) 2 and Na 2 S were prepared in methanol.
  • Example 16c The polymeric conjugate compositions of this invention can also be used to stabilize a variety of other semiconductor materials. For instance, core-shell nanoparticles can be surface stabilized in accordance herewith.
  • Example 17 The optimization experiments of Examples 17-20 were performed with mPEG-DOPA-5K. Several parameters were examined to optimize the adsorption of mPEG-DOPA onto gold from solution, including type and pH of solvent, time of adsorption, and mPEG-DOPA solution concentration.
  • Example 20 [00188] The morphology of fibroblasts cultured on both unmodified and PEG- modified surfaces was examined via electron microscopy (Hitachi 3500 SEM). Fibroblasts on unmodified Au and mPEG-OH-modified Au were generally flat and well spread, while those cultured on mPEG-DOPA modified Au were far less spread ( Figures 14A-C).
  • FIG. 13 illustrates the differences in attachment and spreading of fibroblasts on bare Au, mPEG-OH-treated Au, and Au modified with mPEG-DOPA 5K, mPEG-MAPd 2K, or mPEG-MAPd 5K under optimal conditions (50 mg/ml for 24 hours).
  • the surfaces modified with DOP A-containing conjugates have significantly less cellular adhesion and spreading than either of the other two surfaces.
  • the mPEG-MAP 5K modification though, accounted for a 97% reduction in total projected cellular area and a 91% reduction in density of cells on the surface, a far greater reduction than that achieved by mPEG-DOPA 2K.
  • N-hydroxysuccinimide (0.110 g, 0.95 mmol) was added to a solution of Boc-DOPA(TBDMS) 2 (0.500 g, 0.95 mmol) in dry dichloromethane (DCM) (8.0 mL). The solution was stirred on an ice bath, and 1,3-dicyclohexylcarbodiimide (DCC) (0.197 g, 0.95 mmol) was added under 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 byproduct and subsequently evaporated to 1/5 of its original volume.
  • DCM dry dichloromethane
  • Boc-DOPA(TBDMS) 2 -OSu (0.567 g, 0.91 mmol) was dissolved in dry dimethylformamide (DMF) (2.5 mL), and DOPA(TBDMS) 2 (0.405 g, 0.95 mmol) was added at once under a nitrogen atmosphere. The mixture was stirred on an ice bath, and diisopropylethylamine (DIEA) (158 ⁇ L, 0,91 mmol) was added dropwise via a syringe.
  • DIEA diisopropylethylamine
  • Example 21 The procedure of Example 21 was repeated using Boc-DOPA 2 (TBDMS) to obtain Boc-DOPA 2 (TBDMS) 4 -OSu.
  • Example 24 Synthesis ofBoc-DOPA 3 (TBDMS) 6
  • Example 22 The procedure of Example 22 was repeated using Boc-DOPA 2 (TBDMS) 4 -
  • Boc-DOPA 2 (TBDMS) 4 (0.5 g, 0.54 mmol) was dissolved in saturated
  • Boc-DOPA 3 (TBDMS) 6 (1.06 g, 0.79 mmol) was dissolved in saturated
  • Solid metal substrates Al, 316L stainless steel and NiTi
  • Si wafers were ground and polished, ultimately with 0.04 m colloidial silica (Syton, DuPont).
  • Si wafers were evaporated with either 20 nm TiO 2 or 10 nm TiO 2 /40 nm Au using an Edwards FL400 electron beam evaporator at ⁇ 10 "6 Torr and were subsequently diced in 8 mm x 8 mm pieces. All substrates were cleaned ultrasonically for 20 minutes in each of the following: 5% Contrad70 (Fisher Scientific), ultrapure H 2 O, acetone, and petroleum ether.
  • TiO 2 substrates were modified under cloud point conditions by immersion in mPEG-DOPA 1-3 solutions in 0.6 M K 2 SO 4 buffered with 0.1 M N- morpholinopropanesulfonic acid (MOPS) at 50°C for 24 hours. Modified substrates were rinsed with ultrapure H 2 O and dried under a stream of nitrogen.
  • MOPS N- morpholinopropanesulfonic acid
  • 316L Stainless Steel (Goodfellow, Devon PA) was modified under cloud point conditions by immersion in mPEG-DOPA ⁇ -3 solutions in 0.6 M K 2 SO 4 buffered with 0.1 M N-morpholinopropanesulfonic acid (MOPS) at 50°C for 24 hours. Modified substrates were rinsed with ultrapure H 2 O and dried under a stream of nitrogen.
  • MOPS N-morpholinopropanesulfonic acid
  • Al 2 O 3 (Goodfellow, Devon PA) was modified under cloud point conditions by immersion in mPEG-DOPA 1-3 solutions in 0.6 M K 2 SO 4 buffered with 0.1 M N-morpholinopropanesulfonic acid (MOPS) at 50°C for 24 hours. Modified substrates were rinsed with ultrapure H 2 O and dried under a stream of nitrogen.
  • MOPS N-morpholinopropanesulfonic acid
  • SiO 2 (1500A thermal oxide, University Wafer, South Boston, MA) was modified under cloud point conditions by immersion in mPEG-DOPA 1-3 solutions in 0.6 M K 2 SO buffered with 0.1 M N-morpholinopropanesulfonic acid (MOPS) at 50°C for 24 hours. Modified substrates were rinsed with ultrapure H 2 O and dried under a stream of nitrogen.
  • MOPS N-morpholinopropanesulfonic acid
  • NiTi alloy (10mm x 10mm x 1mm) was obtained from Nitinol Devices &
  • Au electro beam evaporated onto Si Wafer from University Wafer
  • mPEG-DOPA 1-3 solutions in 0.6 M K 2 SO buffered with 0.1 M N-morpholinopropanesulfonic acid (MOPS) at 50°C for 24 hours.
  • Modified substrates were rinsed with ultrapure H 2 O and dried under a stream of nitrogen.
  • Au 2 O 3 (Au samples as described in Example 28f were exposed to an oxygen plasma to form Au2O3) was modified under cloud point conditions by immersion in mPEG-DOPA ⁇ -3 solutions in 0.6 M K 2 SO buffered with 0.1 M N- morpholinopropanesulfonic acid (MOPS) at 50°C for 24 hours. Modified substrates were rinsed with ultrapure H 2 O and dried under a stream of nitrogen.
  • MOPS N- morpholinopropanesulfonic acid
  • GaAs Universal Wafer, South Boston, MA
  • MOPS N-morpholinopropanesulfonic acid
  • 3T3 Swiss albino fibroblasts (ATCC, Manassas, VA) of passage 12-16 were cultured normally at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (FBS) (Cellgro, Herndon, VA), 100 g/mL penicillin, and 100 U/mL steptomycin. Prior to cell adhesion assays, fibroblasts were harvested using 0.25% trypsin-EDTA, resuspended in growth medium, and counted with a hermacytometer.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • DMSO DMSO
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,r-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil 5 ⁇ M l,r-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • Example 29b (depending on substrate size) from random locations on each substrate using a Leica epifluorescent microscope equipped with a SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, MI). The resulting images were quantified in terms of total projected cellular area using thresholding in MetaMorph. (Universal Imaging CorporationTM, a subsidiary of Molecular Devices Corporation, Downington, PA). The mean and standard deviation of the measurements are reported.
  • Example 29b Example 29b
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,l '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil 5 ⁇ M l,l '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,r-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil 5 ⁇ M l,r-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,l '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil 5 ⁇ M l,l '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • DMSO DMSO
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,l '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil 5 ⁇ M l,l '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • DMSO DMSO
  • Test substrates were prepared in 12-well tissue culture polystyrene plates with 1.0 mL DMEM with FBS for 30 minutes at 37°C and 5% CO 2 . Cells were seeded onto 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% CO 2 for 4 hours.
  • adherent cells were fixed in 3.7% paraformaldehyde for 5 minutes and subsequently stained with 5 ⁇ M l, -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR) in DMSO for 45 minutes at 37°C.
  • Dil -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
  • DMSO DMSO
  • Each surface was modified for 24 hours in a solution of 1.0 mg/mL mPEG-DOPA 3 (or mPEG-OH as a control) at 50°C at the pH values shown in Figure 24.
  • a four-hour cell adhesion and spreading assay was conducted as described above in Example 9. Results are shown in Figure 24.
  • Cell adhesion resistance was conferred to all substrates treated with mPEG-DOPA 3 .
  • Cell adhesion and spreading on substrates treated with mPEG-OH did not differ from the unmodified surfaces (data not shown).
  • Example 31 Surfaces and Surface Preparation.
  • Silicon wafers (WaferNet GmbH, Germany) were coated with TiO 2 (20 nm) by physical vapor deposition using reactive magnetron sputtering (PSI, Villigen, Switzerland). Metal oxide coated wafers were subsequently diced into 1 cm x 1 cm pieces for ex-situ ellipsometry measurements.
  • Optical waveguide chips for OWLS measurements were purchased from Microvacuum Ltd. (Budapest, Hungary) and consisted of a AF45 glass substrate (8 x 12 x 0.5 mm) and a 200 nm-thick Sio .25 Tio .75 O 2 waveguiding surface layer.
  • TiO 2 -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 a 3 minute exposure to O 2 plasma (Harrick Scientific, Ossining, USA) to remove all organic components from the surface.
  • O 2 plasma Hard Scientific, Ossining, USA
  • waveguides were regenerated for reuse by sonication (10 minute) in cleaning solution (300 mM HCl, 1% detergent; Roche Diagnostics, Switzerland) and subsequent rinsing with ultrapure water to remove adsorbates.
  • XPS X-ray Photoelectron Spectroscopy
  • Measured intensities were converted to normalized intensities by atomic sensitivity factors, from which atomic compositions of surfaces were calculated. Average values obtained from three substrate replicates is reported in Tables 7-8. Standard deviations were typically ⁇ 10% of the mean and are omitted for clarity.
  • OWLS Optical Waveguide Lightmode Spectroscopy
  • 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 yield 5 as a white solid. The yield was 63%.
  • Precursor solutions of PEG-DA, 1, 7, and photoinitiator were prepared and mixed immediately before photopolymerization.
  • Stock solutions of PEG-DA (200 mg/mL) and! (40 mg/mL) were dissolved in N 2 -purged phosphate buffered saline (PBS, pH 7.4), where 7 (60 mg/mL) was dissolved in 50:50 PBS/95% ethanol previously purged with N 2 .
  • PBS N 2 -purged phosphate buffered saline
  • 7 60 mg/mL was dissolved in 50:50 PBS/95% ethanol previously purged with N 2 .
  • 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.
  • the final VP concentration was adjusted to be between 135 and 300 mM.
  • the amount of DOPA incorporated into the photopolymerized gel was determined using a modification of the colorimetric DOPA assay developed by Waite and Benedict. Photocross-linked gels were stirred in 3 mL of 0.5 N HCI to extract DOPA monomers that were not incorporated into the gel network. 0.9 mL of the nitrite reagent (1.45 M sodium nitrite and 0.41 M sodium molybdate dihydrate) and 1.2 mL of IM NaOH were added to 0.9 mL of the extraction solution, and the absorbance (500 nm) of the mixture were recorded using a Hitachi U-2010 UV-Vis spectrophotometer with 2 to 4 minutes of NaOH addition. Standard curves were constructed using known 1 to 7 concentrations.
  • the other end of the cylinder was attached to a piezoelectric stepping 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 mN.
  • a fiber optic displacement sensor (RC100-GM2OV, Philtec, Inc., MD) measured the axial movement of the steel rod.
  • a TiO 2 -coated Si wafer was positioned below the hydrogel, and the Tio 2 surface was flooded with PBS in order to maintain the hydration of the gel. The indenter was advanced at 5 ⁇ m/s until a maximum compressive load of 4 mN was measured.
  • Elastic moduli were calculated by assuming Hertzian mechanics for the specific case of non-adhesive contact between an incompressible elastic hemisphere and a rigid plane, in which case the Hertzian relationship between load (E h ) and displacement ( ⁇ n ) becomes: 16R //2 E (1) 9 where R and E are the radius of curvature and the elastic modulus of the hemispherical gel, respectively. The radius of curvature of the gels was determined from height and width measurements obtained from a photograph of the gel.
  • Example 43 Chemical oxidation of PEG-DOPA into a hydrogel
  • Triethylamine (Et 3 N), hydrogen peroxide (30 wt%, H 2 O 2 ), sodium molybdate dihydrate, and sodium nitrite were purchased from Aldrich Chemical Company (Milwaukee, WI). L-Dopa was purchased from Lancaster (Windham, NH). 1-Hydroxybenzotriazole (HOBt) was obtained from Novabiochem Corp. (La Jo 11a, CA) and O-(Benzotriazol-l- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) was acquired from Advanced ChemTech (Louisville, KY).
  • the solution was successively washed with saturated sodium chloride solution, 5% NaHCO 3 , diluted 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 precipitation in cold methanol three times, dried in vacuum at room temperature, and stored under nitrogen at -20°C.
  • N-Boc-L-DOPA dicyclohexylammonium salt (5.9 mmoles), HOBt (9.8 mmoles), and Et ⁇ (9.8 mmoles) were dissolved in 50 mL of a 50:50 mixture of DCM and DMF.
  • HBTU (5.9 mmoles) in 25 mL of DCM was then added, 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.
  • PEG-DOPA aqueous solutions 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.
  • nitrite reagent (1.45 M sodium nitrite and 0.41 M sodium molybdate dihydrate)
  • the absorbance (500 nm) of the mixture was recorded using a Hitachi U-2010 UV/vis spectrophotometer, within 2 to 4 minutes of NaOH addition.
  • a standard curve was constructed using solutions of known DOPA concentration.
  • PEG-DOPA hydrogels [00258] To form PEG-DOPA hydrogels, sodium periodate (NaIO 4 ), horseradish peroxidase and hydrogen peroxide (HRP/H 2 O 2 ), or mushroom tyrosinase and oxygen (MT/O 2 ) were added to solutions of PEG-DOPA (200 mg/mL) in phosphate buffered saline (PBS, pH 7.4). For gelation induced by MT, the PBS was sparged with air for 20 minutes prior to adding MT. Gelation time was qualitatively determined to be when the mixture ceased flowing, as measured by inversion of a vial containing the fluid.
  • NaIO 4 sodium periodate
  • HRP/H 2 O 2 horseradish peroxidase and hydrogen peroxide
  • MT/O 2 mushroom tyrosinase and oxygen
  • Oscillatory rheometry was used to monitor the process of gelation and to determine the mechanic properties of the hydrogels.
  • Cross-linking reagent was added to aqueous solution of PEG-DOPA and the well-mixed solution was loaded onto a Bohlin VOR rheometer. The analysis was performed at a frequency of 0.1 Hz, a strain of 1%, and a 30 mm diameter cone and plate fixture with a cone angle of 2.5°.
  • DOPA-modified PEG was dissolved in 10 mM PBS solution (bubbled with argon for HRP/H2O2 and NaIO 4 or air for MT experiments). After adding the oxidizing reagent, the time-dependent UV/vis spectra of the solution were monitored at wavelengths from 200 to 700 nm at a scan rate of 800 nm/min. All samples were initially blanked against PBS buffer and recorded at room temperature using a Hitachi U- 2010 UV/vis spectrophotometer.
  • Molecular Weight Analysis [00261] Molecular weights were determined by GPC-MALLS on a DAWN EOS
  • PEG polyethylene glycol
  • Fmoc- PEG-NHS provides an amine for Boc- DOPA conjugation after Fmoc cleavage.
  • Piperidine (20% v/v in NMP) was used to deprotect Fmoc for 5 min and subsequently cantilevers were transferred to BOP/HOBt/DOPA (a molar ratio of 1 :1:1, final 8mM in NMP) solution with 10 ⁇ L DIPEA. The same procedure was used for Tyrosine modification.
  • Loading rate dependent force measurement revealed the energy landscape of DOPA binding (17).
  • the binding energy barrier is calculated by the force of logarithmic intercept at zero loading rate from the force transition occurred by pulling rate change and xb from the slope.
  • Silicon nitrite AFM cantilevers (Bio-Levers, Olympus, Japan) were used because of their small string constants (-5 pN/nm and -28 pN/nm).
  • XPS X-ray photoelectron microscopy
  • Silicon nitride surfaces (0.7 x 0.7 cm2) prepared in the high temperature chamber (ask to Keun Ho) were cleaned and modified as the same procedures described in AFM tip modification.
  • the photoelectron signal from carbon Is orbital was the major indicator for surface modification considering all abundant species of Si, O, and N in Si3N4 surfaces.
  • the present invention can be used with various other synthetic techniques well known to those skilled in the art to functionally modify a particular polymeric component for a subsequent coupling and preparation of the corresponding DOPA conjugate.

Abstract

La présente invention se rapporte à des compositions polymères adhésives qui peuvent comporter des fractions dihydroxyphényle ainsi que des dérivés de ces fractions. L'invention se rapporte également à des procédés associés d'utilisation de ces compositions.
PCT/US2005/006418 2004-02-27 2005-02-28 Compositions poylmeres et procedes associes d'utilisation WO2005118831A2 (fr)

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See also references of EP1735456A4
SEVER; WILKER, TETRAHEDRON, vol. 57, no. 29, 2001, pages 6139 - 6146

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US8916652B2 (en) 2008-09-28 2014-12-23 Kensey Nash Corporation Multi-armed catechol compound blends
EP2348835A1 (fr) * 2008-09-28 2011-08-03 KNC NER Acquisition Sub, Inc. Melanges de composes de catechol a branches multiples
EP2348835A4 (fr) * 2008-09-28 2014-01-22 Knc Ner Acquisition Sub Inc Melanges de composes de catechol a branches multiples
WO2010037045A1 (fr) 2008-09-28 2010-04-01 Nerites Corporation Melanges de composes de catechol a branches multiples
EP2324810A1 (fr) * 2009-11-19 2011-05-25 Ivoclar Vivadent AG Matériau dentaire plein à base de dérivés de dihydroxyphénylalanine résistants à la polymérisation
US8404760B2 (en) 2009-11-19 2013-03-26 Ivoclar Vivadent Ag Filled dental material based on polymerizable dihydroxy-phenylalanine derivatives
EP2428503A1 (fr) * 2010-09-10 2012-03-14 Justus-Liebig-Universität Gießen Synthèse de dérivés de catéchol tripodaux dotés d'un équipement de base flexible pour la fonctionnalisation de surfaces
WO2012032085A1 (fr) * 2010-09-10 2012-03-15 Justus-Liebig-Universität Giessen Synthèse de squelettes flexibles trivalents avec des ligands comprenant des unités catéchol pour la fonctionnalisation de surfaces
US8912332B2 (en) 2010-09-10 2014-12-16 Justus-Liebig-Universitaet Giessen Synthesis of trivalent flexible frameworks with ligands comprising catechol units for functionalizing surfaces
US9447407B2 (en) 2011-02-02 2016-09-20 Agency For Science, Technology And Research Double coating procedure for the membranes of bioartificial kidneys
US10177383B2 (en) 2014-03-31 2019-01-08 National Institute For Materials Science Nano-coating material, method for manufacturing same, coating agent, functional material, and method for manufacturing same
WO2021110063A1 (fr) * 2019-12-02 2021-06-10 Jiangyin Usun Biochemical Technology Co., Ltd. Nouveaux conjugués de peptides et de polysaccharide

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EP1735456A2 (fr) 2006-12-27
WO2005118831A3 (fr) 2007-06-28
EP1735456A4 (fr) 2011-11-16
AU2005250314A1 (en) 2005-12-15
CA2557330A1 (fr) 2005-12-15
JP2007527871A (ja) 2007-10-04
JP5133048B2 (ja) 2013-01-30

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