CN101065134A - 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 are disclosed.

Description

Polymer composition and relevant using method

The cross reference of related application

The application is the U.S. Patent application of submitting on July 19th, 2,002 10/199,960 partial continuous application, U.S. Patent application 10/199,960 require the U.S. Patent application 60/306 respectively at July 20 calendar year 2001 and submission on April 19th, 2002,750 and 60/373,919 priority.The application also requires the priority of U.S. Patent application of submitting on February 27th, 2,004 60/548,314 and the U.S. Patent application of submitting on March 2nd, 2,004 60/549,259.Each patent is all included this paper in as a reference in full.

About the research of federal government's patronage or the statement of exploitation

Give the fund NCC-1-02097 that fund DE13030, the DE12599 of Northwest University and DE14193 and NASA give Northwest University according to the national health academy, U.S. government enjoys some right of this invention.

Background technology

Sea-mussel mucin (mussel adhesive protein, MAP) is an adhesion material in the very important water, and it forms tough connection between the resident surface of mussel and mussel.In the process that is connected to the surface, MAP comes out as liquid secretion, forms the solid speckle through crosslinked or sclerous reaction.One of unique features of MAP is to contain L-3, and 4-dihydroxyphenylalanine (DOPA)-a kind of rare aminoacid it is believed that this aminoacid has caused the bonding of MAP to base material by mechanism to the small part of also not knowing fully.Mussel adheres to kinds of surface, comprises metal, metal-oxide, polymer, plastics and timber.

For biosensor, medical diagnostic prods, all need handle the healing of medical treatment device, the operative incision of localized drug delivery, implantation in the equipment of serum and other human body/animal body fluid and test, organizational project, the body, be the tissue adhesion bonding and nanosecond science and technology (based on the treatment and the diagnostic tool of nano-particle) of bone and cartilage for example of purpose with the rehabilitation, control cell and protein are all most important to the adhesion on surface.In a lot of commercial Application, control cell and protein are also very important to the adhesion on surface.These application comprise and prevent that mussel from adhering on other used in ship, ship, bridge pier and ocean and fresh water structure, prevent that Sargassum and antibacterial are on the water pipeline of water for industrial use and drinking water and be used to detect on the pick off of water quality and purity and grow.

At medical domain, adopted the physics or the chemical fixation of some materials to be adsorbed onto lip-deep strategy as limit protein and cell, these materials as poly-(alkylene oxide) (PAO), as Polyethylene Glycol (PEG), poly(ethylene oxide) (PEO) and PEO-PPO-PEO block copolymer, copolymer as commodity PLURONICS by name, and polymer for example PEG/ tetraethylene glycol dimethyl ether, poly-(methacrylate methoxyl group ethyl ester) be (PMEMA) and poly-(methacrylyl phosphatidylcholine) (poly-MPC) (E.W.Merrill, Ann.NY Acad.Sci., 516,196 (1987); Ostuni etc., Langmuir 2001,17,5605-20 includes this paper in as a reference).Therefore the method on the polymer-modified surface of usefulness of current employing must need different chemistry strategies according to every kind of material type customization.For example, for example platinum, silver and golden surperficial availablely contain sulfydryl (molecular modification SH), metal-oxide are then usually modified with silane coupled chemistry of noble metal.There is not a kind of finishing strategy can be common to dissimilar materials.And much existing method depends on expensive equipment or complicated synthetic method, perhaps depends on both.

Summary of the invention

For example the present invention relates to basic in the environment of aqueous as the compositions of binding agent.Preferred compositions generally comprises viscosity part and polymer moieties, and described polymer moieties has required surface activity effect (or other desirable characteristics).

On the one hand, the viscosity of the present composition partly contains the dihydroxy benzenes radical derivative, comprises two (DHPD), and wherein second DHPD is

That is the methylene derivatives of dihydroxy phenyl.

On the other hand, polymer moieties contains poly-(alkylene oxide).In highly preferred embodiment, viscosity partly contains DHPD, for example, and DOPA (as described herein), and polymer moieties contains PEO-PPO-PEO block polymer (as mentioned above).

In another preferred implementation, viscosity partly contains DHPD, and it comprises (vinylic) the unsaturated part that contains thiazolinyl (ethylenic) or vinyl, for example side chain of alkyl acrylate.

Detailed Description Of The Invention

More particularly, the present invention includes dihydroxy phenyl (DHPD) viscous compound of formula (I),

In the formula, R ₁And R ₂Can be identical or different, be selected from independently of one another hydrogen, saturated and undersaturated, straight chain and side chain, replacement and unsubstituted C _1-4Alkyl;

P separately and be independently selected from-NH ₂,-COOH ,-OH ,-SH,

R in the formula ₁And R ₂As defined above.

Singly-bound, halogen,

A in the formula ₁And A ₂Separately and be independently selected from H, singly-bound;

Blocking group,

Poly-(alkylene oxide) roughly,

N is 1 to about 3 in the formula,

A ₃Be

R ₄Be H, C _1-6Low alkyl group, or

Polyalkylene oxide

R ₃As defined above, D is suc as formula shown in (I).

In one aspect, described poly-(alkylene oxide) has following structure,

In the formula, R ₃And R ₄Separately and be independently selected from H or CH ₃, the value of m between 1 to about 250, A ₄Be NH ₂, COOH ,-OH ,-SH ,-H or blocking group.

DHPD form very preferably is

R ₁, R ₂With P as defined above.

Further preferred DHPD form has following array structure:

In the formula, A ₂Be-OH A ₁Under roughly being poly-(alkylene oxide) of array structure,

R ₃, R ₄With m such as claim 2 definition.Generally, poly-(alkylene oxide) is the block copolymer of oxirane and expoxy propane.

A method of the present invention comprises base material is adhered on the another one base material that this method comprises the step that the DHPD with following array structure is provided:

R in the formula ₁And R ₂As defined above; The DHPD of said structure is applied to wants on adherent one or another or two base materials; Will adherent base material contact so that described base material is adhering to each other with the DHPD of said structure between them, thereby randomly by base material separately with use the DHPD of the said structure between base material that base material is contacted with each other once more to make relative to each other transposition of base material (reposition).

In a preferred method, R ₁And R ₂Be hydrogen.

Definition: be used for the application's purpose, the dihydroxy benzenes radical derivative of the following structure of dihydroxy benzenes radical derivative of the present invention (DHPD) expression:

P, R in the formula ₁And R ₂As described below, n is 1 to about 5.In one embodiment, R ₁And R ₂Be hydrogen, P self is a dihydroxy phenyl.Preferred DHPD is L-3 in embodiment of the present invention, 4, and Dihydroxyphenylalanine (DOPA), (usually),

A in the formula ₁And A ₂As defined above.

" roughly poly-(alkylene oxide) (substantially poly (alkyleneoxide)) " used herein refers to that the main or overwhelming majority is alkyl oxide (alkyloxide) or alkyl ether in the compositions.The existence of hetero atom and functional group has been considered in this definition, and hetero atom is N, O, S, P etc. for example, functional group for example-COOH ,-NH ₂,-SH and thiazolinyl or vinyl unsaturated group.It should be understood that the amount that these non-alkylene oxide (non-alkyleneoxide) structures exist can only be abundant relatively, thereby can not reduce suitable total surface activity, avirulence or the immune response characteristic of polymer widely.

The accompanying drawing summary

Fig. 1 shows that PLURONIC  F127, its carbonic ester intermediate (SC-PAO7) and DME-PAO7 are at CDCl ₃In ¹H NMR spectrum.

Fig. 2 provides the differential scanning calorimetric figure of the PLURONIC  F127 aqueous solution of DME-PAO7, the DOPA-PAO7 of 30 weight % and unmodified.Arrow is represented the position of gelation heat absorption.

The shearing of 22 weight %DME-PAO7 aqueous solutions storage modulus (storage modulus) G ' was as the function construction of temperature when Fig. 3 represented 0.1Hz and 0.45% strain.Be the rheology characteristic figure of 22 weight % aqueous solutions of the PLURONIC  F127 of unmodified shown in the illustration, rheology characteristic is the function of temperature.

The shearing of 50 weight %DME-PAO8 aqueous solutions storage modulus G ' was as the function construction of temperature when Fig. 4 represented 0.1Hz and 0.45% strain.Be the rheology characteristic figure of 50 weight % aqueous solutions of the PLURONIC  F68 of unmodified shown in the illustration, rheology characteristic is the function of temperature.

When Fig. 5 represented 0.1Hz and 0.45% strain, the storage modulus of the DME-PAO8 aqueous solution of 45 weight % or 50 weight % was mapped respectively as the function of temperature.

(A) DOPA-PAO7 of variable concentrations and (B) the differential scanning calorimetric figure of DME-PAO7 when Fig. 6 A 6B represents to heat.Arrow is represented only in the observed gel heat absorption of higher polymer concentration position.

Fig. 7 A-C represents the Au of (A) unmodified, (B) m-PEG-OH and (C) high-resolution C (ls) the XPS peak of m-PEG-DOPA.The remarkable increase at the ether peak (C) during 286.5eV shows and has PEG.

Fig. 8 A-C provides TOF-SIMS positive spectrum, shows and represents gold and the bonded peak of catechol.With Au peak (m/z～197) this spectrum of standardization.

Fig. 9 provides TOF-SIMS spectrum, shows Au base material, the contacted mPEG-OH of unmodified, the positive secondary quasi-molecular ions of the Au of mPEG-DOPA powder or mPEG-DOPA in quality m/z～43 o'clock.

Figure 10 represents TOF-SIMS spectrum, shows the positive secondary quasi-molecular ions that carries out the Au base material of chemisorbed with mPEG-DOPA.Observe gold and the combining of catechol at m/z～225 (AuOC), 254 (AuOCCO) and 309.In m/z～434,450,462 and 478 observe the more weak AuO of intensity _aC _bThe peak.Observed periodicity triplet is corresponding to being incorporated into DOPA-(CH in m/z is the scope of 530-1150 ₂CH ₂O) _nAu, wherein each inferior peak by 14 or 16amu divide and to open, represent the CH in the PEG chain ₂, CH ₂CH ₂And CH ₂CH ₂O.When n=1-15, observe this pattern.

Figure 11 shows the SPR spectrum of the albumen (0.1mg/ml BSA) of the gold surface that is adsorbed onto modification and unmodified.Compare with the surface that mPEG-OH modifies with exposed gold, the surface that mPEG-DOPA and mPEG-MAPd modify demonstrates reduces proteic absorption.

Figure 12 shows that the mPEG-DOPA concentration of antifouling behavior relies on.Shown in modified gold surface 24 hours in the mPEG-DOPA concentration, analyze the density and the area of adherent cell then.( ^*=p＜0.05, ^*=p＜0.01, ^{* *}=p＜0.001; Black rod=always outstanding area (total proj.area), grey rod=superficial cell density)

Figure 13 relatively under optimum condition (50mg/ml, 24 hours), the adhesion and the diffusion of cell on the gold that exposed gold, mPEG-OH handle, the gold surface handled with mPEG-DOPA 5K, mPEG-MAPd 2K and mPEG-MAPd 5K.(black rod=always protect area, grey rod=superficial cell density, ^{* *}=p＜0.001).

Figure 14 A-C is a series of SEM microphotograpies, is presented at the Au of (A) unmodified, (B) Au that handles with mPEG-OH and (C) Au that modifies of mPEG-DOPA go up the fibroblastic form of NIH 3T3.All processing all are to handle 24 hours in 50mg/ml in DCM.

The UV/vis absorption spectrum of the magnetic nanoparticle that the mPEG-DOPA shown in Figure 15 demonstration is suspended in the NaCl aqueous solution of several concentration is stable.Add NaCl and do not cause nanoparticle deposition.

Figure 16 shows that adding salt in untreated Au nano-particle induces gathering.Be depicted as and be suspended in the be untreated UV/vis scintigram of Au nano-particle of 10nm in (shown in concentration) NaCl aqueous solution.Along with NaCl concentration increases, the reduction of 520nm absorption band and skew have reflected the gathering of nano-particle.

Figure 17 shows that adding salt in the stable Au nano-particle of mPEG-DOPA does not induce gathering.Be depicted as and be suspended in the UV/vis scintigram of the stable Au nano-particle of 10nm mPEG-DOPA in (shown in concentration) NaCl aqueous solution.Along with NaCl concentration increases, reduction and skew do not take place in the 520nm absorption band, have reflected effective stabilisation of nano-particle.

Figure 18 shows and to be suspended in the UV/vis absorption spectrum of the CdS nano-particle that the mPEG-DOPA in (shown in concentration) NaCl aqueous solution is stable.

Figure 19 shows the TiO of unmodified ₂With use mPEG-DOPA _1-3The TiO that handles ₂XPS detect scintigram (survey scan).

Figure 20 shows TiO ₂With through mPEG-DOPA _1-3The TiO that handles ₂The adherent long-term resistance of last pair cell.The length of no dirt reaction duration and DOPA peptide anchoring group (anchoring group) is proportional.Make adherent cell imaging with calcium AM.

Figure 21 shows through mPEG-DOPA _1-3The TiO that modifies ₂The high-resolution XPS scanning in the Cls zone of base material.Attention is along with the length of DOPA peptide anchoring group increases, and ether carbon peak (286.0eV) increases.

Figure 22 shows through mPEG-DOPA _1-3The TiO that modifies ₂The high-resolution XPS scanning in the Ols zone of base material.Along with the increase of DOPA peptide length, on behalf of the peak of polymeric oxygen, 532.9eV the place increase, and Ti-O-H peak (531.7eV) reduces.

Figure 23 is presented at the ad hoc meter experimental result picture of Luo Basi on the 316L rustless steel.

Figure 24 be presented at 50 ℃ and shown in use mPEG-DOPA under the pH value _1-3Various lip-deep 4 hour cells of modifying 24 hours adhere to figure.

The percentage ratio that Figure 25 shows the gel conversion ratio to the UV irradiation time (minute) mapping.

The molar fraction that Figure 26 shows the DOPA mix is to 1 or 7 mole % mapping in the precursor solution.

The percentage ratio that Figure 27 shows the gel conversion ratio is to 1 or 7 mole % mapping in the precursor solution.

Figure 28 shows the x-ray photoelectron spectroscopy XPS analysis of silicon nitride surface.

Figure 29 shows freely the monitoring of functionalized silicon nitride cantilevers (free monitoring).

Figure 30 is that the entropic elasticity (entropic elasticity) of Polyethylene Glycol is analyzed.

Figure 31 is that the power of the DOPA of side chain modification detects.

Figure 32 DOPA-T ₁O ₂The possible model of binding mechanism.

Figure 33 is that the atomic force microgram is arranged.

Figure 34 is the relevant data of force measurement.

Figure 35 is the viscosity data.

Figure 36 is synthesis path and data analysis.

These dihydroxy benzenes radical derivatives (" DHPD ") binding agent plays a role in aqueous environments.In order to form polymer composition, provide the functional DHPD of viscosity partly to be coupled to the polymer that required surface activity effect is provided usually.Below these components will be described in more detail.

These viscosity, polymer composition have a lot of purposes, are included in to prevent in various medical science, industry and the consumer applications that protein and/or cell adhesion are to the surface.DHPD also can be used as the substitute of wound sutures, auxiliary fracture or cartilage-and to the healing of-bone injury.These and other application will be described below in more detail.

Preferred polymeric compositions of the present invention has following array structure:

Wherein, for each chemical compound of formula (1a), R ₁And R ₂Separately and independently as defined above,

P ₁And P ₂Separately and define suc as formula the P in (I) independently;

N and m independently are 0 to about 5, and condition is that among n or the m at least one is 1 at least;

The viscosity part

Viscosity of the present invention partly is the dihydroxy benzenes radical derivative (" DHPD ") with following preferred structure:

R wherein ₁, R ₂With P as defined above, t is 1 to about 10, preferably is about 1-5, most preferably 1 to about 3.DHPD is bonding can to play a role in aqueous environments.In this article, aqueous environments is any medium that contains water.It includes but not limited to water, comprises saline and fresh water, cell and bacterial growth media solution, and the water buffer, other is based on aqueous solution and body fluid.The DHPD part can be by derivatization.It will be appreciated by those skilled in the art that this derivatization is subjected to the restriction of the required adhesion characteristics that will keep.

Polymers compositions

Know the various polymers compositionss that those skilled in the art of the present invention will know provides surface activity effect and other desirable characteristics.Required surface activity effect is relevant with the antibiont dirt with the particle aggregation of minimizing, and described antibiont dirt comprises anti-cell and/or protein adherence.For example, according to final purposes, polymers compositions can be water miscible, and/or according to multiple other final use, polymers compositions can form micelle.Can be used for polymer of the present invention and include but not limited to, Polyethylene Glycol (PEG), poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO), PEO-PPO-PEO block copolymer, polyphenylene oxide, PEG/ tetraethylene glycol dimethyl ether, PMEMA, poly-MPC and fluoridized (perfluorunated) polyethers.Can be by several approach synthetic polymer compositions.For example, can come synthetic polymer compositions by the general synthetic method of activated polymer end group.Available carbonic ester chemistry activates various polymer or monomer whose component.Specifically, activatory polymers compositions of succinimdyl carbonate and DHPD partial reaction can provide stable ammonia ester (urethane) conjugate (conjugate).In much may approach two kinds, i.e. approach (a) among figure below scheme 1a, the 1b and (b) has shown in aqueous and non-aqueous solvent and the coupling that gathers (alkylene oxide), does not damage required bioadhesive simultaneously.For example, by forming ammonia ester or amido link, the DHPD residue can be coupled on the polymers compositions so that required coupling compositions to be provided.These route of synthesis are shown in scheme 1a and 1b, and describe in more detail hereinafter.

Scheme 1a

Scheme 1b

More specifically, if be coupled to polymers compositions by forming ammonia ester bond, available multiple other functional group comes esterification or the hydroxy-acid group of the DHPD component of deriving.Perhaps, the DHPD component can be coupled to polymers compositions (for example, according to polymer end groups ,-NH ₂Or-OH, amidatioon or esterification can take place), the deutero-DHPD of available various known blocking groups is provided functional group, described blocking group includes but not limited to, Boc, Fmoc, borate, phosphate ester and tributyl dimetylsilyl.The N-end protection of DHPD component can make hydroxy-acid group can be used in multiple functionalized deriving and/or the more highdensity polymers compositions of coupling.

Therefore, a part of the present invention also provides a kind of and with ammonia ester synthetic method the DHPD residue is incorporated into method in the polymer system.This method comprises that (1) provides terminal a plurality of polymer of monomers components that are, each monomer has terminal functional group; (2) carbonic acid ester derivative of the described polymers compositions of preparation; (3) described carbonic acid ester derivative and at least one DHPD partial reaction prepare the ammonia ester moiety.As mentioned above, the used polymers compositions of this method can comprise the polymers compositions with terminal monomer, wherein terminal monomer can provide required carbonic acid ester derivative with certain reagent reacting, and finally provides polymers compositions and the link coupled ammonia ester moiety of DHPD component.Also can adopt other multiple coupling agent and/or C-terminal polymers compositions that required ammonia ester moiety is provided.

A part of the present invention also provides the method that keeps mixing the catechol functional group of the polymer composition of DHPD and/or system with the carbonic ester intermediate, perhaps strengthens its adhesiveness.This method comprises that (1) provides terminal a plurality of polymer of monomers components that are, each monomer has terminal functional group; (2) described polymers compositions and certain reagent reacting are to provide the carbonic ester intermediate; (3) described carbonic ester intermediate and at least one DHPD partial reaction.Be not subjected to the restriction of any theory and embodiment, method of the present invention can be thought a kind of reactive method of end group that strengthens polymers compositions by suitable carbonic ester intermediate.The subsequent reactions that takes place on the amino nitrogen of DHPD part provides corresponding conjugates to keep catechol functional group simultaneously.

According to the present invention, shown in scheme 1a, available various route of synthesis partly are coupled to DHPD on the activatory intermediate of described carbonic ester.The DOPA methyl ester (DME) that the reaction of DOPA and methanol obtains in the presence of thionyl chloride can use in organic solvent.Available TLC and NMR monitoring reaction process, in fact coupling reaction was finished (representative conjugate is DME-PAO7 (from PAO PLURONIC  F127) and DME-PAO8 (from PAO PLURONIC  F68)) in 1 hour.Behind the cold methanol purification, obtained high product yield.

Can be in aqueous alkali the DOPA of free carboxy form be coupled to the carbonic ester intermediate.As everyone knows, the main difficulty of operation DOPA is that it is easy to oxidation (becoming DOPA quinone and other product), and oxidation is easy to take place in aqueous alkali.In order to prevent undesirable oxidation of DOPA catechol side chain in the coupling process under the alkali condition, can earlier DOPA be added the DOPA (scheme 1b) that forms the borate protection in the sodium borate aqueous solution.The complex that produces is highly stable in neutrality or alkaline solution, and is easy to protection under acid condition.Utilize DOPA and boratory complexation, under alkali condition,, produce DOPA-PAO7 and DOPA-PAO8 the terminal coupling of DOPA and several commercially available PAO.The range estimation reaction solution finds not exist the DOPA-quinone of strong absorbent, and it is not oxidized to be illustrated in the course of reaction DOPA.When reaction finishes, go to protect with the DOPA end group of HCl acidify with block copolymer.

¹H NMR spectrum and colorimetric determination have confirmed the composition of the PAO that 4 kinds of DOPA-of the activatory reaction intermediate of butanimide and all modify in the scheme 1.Shown in Figure 1 is that PAO PLURONIC  F127, the activatory intermediate of succinimdyl carbonate (SC-PAO7) and the corresponding D OPA methyl ester PAO that modifies is (with PLURONIC  F127, DME-PAO7) ¹H NMR spectrum.The PAO's that contains DOPA ¹In the H NMR spectrum, from the succinimidyl carbonate group-CH ₂The spike that-proton forms at～2.8ppm and-CH ₂The spike complete obiteration that-O-proton (from the unique ethylene oxide group that adjoins carbonate group among the activatory PAO) forms at～4.4ppm, and because DOPA partly is incorporated in the copolymer, a series of new peaks have appearred.Contain the PAO's of DOPA ¹A feature of H NMR spectrum be in the 6.5-6.9ppm scope, occurred (corresponding to three protons of DOPA phenyl ring) one unimodal and two bimodal.Synthetic DOPA-PAO conjugate in the aqueous solution ¹H NMR spectrum (not shown) is also observed similar feature.

Suppose in corresponding carbonic ester intermediate SC-PAO7 and SC-PAO8 and have two succinimidyl carbonate groups, according to this hypothesis, with the colorimetric analysis quantitative assay DOPA methyl ester and DOPA be 76%-81% (table 1) to the coupling efficiency of this two kinds of POA.The coupling efficiency of report is to repeat synthetic meansigma methods at least 3 times under the same terms, and when the more excessive DOPA of use in the reaction, does not observe the remarkable increase of coupling efficiency.DOPA-PAO7 for preparing from aqueous solution and DOPA-PAO8 also observe similar coupling efficiency, illustrate containing Na ₂B ₄O ₇Aqueous alkali in, the hydrolysis of the activatory PAO of succinimidyl carbonate is slow.

Opposite with coupling efficiency, the product yield (as shown in Figure 1) of the PAO that synthetic optionally DOPA-modifies in aqueous solution is lower than the product yield of the PAO that synthetic optionally DOPA-in the organic solvent modifies.This may be because the surfactant properties of initial PAO material, causes when extract the PAO of DOPA-modification with dichloromethane from water efficient low.The chemistry of peptides that it should be noted that available standards is with the further functionalized performance with the customization block copolymer of the free carboxy acid of DOPA-PAO7 and DOPA-PAO8.The PAO that 4 kinds of DOPA-in the table 1 modify can be stored in-20 ℃, variable color or character may not can take place change.

Table 1

DOPA modifies coupling efficiency and the product yield of PLURONIC 

	Coupling efficiency (%) *	Product yield (%)
			DME-PAO7 DOPA-PAO7 DME-PAO8 DOPA-PAO8	78.0±4.0 80.0±4.0 76.0±2.0 81.0±2.0	75.0±5.0 52.0±3.0 76.0±4.0 49.0±2.0

^*With Waite and Benedict (Waite, J.H.﹠amp; Benedict, the mensuration of dihydroxyphenylalanine (DOPA) in the C.V. invertebrates structural protein, Methods in Enzymology 107,397-413 (1984) includes this paper in as a reference) colorimetric method for determining that disclosed.

For the performance of biosensor, medical diagnostic prods, all need handle the healing of medical treatment device, the operative incision of localized drug delivery, implantation in the equipment of serum and other human body/animal body fluid and test, organizational project, the body, be the tissue adhesion bonding and nanosecond science and technology (based on the treatment and the diagnostic tool of nano-particle) of bone and cartilage for example of purpose with the rehabilitation, control cell and protein are all most important in the adhesion on surface.In a lot of commercial Application, control cell and protein are also very important in the adhesion on surface.These application comprise and prevent that mussel from adhering on other used in ship, ship, bridge pier and ocean and fresh water structure, prevent that Sargassum and antibacterial are on the water pipeline of water for industrial use and drinking water and be used to detect on the pick off of water quality and purity and grow.

Polymer composition of the present invention can be used as coating to prevent that protein and cell adhesion are to the device that is used for medical science and research purposes, include but not limited to as the coating of medical implant, surgery device coating, handle device coating, the coating of medical diagnostic apparatus and the coating of biosensor of serum and other animal or human's syntaxy material.Perhaps, described polymer composition can be to be used for the tissue adhesion polyalcohol hydrogel that sealing is for example organized in medical application, the gel of bonding (scar tissue formation) is used to prevent to perform the operation, bone and cartilage binding agent, organizational project, the medicine of specific site flows out, and is used to study purposes, and for example protein (comprising antibody) and micromolecule analyte (comprising medicine) is fixing.In addition, these coatings and hydrogel also have a lot of purposes in industry and consumer products, include but not limited to, biofouling (Sargassum in preventing to transport by sea, antibacterial and mussel adhere to underwater surface), prevent to flow to for example germ contamination in the current of electric power and pharmaceutical factory of factory, prevent the germ contamination in the drinking water, as tooth and Denture adhesive, the underwater adhesive of transfer indicator, the coating of water purity and detecting sensor, be used to prevent the paint of biofouling, be used for cosmetics required flavouring agent and coloring agent are adhered to hair, eyelid, on lip and the skin, with the temporary dye of giving,, and be used for the resealable binding agent of consumer products as the storage bag as tattoo etc.Method of the present invention can be used for preparing the surface that multiple polymers is modified, and is used for medical science (diagnostics, device, based on the treatment of nano-particle) and non-medical (paint and other particle dispersion, MEMS, quantum dot (quantum dot), no dirt surface) technology.

Also available method of the present invention forms cohesive hydrogel.The DHPD binding agent is connected in can be in vivo or the polymer of external formation hydrogel.Available a lot of method forms these hydrogels, comprise employing higher temperature for example the blood heat form the self-assembling polymers of gel, adopt the polymer of available enzyme cross-linking reaction, employing can make it the polymer that oxidation forms cross-linked hydrogel, and employing can make it photoactivation to produce the polymer of cross-linked hydrogel.

Antibiont dirt coating

Antibiont dirt coating of the present invention can put on medical apparatus, as blood vessel or arterial bracket, pacemaker, cardiac valve, glucose monitor and other biosensor, blood vessel crust, defibrillator, orthopedic device and operation device, comprise suture and conduit.Polymer composition of the present invention can be used as coating to prevent that protein and/or cell adhesion are to being used for the device that medical science or research are used.These application include but not limited to, as the coating of the coating of medical science implant, operation device, handle device coating, medical diagnosis device and the biosensor of serum and other animal or human's syntaxy material.Comprise that generation is enough highdensity, can repel the polymer of protein and cell with the adherent challenge of anti-cell with polymer-modified biomaterial surface, and produce the coating that covers described surface fully.This especially is a problem in the device that contains the component that multiple different materials makes.Can be through any number of ways with polymer poly compound modification of surfaces of the present invention.For example, polymer composition can be adsorbed onto the surface, the DHPD that maybe will contain polymerization initiator partly is adsorbed onto on the surface, and the growth of polymer is from this surface.In the latter event, can adopt multiple polymerization technique, include but not limited to, radical polymerization, free radical polymerisation process, ionic polymerization, ring-opening polymerisation and photo polymerization are caused in the surface.

For example, utilize the dissolubility of PEG uniqueness, can use PEG solution-treated surface to increase the density of polymer near the temperature of critical solution temperature lower limit (LCST) or cloud temperature (cloud point).Though be not bound by any theory, but the applicant believes, under the temperature conditions of used in the present invention high ionic strength and rising, the hydraulic pressure radius of PEG molecule (hydrodynamic radius) reduces, in principle than allowing more highdensity PEG chain to be stacked on the surface under the standard conditions.This method is used in and occurs the polymer that dissolubility reverses under high temperature and the high ionic strength, and for example, poly-(acrylamide) that Polyethylene Glycol, poly-(N-N-isopropylacrylamide) and other N-replace etc. shows the polymer that dissolubility reverses.

By with various polymer composition modification of surfaces of the present invention, the resistance that makes pair cell and protein adherence up to 7 days, 14 days, 21 days, 30 days, 60 days, 90 days and 120 days or longer.The overwhelming majority variation that modified material pair cell and/or albumen adhere to resistance is that the number of DHPD part in the sticky ingredient and the pH value of modifying buffer cause.For the surface of modifying with absorption method, the concentration of adsorption time and polymer composition is very little to the influence of the variation of modified material pair cell and/or protein adherence resistance.The number of DHPD partial monosomy is many more in the sticky ingredient, and pair cell and/or proteinic adhesion resistance are big more.On the surface density of polymer composition and pair cell and/dependency of protein adherence resistance is very big.The thickness of coating can be about 20 -100 μ m, comprises 30 , depends on the pH of used polymer composition and modification buffer.

The concentration that is used for the polymer composition of modification of surfaces can be about .1mg/ml-75mg/ml.The pH that modifies buffer can be about 3-9.The modification time can be about 10 minutes-Yue 72 hours.The used temperature of modification can be about 25 ℃-60 ℃.

As shown in figure 19, unmodified TiO ₂XPS detect scanning～458eV (Ti2p) and～530eV (Ols) has strong peak, this represents the feature of initial oxide, also at 248.7eV (Cls) small peak is arranged, and is external hydrocarbon contamination.But under the cloud temperature condition, use mPEG-DOPA _1-3The TiO that handles ₂The carbon that demonstrates surface combination significantly increases (shown in Cls), illustrates to have PEG on the surface.And, use mPEG-DOPA _1-3The increase of handling observed Cls peak, back is directly proportional with the number of the terminal DOPA that exists.In addition, use mPEG-DOPA _1-3The TiO that modifies ₂Observing at 400eV (Nls) in the spectrum on surface has a small peak, represents the amide nitrogen among the DOPA.

The quantitative analysis of the high-resolution XPS data of substrate surface can provide the useful information of the PEG relative quantity that is incorporated into the surface.Table 2 shows mPEG-DOPA _1-3The TiO that modifies ₂The atom of middle titanium, oxygen and carbon is formed calculating.Oxygen signal further is divided into metal-oxide (Ti-O-Ti), surperficial hydroxide (Ti-O-H) and organic oxygen and paired water (coupled water) (C-O, H ₂Subclass such as O).

Table 2

The atom of titanium, oxygen and carbon is formed calculating

	Atom is formed a(wt％)
								O
The surface	Ti	Ti-O-Ti	Ti-O-H	C-O，H 2O	C
						The TiO of unmodified 2	31.0	47.0	7.1	3.9 b	11.1
MPEG-DOPA	15.5	24.5	6.2	14.9	38.9
						MPEG-DOPA 2	12.3	19.4	5.4	18.3	44.6
MPEG-DOPA 3	11.5	17.3	4.7	20.6	45.9

^aThe nitrogen of having ignored trace.

^bBe assumed to the water that is incorporated into the surface.

The Ti:Ti-O-Ti ratio of all base materials all differs greatly with 2.0 theoretical chemistry variable; This difference may be because depth selection has surpassed the degree of depth (3-4mm) of oxide on surface.Consider 5000M _wThe XPS depth selection of the Flory radius of PEG (2.8mm) and common 5-10mm, this result is in expection.Through mPEG-DOPA _1-3On the surface of modifying, along with the increase of DOPA peptide length, Ti: the C atomic ratio lowers greatly, corresponding to the increase of the PEG number that adsorbs.Observe C: the ratio of organic oxygen (C-O) has surpassed the theoretical value 2.0 of pure PEG, illustrates on the surface of modifying to still have external hydrocarbon contamination.These the results are shown in table 3.

Table 3

The atomic ratio of the polymer composition of absorption

	Atomic ratio
				The surface	C/Ti	Ti/Ti-O-Ti	C/C-O
The TiO of unmodified 2	0.36	1.51	2.84
				MPEG-DOPA	2.51	1.58	2.62
MPEG-DOPA 2	3.62	1.57	2.43
				MPEG-DOPA 3	4.01	1.51	2.24

DOPA and TiO ₂Produce very strong, reversible key.The bond energy of this key is 30.56kcal/mol, could be from TiO in the power that the individual molecule level need about 800pN ₂Last separation, stronger 4 times than the interaction of avidin and biotin.DOPA-TiO ₂Interaction force be in approximately avidin-biotin interaction force (in biology based on one of pretending most firmly of hydrogen bond (0.1-0.2nN)) and covalent bond (＞2nN) between.

In order suitably to study the adhesion of DOPA, adopted following condition: the unimolecule method, aqueous environments is used for fixing the platform of DOPA.Selecting atomic force microscope (AFM) as research tool, because it can satisfy above-mentioned 3 conditions, and is enough sensitive for the viscoelasticity property that detects soft material, the protein of described soft material such as single molecules level, DNA and synthetic polymer.Amine moiety is incorporated into cantilevered distal end (Si3N4), then coupling methoxyl group-poly-(ethylene glycol, mPEG) and the mixture of the end capped PEG of Fmoc-(Fmoc-PEG) derivant.(Boc-) DOPA is coupled to the amino (Figure 33 C) that produces owing to the Fmoc fracture.Used mPEG with respect to the excessive 5-10 mole of DOPA-PEG, thus separable single, loose DOPA4 PEG.This molecular configuration has spatially hindered the molecular dynamics of DOPA-PEG, thereby provides a kind of explanation (Figure 36) for the DOPA oxidation test result of back.(XPS) comes the monitoring chemical reaction step with x-ray photoelectron spectroscopy, demonstrates in (the PEG modification (Figure 28) of the success on 1 * 1cm2) of smooth silicon nitride surface.The chemical group that is incorporated into silicon nitride end (tip) surface has changed static characteristic, and this also is a well indication (Figure 29-A) of finishing.Note having any it is important, between exposed and modified cantilever, detect the difference of approaching signal (approaching signal), represented because the resistance that molecular layer causes (Figure 29-B).

The link coupled cantilever of DOPA shows the very big viscosity (Figure 34) with the entropic elasticity of PEG chain.The block diagram that power distributes is single entry type (uni-modal shape), illustrates the incident that once adheres to has only taken place, and is different with multivalence albumen avidin 12.The power of having collected-distance (F-D) measurement result and carried out statistical analysis (Figure 34, n=105).When speed under load was 180nN/s, the mean force in water was 785pN.The most important thing is, profile (contour) length of PEG molecule of the length of the PEG of elongation (36nm) and expectation consistent (37nm, Figure 30).These data all are used for illustrating the unit price combination of molecular events a: DOPA, polydispersity and most advanced and sophisticated how much (the tip geometry) of PEG.One DOPA is coupled to the end of polymer chain, as TiO ₂A lip-deep combining unit (Figure 33).This unit price connects with different during metal-6 histidine (metal-(His) 6) is studied, and (His) 6 that links among the latter provides 3 metal-chelating sites (3 * metal/(His) 2) 13-15.In addition, because the polydispersity of PEG (tree) and ball tip (mountain) are (r=25nm), " mountain-tree " sample configuration of PEG5 cantilever can separate two different DOPA separation signals.

The z-translocation distance (Figure 34 C) of piezo-electric device when " d " is defined as single DOPA-PEG molecule full extension in the retraction process.In repeated circulation,, seem it is constant (Figure 34 A) substantially though ' d ' value has slight variation.This little variation may be because DOPA is attached to the surface with different angles.One of our test very important characteristic is that binding signal is not the repetition of being carried out with ' same ' DOPA molecule.This and traditional unimolecule lift in the test (single molecule pulling experiment) most advanced and sophisticated (tip) and mention a molecule randomly and form contrast.This proves that also the bonding chemistry of DOPA is a completely reversibility.This reversibility makes us obtain such conclusion, and promptly (the most weak chemical bond of TiO2～Si3N4) is Ti (surface)-O (DOPA) key base material and tip.

This presentation of results, when～0.8 nN,, from mechanical angle, the interaction force of DOPA-TiO2 be in avidin-biotin interaction force (a kind of (0.1～0.2nN)) and the covalent bond of the strongest interaction force based on hydrogen bond in the biology (＞2nN) between.From the strength data that obtain by change speed under load (power that time per unit applies), know energy information.The change of 4 orders of magnitude of speed under load produces 4 kinds of different power and distributes, to determine the bonded energy situation of DOPA.The power of Figure 34 D neutral line provides binding energy and distance to the logarithm mapping of speed under load, removes combination along application of force direction then.The energy barrier of DOPA is 28.1kcal/mol, and the distance that reaches maximum activation energy is 1.27  (Figure 34 E).

DOPA in conjunction with the orientation it is believed that be with aromatic ring on two hydroxyls point to the surface downwards.Therefore, determine in AFM, to cause that chemical group that unimolecule adheres to signal is extremely important in conjunction with chemistry for getting rid of other that caused by different orientation.Two kinds of methods have been adopted.

The first, uncle (ertary) butyl dimethyl siloxane (TBDMS) is modified the covalent chemical that hydroxyl carries out, and causes not taking place in conjunction with (Figure 31, article one line) in 200 circulations of advancing-bounce back (approach-retraction cycle).Yet, the TBDMS group go to protect the binding ability (Figure 31, two lines in bottom) that has produced DOPA again.

The second, with the borate ion complexation protect the strong adhesion that also suppressed DOPA fully (Figure 31, n=200).These data have determined that clearly the dihydroxy of DOPA is strong, reversible bonded real structure source.

Mussel develops a kind of very interesting mode and produce a kind of like this strongly bonded in water, and this interesting mode is carried out post translational modification with tyrosine hydroxylase to tyrosine exactly.With tyrosine is substrate, and this kind of enzyme catalysis adds the reaction of a hydroxyl.A large amount of this kind of enzyme is found among the threads and plaques, and DOPA also exists among the threads and plaques.It is shocking that as if (OH) having produced huge adhesive capacity changes in very little post translational modification.Therefore, designed test to show the relation between post translational modification and the binding ability.

Prepared with tyrosine but not the cantilever of DOPA link has been studied the adhesion of tyrosine on TiO2.Except the very low non-specific adhesion of some probability, do not observe detectable force signal (Figure 35 A).In order to get rid of a kind of like this hypothesis, promptly any tyrosine molecule is not contained at used tip in this test, has replaced the TiO2 surface with the Jin Dynasty.Interact by π-pi-electron, the aromatic ring of tyrosine is incorporated into gold surface with the orientation parallel with the surface, and this mechanism is well-known in surface adsorption chemistry 18,19.Identical cantilever used among the TiO2 repeatedly produces intensive bonding (Figure 35 B) in gold surface.The statistical analysis that power distributes shows that π-pi-electron adhesion is 398 (± 98) pN, is about 50% (Figure 35 C) of DOPA-TiO2 interaction force.Also demonstrate and the same feature of DOPA-TiO2 interaction that illustrates previously from the bonded force signal of tyrosine-Jin: have the PEG elastic elongation of expection contour length and have the similarly repeating signal shape (Figure 33 C) of ' d ' value.In this test, prove that clearly the post translational modification of tyrosine hydroxylase mediation has improved the binding ability of DOPA greatly, from almost being the zero 800pN that brings up to.

The biological agent of DOPA does not just have viscosity after oxidation: it also can be crosslinked with polypeptide chain, produces than the more hard material of finding in threads and pads.Crosslinked mechanism has number of ways, and these approach originate in the DOPA quinone structure of chemical instability.At sea-mussel mucin ₂₀In found the coupling of aryl-aryl ring (two-DOPA), in other species, found Michael addition (quinone-alkylamine adduct) product, but this addition compound product is found (Figure 36 A) in mussel.Therefore, these structures also might take place because of the oxidation in the mussel.This is very clearly from crosslinked angle, but also has dispute for the viscosity after ripe (being oxidation).Proved that the DOPA quinone structure is not the principal element that forms viscosity.Excessive conjugation by methoxyl group-PEG molecule (5～10 molar equivalent) (it is to prevent the further important molecule configuration of reaction of DOPA quinone) makes DOPA quinone-PEG chain spatially and chemically stabilized.

Time-resolved (Time-resolved) monitoring force signal that is increased the unimolecule DOPA quinone that (=9.7) causes by pH has disclosed the interesting fact of hiding so far.The first, detected AFM signal has two significantly distribute (Figure 36 B) according to the magnitude (powerful (high force) and weak power (low force)) of power.The statistical analysis of data has produced two clearly block diagrams, and weak power is 178 ± 62pN, and brute force is 741 ± 110pN (Figure 36 C).The combination of quinone can belong to weak power zone, because should only after oxidation begins, just occur in the zone, and subsequently along with the progress of time more frequent (Figure 36 D).The slow dynamic characteristic of DOPA oxidation has been facilitated the initial altofrequency of DOPA signal.This is to detect tested by first unimolecule that small molecule structure changes behind the environmental stimuli.Based on these results, can get rid of the probability that the DOPA quinone structure causes high viscosity.Therefore, be not subjected to the restriction of any theory, in the oxidizing process reduction form be DOPA two-oh group produce to it is believed that it is a very important requirement keeping or change the viscosity that contains the DOPA material at the interface again.

In addition, the DOPA anchoring system can be for example polysaccharide, DNA and a proteinic new platform of other ductility biomacromolecule of research.In the research of being carried out, shown the elastic property of PEG (Mw 3400), this PEG length it is believed that it is the shortest chain length of studying so far.Why can realize this research for no other reason than that used two kinds of definite anchorage methods at two ends: the covalent bond between (1) PEG and the cantilever, and the DOPA grappling between (2) PEG and the base material.This method is tested with conventional unimolecule and is also formed degree of contrast, in the unimolecule test of routine, and most advanced and sophisticated " seeing " different molecule in the motion each time of cantilever.For of the reaction of research molecule, if the stimulation that gives not is absolutely effectively to outside stimulus ₂₃, will be the very big obstacle of studying.Can be used as substitute technology based on the anchoring system of DOPA overcomes existing unimolecule and lifts these problems in the test.

Current, the model of action that does not also have clear answer to be illustrated as what DOPA is similar to " Sticky Note " as a kind of reversible glue.Two kinds of molecule combination models are arranged, monokaryon bidentate (Figure 32 A, the right side) and double-core bidentate (Figure 32 A, a left side), but these two kinds of models are not all considered the reversible combination of DOPA, because these researchs only are absorbed in adsorption process but not desorption process 25.Therefore, suppose that this chemically combined essence mainly is covalent bond, irrelevant with the factor that produces owing to the absorption removal of hydrone afterwards.One is utilized studies show that FTIR carries out, the bonded essence of DOPA-TiO2 may 60% is ions binding, and the 40%th, covalent bond.Based on this discovery, revised a kind of Molecular Adsorption model to add reversibility, wherein form a plurality of hydrogen bonds (Fig 32B) in the water.

Figure 33 EXPERIMENTAL DESIGN and unimolecule DOPA adhere to

One width of cloth figure has described blue mussel (Mytilus Edulis) and how to have adhered on the metal oxide surface.Circle comprises a speckle, has wherein found rare aminoacid DOPA.

(B) two kinds of main protein components finding in the mussel speckle, Mefp-3 and Mefp-5.The content of DOPA is very high in these sea-mussel mucins, is 27 mole% in Mefp-5, is 21% in Mefp-3.Runic Y (Y): DOPA, italic S (S): phosphoserine, italic R (R): hydroxyl arginine.

(C) AFM is most advanced and sophisticated modifies.The polymerization of 3-TSL 8330 (APTMS) is incorporated into Si3N4 tip end surface (not shown) with amino group.Long-chain is described terminal and the link coupled PEG molecule of unimolecule (Boc)-DOPA.With molar ratio is that the mixture of 5～10: 1 mPEG-NHS (2k) and Fmoc-PEG-NHS (3.4k) is stablized DOPA-PEG molecule (see and replenish the part more detailed description).

Figure 34. measurement of unimolecule power and DOPA are attached to the energy situation on TiO2 surface and measure

4 representative AFM unimolecule DOPA are from a dissociated signal of cantilever.These 4 signals are not (deleted and do not shown any adherent signal) that produces continuously.Though detect the adherent probability of DOPA very little (5～10%), detected signal demonstrates similarly ' d ' value (referring to Figure 34 C).

(B) block diagram is described the distribution of power.Speed under load be 180nN/ during second meansigma methods be 781 ± 151pN (n=105).

(C) definition of distance ' d ', the z-of piezo-electric device is to move distance when DOPA-PEG molecule full extension.

(D) adhesion (straight line) is mapped to speed under load (log).Speed under load is the spring constant of cantilever and the product of pull rate.4 different speed under loads have been selected: 1500,180.7,28.4 and 2nN/ second.Illustrate the mean force under each given speed under load with standard deviation.Power is 846.48 ± 157pN (1500nN/s), 781 ± 151pN (180nN/s), 744 ± 206pN (28.4nN/s) and 636.2 ± 150 (2nN/s).

(E) the bonded energy situation of diagram DOPA.External force makes Energy distribution that inclination take place, and has reduced the energy barrier of reaction coordination compound.The slope of linear regression graph (=kBT/xb) be 32.31, activated disfunction (activationbarrier) distance (xb) is 1.27  like this.The energy barrier height is determined (Eb=28.1kcal/mol) by extrapolation when speed under load equals zero.

The Molecular Identification of Figure 35 .DOPA viscosity origin

The tyrosine unimolecule is incorporated into the TiO2 surface.Except initial electrostatic interaction (being inevitable in some cantilever), useless detect clearly binding signal (upper limit representation signal, 700 repetitions, n=639).Do not have the non-specific adsorption signal (the lower limit representation signal, 700 repetitions, n=61).

(B) conclusive evidence tyrosine exists in tip end surface.The pi-electron of tyrosine phenyl interacts with the pi-electron specificity of gold.

(C) power that is incorporated into the tyrosine of gold surface distributes.Tyrosine demonstrates 398 ± 98pN bonding force of (180nN/ second).

Figure 36. the DOPA after the oxidation (DOPA quinone) viscosity change

The chemistry route of illustrated formation and oxidation DOPA.Effect by tyrosine hydroxylase has generated DOPA, becomes the DOPA quinone by pH with this oxydasis then.Owing to easily form free radical, DOPA quinone instability also has reactivity.It can be molecule crosslinked with other DOPA (two-DOPA), also can react with lysine amino.Arrow is at other species but may reacting of not finding in sea-mussel mucin.

(B) representational force signal (n=16), have similar ' d ' value shown in Fig. 1 C (～50nm).Collect the AFM test (1800 repetitions) that they under alkali condition (20mM Tris-Cl, pH 9.7) carried out 1 hour.The top of this figure is to the passing of bottom express time.Danger signal is represented DOPA-TiO2, and black signal is represented DOPA quinone-TiO2.

(C) 1800 F-D curves are carried out the block diagram of the power that obtains after the aggregate analysis.The block diagram in weak power zone shows 178 ± 82pN (n=143), and the block diagram in powerful zone shows 741 ± 110pN (n=51).

(D) scatter diagram of event number in official hour window (10 minutes).DOPA signal (circle, left y-axle) reduces to only 3 incidents of last time window from first 22 incidents of 10 minutes.Yet the signal of quinone (triangle, right y-axle) is increased to 42 incidents of last time window (50-60 minute) from an incident of first time window.

Generally speaking, successfully detect DOPA the unimolecule adhesion (～0.8nN), and show its reversible in conjunction with the chemistry.This strongly bonded is produced by post translational modification, then greatly reduces cohesive but DOPA is oxidized to the DOPA quinone.

Anti-soil of the present invention dirt coating can be basic permanent type, promptly continue 120 days or more, or biodegradability, and this depends on the number of DOPA in the sticky ingredient or DOPA derivative moiety.Figure 20 demonstration mPEG-DOPA _1-3The TiO that handles ₂Last the 28th day 3T3 fibroblast adheres to and diffusion test.Time point in early days (promptly being less than 7 days), the dependency of protein and cell adhesion resistance and DOPA peptide anchoring group length is fine, and the order that resistance increases is mPEG-DOPA＜mPEG-DOPA ₂＜mPEG-DOPA ₃Use mPEG-DOPA ₂And mPEG-DOPA ₃The TiO that handles ₂Base material kept the minimizing (or being called the cell adhesion resistance) of cell adhesion in 21 days.

Adopt the ad hoc meter of Luo Basi (Robust design) method to determine that DOPA peptide anchoring group length and modification condition (pH, concentration and time) are to the dirty Effect on Performance of the anti-soil of PEG area density and metal, metal-oxide, quasiconductor and polymer surfaces.9 tests that each base material adopts are shown in the following table 7.Detect and the detection of fibroblastic adhesion of 3T3 and diffusion is shown through XPS, for nearly all surface, the length of DOPA peptide and the pH that modifies buffer cause the variation maximum of the PEG quantity of adsorbing.The test of summing up in the table 7 makes us can be defined as various materials provides optimum cell to adhere to the modification condition of resistance, detects as 4 hour cell adherence tests.After carrying out these 9 tests for each base material, data are carried out the ad hoc score of Luo Basi and are analysed.To have big error amount when drawing the ad hoc meter datagram of Luo Basi be the characteristics of this technology because the individual data point on the factor level comprised other factors by all factor levels the deviation after average.

Polymeric composition of the present invention also can be used for applying and is used to handle the surface that body fluid comprises the device and the equipment of serum.Coating on these devices or the equipment surface stops protein bound to its surface, washs in a large number between each time use or necessity of cleaning device or equipment thereby reduced or eliminated.These devices need thorough cleaning with the cross-contamination between the body fluid sample that prevents to be applied to this apparatus surface.Current, between each time used these equipment of cleaning and device need with caustic for example the temperature of 50% bleach and/or rising carry out a large amount of washings.Painting method of the present invention is to make the DHPD aqueous solutions of polymers of 1mg/ml cycle through the device several hrs in room temperature.

Coating of the present invention can be used on the multi-purpose medical science implant.For example, thereby these coatings can be used for stoping bacterial adhesion to stop antibacterial to grow on implanting device thereon, reduce the implant site possibility of infection.By reducing combination and cell the adhesion to install of protein to device, these coatings can be used for reducing these devices acutely inflamed quantity upward take place.Coating of the present invention also can be used as nano-particle, prevents these particulate coagulations in the presence of serum.

Hydrogel

Polymer composition of the present invention also can be used as the surgical operation binding agent that is used for medical science and tooth application and is used as the carrier that delivers drugs into mucomembranous surface.Described polymer composition can be to be used for the tissue adhesion polyalcohol hydrogel that sealing is for example organized in medical application, the gel of bonding (scar tissue formation) is used to prevent to perform the operation, bone and cartilage binding agent, organizational project, the locus specificity medicine flows out, and be used to study purposes, for example protein (comprising antibody) and micromolecule analyte (comprising medicine) is fixing.Polymer composition of the present invention also can be used as the interfacial adhesion agent, wherein clean (neat) monomer or monomer solution put on the surface, as priming paint (primer) or the binding agent between tissue surface or metal or metal-oxide implant/apparatus surface and number of polymers or the polyalcohol hydrogel.By selecting suitable polymers component (those skilled in the art can determine), polymer composition of the present invention can fluid form injects or sends, and sclerosis forms gel network in position.Can or by heating up naturally after being delivered in the body original position take place by photocuring, chemical oxidation, enzyme reaction hardens.

A part of the present invention provides the method for the non-oxidizable gelation of a kind of polymer composition of the present invention.This method comprises that (1) provides polymer composition of the present invention; (2) mixing water and described polymer composition; (3) temperature that improves mixture is to the polymer that is enough to that described polymer composition gelation, the raising of described temperature can not made and mixes in the described compositions or the oxidation of DOPA or DOPA derivative moiety residue.According to selection and its character of polymers compositions in the compositions, the raising of mixture concentration can reduce the required temperature of realization gelation.According to specific copolymer components selection in the compositions and its character, bigger hydrophilic block copolymers can improve the required temperature of correspondent composition gelation.Can change other a lot of structures and/or physical parameter and customize gelation, these variations can expand to aspect corresponding to other polymer composition and/or the system bigger with the present invention.

Extensively the commercially available PLURONIC  block copolymer of approval is self-assembled into micelle in the mode that concentration and temperature rely on, and these micelles are made up of hydrophobic PPO nuclear and water-swellable hat (being made up of the PEO fragment).During high concentration, some PEO-PPO-PEO block copolymer as PLURONIC  F127 and PLURONIC  F68, is converted into clarifying temperature reversibility gel from low viscosity solution when temperature raises.Be not bound by any theory, the interaction when it is generally acknowledged the temperature rising between the micelle causes the formation of gel phase, and gel phase is owing to the micelle tied up in knots is stablized.Micelleization depends on factors such as block copolymer amount, relative block size, solvent composition, polymer concentration and temperature with gelation process.For example, the length that increases the hydrophobic relatively PPO block of hydrophilic PEO block causes micelleization and gelling temperature (T _Gel) increase.

Variable concentrations aqueous solution to DME-PAO7 and DOPA-PAO7 has carried out differential scanning calorimetry (DSC) detection, becomes micelle to detect the block copolymer coagulation.PLURONIC  F127, DME-PAO7 that obtains and the DSC feature of DOPA-PAO7 are similar in nature, and feature is the significantly heat absorption conversion that forms corresponding to micelle, is at T then _GelA small amount of heat absorption (Fig. 2) at place.Find the T of inversion temperature and rheometry and bottle inversion (vial inversion) method mensuration of small peak _GelRelatedness very strong (table 4).

Table 4

The DME-PAO7 of 22 weight %, DOPA-PAO7 and PLURONIC  F127 solution obtain gelling temperature by bottle anastrophe, rheometry or differential scanning calorimetry.

	Gelling temp (℃)
				The bottle anastrophe	Rheology	DSC
	DME-PAO7 (22 weight %) DOPA-PAO7 (22 weight %) PLURONIC  F127 (22 weight %)	22.0±1.0 22.0±1.0 17.0±1.0	20.3±0.6 20.4±0.5 15.4±0.4				20.9±0.1 21.7±0.2 17.5±0.4

The concentration for preparing the DOPA-PAO7 copolymer with cold process is that the aqueous solution of 10-30% (w/w) and the concentration of DOPA-PAO8 copolymer are the aqueous solution of 35-54% (w/w), wherein the DOPA conjugate is dissolved in about 4 ℃ distilled water, intermittently stirs up to obtaining clear solutions.The initial Thermogelling of estimating concentrated solution with the bottle anastrophe.In this method, the no longer mobile temperature of solution is as gelling temperature.

Find that composition that gelling temperature depends on copolymer concentration and block copolymer strongly (that is, PAO7vs.PAO8).For example, find that the DOPA-PAO7 solution of 22 weight % and DME-PAO8 solution form clear gel at about 22.0 ± 1.0 ℃; Polymer concentration is reduced to 18 weight % then to be obtained gelling temperature and is about 31.0 ± 1.0 ℃.Yet concentration does not form gel when joining 60 ℃ less than the DOPA-PAO7 solution of 17 weight %.DOPA-PAO7 demonstrates slightly higher gelling temperature than the PLURONIC  F127 (17.0 ± 1.0 ℃) of unmodified.The gelation behavior of finding DOPA-PAO8 is similarly in nature, but needs higher polymer concentration to form gel.The DOPA-PAO8 solution of 54 weight % and DME-PAO8 solution form gel at 23.0 ± 1.0 ℃, and the DOPA-PAO8 solution of 50 weight % forms gel at 33.0 ± 1.0 ℃.Yet concentration does not form gel when being heated to 60 ℃ less than the DOPA-PAO8 solution of 35 weight %.DOPA-PAO8 demonstrates much higher gelling temperature than the PLURONIC  F68 (16.0 ± 1.0 ℃) of unmodified.Find that these gels do not flow in a very long time.By this test, we find that also the DOPA of same commercially available prod PLURONIC  PAO and DOPA methyl ester derivation demonstrate gelling temperature much at one, and the gel that room temperature is made with the solution of the DME-PAO8 solution of 54 weight % or DOPA-PAO8 is harder than the gel made from the solution of the DME-PAO7 solution of 22 weight % or DOPA-PAO7.

Further studied the viscoelasticity behavior of the PLURONIC  solution of DOPA-modification with the vibration rheometry.Fig. 3 has shown the elastic storage modulus of aqueous solution of the 22 weight % of the PLURONIC  F127 of unmodified and DME-PAO7, G ', and it is the function of temperature.Below gelling temperature, storage modulus G ' can ignore, but at gelling temperature (T _{The time}), G ' increases sharply, shown in the figure that G ' begins to increase the vs. temperature.The DOPA-PAO7 (not shown) demonstrates similar rheological charactristics.Find the DME-PAO7 solution of 22 weight % and the T of DOPA-PAO7 solution _GelBe identical (20.3 ± 0.6 ℃), approximately than with the isoconcentration unmodified-PLURONIC  F127 (15.4 ± 0.4 ℃) is high 5 ℃.The G ' of DME-PAO7 or DOPA-PAO7 value is near the plateau value of 13kPa, and is suitable with the plateau value of the PLURONIC  F127 of unmodified.

Shown in Figure 4 is the PLURONIC  F68 solution of unmodified of 50 weight % and the rheological charactristics of DME-PAO8 solution, and it is as the function of temperature.The T of 50 weight %DME-PAO8 solution _GelBe 34.1 ± 0.6 ° of e, and the T of the PLURONIC  F68 of the unmodified of same concentrations _GelLow 18 ℃ (16.2 ± 0.8 ℃) approximately.The platform storage modulus of the 50 weight % solution of the PLURONIC  F68 of DME-PAO8 and unmodified does not have a great difference, near the plateau value that is up to 50 kPa.Fig. 5 has shown T _GelConcentration dependent, the rheological charactristics of the DME-PAO8 of two kinds of concentration is as the function of temperature.The T of the DME-PAO8 solution of 45 weight % _GelHigh 12 ℃ than the solution of the DME-PAO8 of 50 weight % approximately.

Because it is hydrophilic that DOPA and DOPA methyl ester all can be thought, with respect to the PLURONIC  PAO of unmodified, the PLURONIC  PAO T that DOPA-modifies _GelThe increase of value may be the increase of the hydrophilic PEO fragment length that causes because DOPA is coupled to end group.Can be clear that by the data shown in Fig. 3 and 4 with respect to PLURONIC  F127, DOPA or DOPA methyl ester are coupled to the T of PLURONIC  PAO end group for PLURONIC  F68 _GelThe value influence is bigger.This total molecular weight that can pass through F68 (about 8,600) and F127 (about 12,600) is explained.With the chemical method shown in the scheme 1 DOPA and DOPA methyl ester being added to two ends makes molecular weight increase by 446 and 474 respectively.This explanation is because the molecular weight basis of F68 is littler, and with respect to F127, the percent that the F68 molecular weight increases is bigger.

Data shown here are studied consistent with the calorimetric of the PLURONIC  PAO of unmodified before, the broad peak that proof low temperature makes is that micelle causes, and only in concentrated solution the small peak during observed high temperature corresponding to gelation, the process that thermal change does not take place basically.As shown in Figure 5, the micelle initial temperature of the PLURONIC  F127 of unmodified, high heat capacity temperature and T _GelBe worth all lower, and basic identical by the determined concrete enthalpy of area (Fig. 2) that transforms the below than DOPA-PAO7.These enthalpys comprise the enthalpy that micelleization and gelation are caused.Yet,, observe enthalpy change and may major part cause by micelleization because the enthalpy of gelation is very little.

Table 5

The comparison of the micelle initial temperature of the 30 weight % solution of the PLURONIC  F127 of the DME-PAO7 that the differential scanning calorimetric test obtains, DOPA-PAO7 and unmodified, high heat capacity temperature, enthalpy and gelling temperature.

	The micelle temperature (℃)	High heat capacity temperature (℃)	ΔH (J/g)	Gelling temperature (℃)
					DME-PAO7 (30 weight %) DOPA-PAO7 (30 weight %) PLURONIC  F127 (30 weight %)	5.2±0.2 4.6±0.2 1.9±0.3	8.3±0.1 8.0±0.6 6.0±0.4	20.3±2.4 19.3±1.4 20.6±1.6	14.0±0.4 14.0±0.2 10.6±0.6

Observe the micelle peak and extend on the gelation initial temperature, illustrate that other monomer coagulation becomes micelle when temperature is higher than gelling temperature.The agglutinative temperature dependency of DOPA-PAO7 and DME-PAO7 is shown in Fig. 6.The DSC pyrolysis curve shows the increase along with concentration, micelle temperature and T _GelReduce.Under the concentration that gelation does not take place, also can be observed wide endothermic peak corresponding to micelleization at solution; Along with the attenuating of copolymer concentration, the characteristic temperature straight line of broad peak rises, and it is consistent with the gelling temperature of concentrated copolymer to observe small peak, but disappears when copolymer concentration reduces.

From as can be known aforementioned, can design and prepare multiple polymer composition of the present invention, so that multiple micelleization and/or Thermogelling performance to be provided.Perhaps, or in addition, can with for example Polyethylene Glycol and lactic acid/glycol acid polymer composition of the present invention be degraded into respectively can excretory polymers compositions and metabolite.Howsoever, polymer composition of the present invention provides improved adhesion property by mixing one or more DHPD residues, and described mixing is to be coupled to described residue by the terminal monomer with polymers compositions to realize.

The another kind of method of the non-oxidizable gelation of polymer composition of the present invention is a photocuring.With photo curable monomer and PEG-DA (PEG-diacrylate) combined polymerization that contains the DHPD part, to form cohesive hydrogel by photo polymerization.Photo polymerization can realize that this depends on used monomer under visible light or the UV wavelength arbitrarily.This can be determined by those skilled in the art.Photo curable monomer partly is made up of viscosity, and viscosity partly is coupled to the polymerisable monomer (as methacrylate based group, the centre is with or without oligomerization oxirane joint or fluorinated ether joint) with thiazolinyl.

Contain photopolymerizable monomer, PEG-DA and the 1.5 μ L/mL light triggers of binding agent of the present invention (as 2 with uviol lamp (365nm) irradiation, 2 '-dimethoxy-2-phenyl-1-Phenylethanone. (2,2 '-dimethoxy-2-phneyl-acetonephenone) (DMPA), camphorquinone/4-(dimethylamino)-benzoic acid (CQ/DMAB), and ascorbic acid/fluorescein sodium salt (AA/FNa ₂)) aqueous mixture more than 5 minutes.Find to exist in the precursor solution binding agent can influence the polymerization process that free radical causes.The catechol binding agent reduced the degree that gelation forms, and reduced binding agent and mixed percentage ratio in the gel network, and prolonged gel formation time.

As shown in figure 25, by detecting quality with the detection of echoes instrument, measured the UV irradiation after 2 minutes the gel conversion ratio reach 75 weight %, irradiation is greater than after 5 minutes, the gel conversion ratio increases to greater than 85 weight %.When adopting visible light initiator, the gelation of PEG-DA takes place in 4 minutes or shorter time that (CQ/DMAB is 4 minutes, AA/FNa ₂It is 3 minutes).

The combined polymerization of PEG-DA and chemical compound 1 or 7 ( chemical compound 1 and 7 the synthetic scheme 2 and 3 that is shown in) in nature with the polymeric type of pure PEG-DA seemingly, cause the gel conversion ratio to reduce (described gel conversion ratio depends on DOPA monomer concentration and initiator system) though chemical compound 1 or 7 is added to the PEG-DA precursor solution.For example, have 2.5mol% or more 1 or at 7 o'clock, in the UV polymerization that DMPA causes, the gel conversion ratio is lowered to less than 85 weight %.Yet the gel transforming degree of these gels does not have difference statistically.To the initiator of visible light-inducing, observe the inhibition that similar DOPA concentration relies on.For AA/FNa ₂With the mixture that CQ/DMAB causes, the chemical compound 1 that adds 33.3mol% increases to more than 8 minutes gelation time.Yet, contain chemical compound 1 and 7 solution even when relative high DOPA mol%, still can carry out photocuring.

Scheme 2

Scheme 3

For example, exist the chemical compound 1 or 7 of 53.8mol% to make gel ease down to 77 weight % in the precursor solution, have only the DOPA of 85mol% to be incorporated in the hydrogel from 88 weight %.The DOPA colorimetric determination (colorimetric determination is by Waite and Benedict exploitation) that the hydrogel of photocuring is carried out shows and has catechol DOPA in the hydrogel.Behind the photocuring, contain the gel of DOPA to extract unreacted DOPA monomer in 0.5N HCl dialysis.For the degree that quantitative measurement DOPA mixes, analyze dialysis solution according to the DOPA colorimetric test of Waite and Benedict, be incorporated into the amount of the DOPA in the gel network with result's calculating of this test.Figure 26 shows the molar fraction that is incorporated into DOPA in the gel network, and it is as the function of the mole % of monomer in the precursor solution 1 and monomer 7.Contain the DOPA mark that mixes in the sample of monomer 1 and 7 and do not have marked difference.The DOPA that is incorporated in the PEG hydrogel is up to 24.9 μ mol/g.

The complete gel through dialysis is immersed in the nitrite reagent, immerse then among the NaOH, obtained to exist in the gel positive evidence of DOPA.Add after the nitrite, initial colourless gel becomes glassy yellow, adds excessive alkali and becomes redness afterwards again.This color transition is the typical change of catechol, shows by the not oxidised form of photo polymerization with DOPA to be incorporated in the hydrogel.Red intensity has also been reacted the concentration of mixing the DOPA in the photocuring hydrogel.

On hemispherical photocuring gel, contact mechanical test, to obtain the mechanical performance information of gel.Hertz mechanics (Hertzian mechanics) by supposing that concrete non-cohesive contacts between incompressible elasticity hemisphere and rigid plane has calculated elastic modelling quantity (E), wherein load (P _h) and displacement (δ _h) between hertz relation be:

$P_h=\frac{{16\mathrm{<figure-callout\; id="1"\; label="R"\; filenames="A20058000618000811.png,A20058000618000862.png"\; state="\{\{state\}\}">R</figure-callout>}}^{1/2}\mathrm{<figure-callout\; id="9"\; label="E"\; filenames="A20058000618000891.png,A20058000618001051.png"\; state="\{\{state\}\}">E</figure-callout>}}{9}{\mathrm{\&delta;}}_{\mathrm{<figure-callout\; id="5"\; label="h"\; filenames="A20058000618000831.png,A20058000618000861.png"\; state="\{\{state\}\}">h</figure-callout>}}{5}^{3/2}---\left(1\right)$

Wherein R is the radius of hemispherical gel cantilever.With the data of equation (1) match load to displacement, but according to the proportionality coefficient calculating elastic modulus of matched curve.As shown in Figure 6, for the gel that contains DOPA, obtained the average Young's modulus (E) of about 50kPa.

Table 6

The average Young's modulus that contains the gel of DOPA

	DOPA content *(μ mol/g gel)		Young's modulus (kPa)
						On average	Standard deviation	On average	Standard deviation
PEG-DA			72.3	7.69
					PEG-DA+1 +	15.0	0.25	47.4 **	8.63
PEG-DA+7 +	14.5	0.056	51.4 **	9.91

⁺In precursor solution 33mol%

^*By the DOPA test determination

^*P＜0.001 is with respect to the PEG-DA gel

These modulus values are low more about 30% than the modulus value of PEG-DA gel, confirmed that DOPA has the inhibition effect to the photo polymerization that free radical causes.Though modulus decreases with respect to the PEG-DA gel, the gel that contains DOPA still demonstrates the modulus that is fit to a lot of biomedical applications.Suitable modulus is greater than 500Pa.The Another application of cohesive hydrogel is a localized drug delivery.For example, cohesive hydrogel can be formed on the mucomembranous surface in mouth or oral cavity.But the hydrogel load has for example antibiotic of medicine, and helps the slow release of medicine in a period of time.But these hydrogels also load have analgesics, are used for reducing pain in localized site.But these hydrogels also load have chemotherapeutics, are inserted into and send treatment of cancer in the tumor tissues.But these hydrogels are load inhibition of cell proliferation medicine also, as applying support or other vascular devices, is used for the implantation position control cell proliferation at vascular devices.

But the internally crosslinked organizational coherence hydrogel of body can replace metal or plastics suture as organizing sealant.Binding agent is incorporated into surrounding tissue in operation or injury site, and polymer formation viscosity connects with wound closure.Hydrogel also can be used for the damage to bone of repair of bone fractures and cartilage.

Other purposes

These coatings and hydrogel also have a lot of purposes in industrial products, comprise the biofouling (Sargassum, antibacterial and mussel adhere to underwater surface) in preventing to transport by sea, prevent to flow to for example germ contamination in electric power and the pharmaceutical factory current of factory, prevent the germ contamination in the potable water system, as tooth and Denture adhesive, binding agent in the water of transfer indicator, the coating of water purity and detecting sensor is used to prevent the paint of biofouling.

These coatings and hydrogel also have a lot of purposes in consumer products and cosmetics, include but not limited to, be used for tooth and Denture adhesive, be used for the binding agent of cosmetics as hair, skin and lower limb, be used for cosmetics for example eye shadow, lipstick and mascara (mascara), be used for temporary tattoo, and the resealable binding agent of effect bag and container.

Be not limited to any concrete synthetic schemes or preparation method, the suitable compositions of the present invention can include but not limited to the ammonia ester moiety between each terminal monomer and the DOPA residue.In greater detail following, this ammonia ester moiety is the synthetic material who is used for the material/reagent of crosslinked DOPA residue and polymers compositions.As know those skilled in the art of the present invention and understand like that, of the present invention extensive aspect multiple other the part of consideration, this depends on the selection of terminal monomer and coupling agent.

Embodiment: describe, in general terms

Below non-limiting example with data interpretation various aspects and the feature relevant with compositions of the present invention and/or method, comprise the preparation of the polymer that mixes one or more DHPD components or altogether-polymer composition, the synthetic method of describing by the present invention as can be known.Though with several polymer or altogether-polymeric system explained application of the present invention, it will be understood by those skilled in the art that with multiple other compositions and/or preparation method to obtain similar result that these are all within the scope of the invention.

PEO ₁₀₀PPO ₆₅PEO ₁₀₀(PLURONIC  F127, weight average molecular weight=12,600), PEO ₇₈PPO ₃₀PEO ₇₈(PLURONIC  F68, weight average molecular weight=8,400), PEG (weight average molecular weight=8000), Pentafluorophenol, 1,3-dicyclohexylcarbodiimide (DCC), 4,7,10-trioxa-1,13-tridecane diamino, fluorescein sodium salt (FNa ₂), and ascorbic acid (AA) is available from Sigma (St.Louis, the Missouri State).L-DOPA, thionyl chloride, isobutene. acyl chlorides, tert-butyldimethylsilyl chloride (TBDMS-Cl), Bis(tert-butoxycarbonyl)oxide, methacrylic anhydride, 2,2 '-dimethoxy-2-phenyl-1-Phenylethanone. (DMPA), acryloyl chloride, 1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene (DBU), tetrabutylammonium (TBAF), 4-(dimethylamino)-benzoic acid (DMAB), l-vinyl-2-pyrrolidone (VP), N, N-two succinimidyl carbonates, sodium borate, two molybdic acid hydrate sodium, sodium nitrite, 4-(dimethylamino) pyridine (DMAP), N-hydroxy-succinamide, N, the N-diisopropylethylamine, dimethylformamide and dichloromethane are available from Aldrich (Milwaukee, the state of Wisconsin).Camphorquinone (GQ) is available from Polysciences, Inc. (Warrington, Pennsylvania).Acetone is used P with the molecular sieve drying of 4  before the use ₂O ₅Distillation.Triethylamine is distillation newly before use.Other chemical reagent uses according to a conventional method.

According to Patel and Price, J.Org.Chem., the method for 1965,30,3575 (including this paper in as a reference) prepares the L-DOPA methyl ester hydrochloride.The activatory PEG of succinyl phosphorons amino propyl acid ester (mPEG-SPA, weight average molecular weight=5000) is available from Shearwater Polymers, and Inc. (Huntsville, AL).With HCl gas about 10 minutes preparation saturated ethyl acetate of HCl of bubbling in ethyl acetate (50mL).According to Sever and Wilker, Tetrahedron, 2001,57, (29), the method for 6139-6146 (including this paper in as a reference) synthesizes 3, two (t-butyldimethylsilyloxy base) L-phenylalanine (DOPA (TBDMS) of 4- ₂) and 3, two (the t-butyldimethylsilyloxy bases-N-tert-butoxycarbonyl-L-phenylalanine (Boc-DOPA (TBDMS) of 4- ₂).

(a kind of anionic and nonionic surfactant are at allealtine aqueous base (Decon Labs for glass cover slide (diameter 12mm) immersion 5%Contrad70 solution used in following examples, Inc., Bryn Mawr, Pennsylvania) cleans the emulsion that ultra sonic bath formed in 20 minutes in), use deionization (DI) water washing then, supersound process is 20 minutes in DI water, in acetone, clean, supersound process is 20 minutes in acetone, cleans in hexane, and supersound process is 20 minutes in hexane, in acetone, clean, supersound process is 20 minutes in acetone, cleans in DI water, and supersound process is 20 minutes in DI water.Coverslip is air-dry in the filtering laminar flow fume hood of HEPA then.Primary in order to generate (pristine) auri material with the clean coverslip of 2nm Cr spraying plating (Cressington 208HR), is used Au (purity is 99.9%) the clean coverslip of spraying plating of 10nm then.

Prepare titanium dioxide (TiO by the following method ₂) surface: with TiO ₂, to silicon (Si) wafer, in plasma chamber, clean before the test then with the electron beam physical evaporation.With Edwards FL400 electron-beam evaporator＜10 ^-6Holder is coated to 100nm Ti on the silicon wafer (MEMC Electronic Materials, St.Peters, the Missouri State, surface orientation (100)).Then silicon wafer is cut into the sheet of 8mm * 8mm, in following medium, uses ultrasonic cleaning: 5%Contrad70, ultra-pure water (ultra-pure water is a deionization and distilled), acetone and petroleum ether.Base material further cleaned (HarrickScientific, Ossining, New York) 3 minutes at＜200 millitorrs and 100W in the oxygen plasma chamber then.

As described below, (XPS) characterizes primary and modified gold surface with x-ray photoelectron spectroscopy.At the x-ray source that disposes unicolor (monochromated) AlK α (1486.8eV) 300-W, the circular point (circular spot) of 1.5mm size, the flood gun and the ultrahigh vacuum (＜10 of counting charge effect ^-8Torr) (Omicron, Taunusstein Germany) go up collection XPS data to Omicron ESCALAB.Fly away from angle (Takeoff angle) and be defined as the angle between base material normal and the detector, this fixed angle is 45 °.With two-sided tape base material is fixed on the standard sample capo spiral shell (stud).All binding energy are all used Au (4f _7/2) Jin Feng (84.0eV) or C (ls) carbon peak (284.6eV) come standardization.Analysis scans (survey scan) (50.0eV is by energy (pass energy)) by wide detection and 10 minutes high resolution scannings (22.0eV is by energy) of C (ls) 270-300eV is formed.Peak deconvolution (deconvolution) and atomic percentage are calculated and are carried out with the EIS analysis software.

Secondary ion spectrum is at TRIFT III ^TM(Physical Electronics, Eden Prairie MN) improve quality and collect in the scope 0-2000m/z flight time secondary ion mass spectrometer (TOF-SIMS).Use Ga ⁺The source, light beam can be 15keV, raster size is 100 μ m.Collect positive and negative spectrum, and overlap the standardization of low quality ion with one with PHI softwareCadence.

In order to detect the relative hydrophilic/hydrophobic on surface, as described below with sessile drop method collection contact angle data.The contact angle goniometer of the customization of humidification sample room is equipped with in use, and (assembly is from Ram é-Hart, and Mountain Lakes NJ) detects ultra-pure water (18.2M Ω-cm on unmodified and the modified base material; Barnstead, Dubuque, the contact angle that moves forward and backward IA) (advancing and receding contactangles).To each surface, done 4 detections in different positions, reported meansigma methods and standard deviation.

With exposed golden pick off tube (sensor cartridges) at BIACORE 2000 (BiacoreInternational AB; Uppsala carries out surface plasma body resonant vibration (SPR) on Sweden) and detects.With 0-100mg/ml NaCl solvent with the resonant reaction standardization.Diluent (H with mPEG-DOPA, mPEG-MAPd and mPEG-OH ₂Among the O, 0.1mM) be expelled in the SPR flow cell (flow cell) 10 minutes, liquid stream (flow) rotates back in the pure DI water then.Detect in the routine tests of albumen to the absorption of modification base material at one, the sensor surface contact that is formed with the PEG film in advance is dissolved in 10mM HEPES buffer (0.15M NaCl, pH=7.2) 0.1mg/ml bovine serum albumin (BSA) solution contacts pure buffer then.

In the test of proof anti-soil dirt effect, (Manassas, NIH 3T3-Switzerland albino fibroblast VA) maintains 37 ℃, 10%CO available from ATCC ₂, and contain Dulbecco ' the s improvement Eagle ' s culture medium (DMEM of 10% (v/v) hyclone (FBS) and 100U/ml penicillin and 100U/ml streptomycin; Cellgro, Herndon, VA) in.

(MA) the gradient liquid with acetonitrile/0.1% trifluoroacetic acid (v/v) aqueous solution carries out the RP-HPLC preparation on Vydac 218TP reversed-phase column for Waters, Milford with Waters HPLC system.(Finnigan, Thermoquest carry out ESI-MS on CA) and analyze in LCQ LC-MS system.(Perseptive Biosystem carries out MALDI-TOF MS on MA) and analyzes at Voyager DE-Pro Mass Spectrometer Method instrument.Alpha-cyano-4-hydroxycinnamic acid is as substrate.The NiTi alloy (10mm * 10mm * 1mm) available from Nitinol Devices﹠amp; Components (Fremont, CA).Si, SiO ₂(1500  thermal oxide) and GaAs wafer available from University Wafer (South Boston, MA).

For test of following cell adhesion and/or diffusion test, modification in 12 hole TCPS plates, contain the DMEM of 10%FBS at 37 ℃, 10%CO with 1.0ml with base material unmodified ₂Middle pretreatment 30 minutes.Trypsin with 0.25%-EDTA gathers in the crops the 12-16 fibroblast in generation, is resuspended among the DMEM that contains 10%FBS, counts with hemocytometer.By suspension being diluted to suitable volume, every then hole adds the 1ml diluent, is 2.9 * 10 thereby make the density of cell inoculation ³Individual cell/cm ²Base material is containing the DMEM of 10%FBS, 37 ℃, 10%CO ₂In keep 4 and disappear, siphon away not adherent cell then.The cell that sticks on the base material is fixed 5 minutes in 3.7% paraformaldehyde, be dissolved in 1 of DMSO with 5 μ M then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (1,1 '-dioctadecyl-3,3,3 ', 3 '-tetramethyl indocarbocyanine perchlorate, DiI; Molecular Probes, Eugene OR) handled 30 minutes for 37 ℃.Siphon away dyestuff then, base material washs 10 minutes (3x) with DMSO, and (Stephens Scientific, Kalamazoo MI) are fixed on the slide to keep fluorescence with Cytoseal.For the statistics purpose, three repetitions are done in these tests.In order to use electron microscope observation, with the EtOH dehydration, critical point drying is with 3nm Au spraying plating in fixing back for some samples.

For quantitative cell adhesion, with Olympus BX-40 (λ _Ex=549nm, λ _Em=565nm) detect base material, with Coolsnap CCD camera (Roper Scientific, Trenton, NJ) record color.For in the in triplicate base material each, clap 5 photos.(PA) threshold value in is quantitative with the photo that obtains for Universal Imaging, Downington with MetaMorph.(SPSS, Chicago IL) come the statistics importance of specified data with the Tukey ' spost-hoc of unidirectional ANOVA and 95% confidence interval test.The meansigma methods and the standard deviation of testing result have been reported.

Embodiment 1

Succinimidyl carbonate PAO, SC-PAO7's is synthetic

PLURONIC  F127 (0.60 mM) is dissolved in the anhydrous two  alkane of 30mL.Add the N that is dissolved in the 10mL anhydrous propanone, N '-two succinimidyl carbonate (6.0 mM).DMAP (6.0 mM) is dissolved in the 10mL anhydrous propanone, and under magnetic agitation, slowly adds.Carry out activation in 6 hours in room temperature, then SC-PAO7 is deposited in the ether.Follow the tracks of the disappearance of initial substance in the reaction with TLC (chloroform-methanol (5: 1) dicyandiamide solution).Be dissolved in the acetone and also come purified product 4 times with ether sedimentation.Product yield is 65%. ¹H NMR(500MHz，CDCl ₃)：δppm 0.96-1.68(br，-OCHCH ₃CH ₂O-)，2.80(s，-COON(CO) ₂(CH ₂) ₂)，3.15-4.01(br，-OCH ₂CH ₂O-；-OCHCH ₃CH ₂O-)，4.40(s，-OCH ₂CH ₂OCOON(CO) ₂CH ₂CH ₂-)。

Embodiment 2

DME-PAO7's is synthetic

The serosity of DOPA methyl ester hydrochloride (1.25 mM) and triethylamine (2.5 mM) is mixed in the 10mL chloroform with SC-PAO7 (0.16 mM).Follow the tracks of the disappearance of initial substance in the reaction with TLC (chloroform-methanol-acetic acid (5: 3: 1) dicyandiamide solution).After the stirring at room 1 hour, evaporation removes and desolvates, and comes purification DME-PAO7 3 times with the cold methanol precipitation.DME-PAO7 has obtained positive Arnow test result, illustrates to have the catechol hydroxyl.Product yield is 75%. ¹H NMR(500MHz，CDCl ₃)：δppm 0.98-1.71(br，-OCHCH ₃CH ₂O-)，2.83-3.06(m，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，3.15-4.02(br，-OCH ₂CH ₂O-；-NHCH(CH ₂C ₆H ₃(OH) ₂COOCH ₃)，4.05-4.35(d，-OCH ₂CH ₂OCONHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，4.55(br，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，5.30(d，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，6.45-6.80(1s，2d，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)。

Embodiment 3

DOPA-PAO7's is synthetic

Under Ar atmosphere, L-DOPA (1.56 mM) is joined the 0.1M Na of 30mL ₂B ₄O ₇(pH=9.32) in the aqueous solution, stirring at room is 30 minutes then.To join in the mixture that obtains stirred overnight at room temperature at the SC-PAO7 in the 5mL acetone (0.156 mM).Keep the pH of solution in the course of reaction with sodium carbonate.Follow the tracks of the disappearance of initial substance in the reaction with TLC (chloroform-methanol-acetic acid (5: 3: 1) dicyandiamide solution).With concentrated hydrochloric acid this solution is acidified to pH2, uses dichloromethane extraction then 3 times.With the dichloromethane extract anhydrous sodium sulfate drying that merges, to filter, dichloromethane is removed in evaporation.Be further purified product from the cold methanol precipitation.DOPA-PAO7 has obtained positive Arnow test result, illustrates to have the catechol hydroxyl.Product yield is 52%. ¹H NMR(500MHz，CDCl ₃)：δppm 0.92-1.70(br，-OCHCH ₃CH ₂O-)，2.91-3.15(m，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH)，3.20-4.10(br，-OCH ₂CH ₂O-；-OCHCH ₃CH ₂O-)，4.1-4.35(d，-OCH ₂CH ₂OCONHCHCH ₂C ₆H ₃(OH) ₂COOH)，4.56(m，-NHCHCH ₂C ₆H ₃(OH) ₂COOH)，5.41(d，-NHCHCH ₂C ₆H ₅(OH) ₂COOH)，6.60-6.82(1s，2d，-NHCHCH ₂C ₆H ₃(OH) ₂COOH)。

Embodiment 4

Succinimidyl carbonate PAO8, SC-PAO8's is synthetic

With preparing SC-PAO8 with above-mentioned synthesizing with the similar method of purification SC-PAO7.Product yield is 68%. ¹H NMR(500MHz，CDCl ₃)：δppm 0.95-1.58(br，-OCHCH ₃CH ₂O-)，2.80(s，-COON(CO) ₂(CH ₂) ₂)，3.10-4.03(br，-OCH ₂CH ₂O-；-OCHCH ₃CH ₂O-)，4.40(s，-OCH ₂CH ₂OCOON(CO) ₂CH ₂CH ₂)。

Embodiment 5

DME-PAO8's is synthetic

With preparing DME-PAO8 with above-mentioned synthesizing with the similar method of purification DME-PAO7 conjugate.Product yield is 76%. ¹H NMR(500MHz，CDCl ₃)：δppm 0.98-1.50(br，-OCHCH ₃CH ₂O-)，2.85-3.10(m，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，3.15-4.01(br，-OCH ₂CH ₂O-；-OCHCH ₃CH ₂O-；-NHCH(CH ₂C ₆H ₃(OH) ₂COOCH ₃)，4.03-4.26(d，-OCH ₂CH ₂OCONHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，4.55(m，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，5.30(d，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)，6.45-6.77(1s，2d，-NHCHCH ₂C ₆H ₃(OH) ₂COOCH ₃)。

Embodiment 6

DOPA-PAO8's is synthetic

With preparing and purification DOPA-PAO8 with the similar method of above-mentioned synthetic DOPA-PAO7 conjugate.Product yield is 49%. ¹H NMR(500MHz，CDCl ₃)：δppm 0.92-1.50(br，-OCHCH ₃CH ₂O-)， 2.91-3.10(m，-NHCHCH ₂C ₆H ₃(OH) ₂COOH)，3.15-3.95(br，-OCH ₂CH ₂O-；-OCHCH ₃CH ₂O-)，4.06-4.30(d，-OCH ₂CH ₂OCONHCHCH ₂C ₆H ₃(OH) ₂COOH)，4.54 (m，-NHCHCH ₂C ₆H ₃(OH) ₂COOH)，5.35(d，-NHCHCH ₂C ₆H ₅(OH) ₂COOH)，6.50-6.80(1s，2d，-NHCHCH ₂C ₆H ₃(OH) ₂COOH)。

Embodiment 7

Colorimetric determination

Measure the coupling efficiency of DOPA methyl ester and DOPA and PLURONICs  F127 and F68 with the colorimetric method of Waite and Benedict.In brief, with 1N HCl dilution standard or unknown solution etc. duplicate samples to final volume be 0.9mL, so triplicate analytic sample.0.9mL nitrite reagent (1.45M sodium nitrite and 0.41M two molybdic acid hydrate sodium) is joined in the DOPA solution, add the 1N NaOH of 1.2mL then immediately.Because absorbance is time dependent, note to guarantee that the interval that adds between NaOH and the record absorbance all is 3 minutes for all standard substance and sample.Write down the absorbance of all standard substance and sample at 500nm.With the standard substance of DOPA as DOPA methyl ester and DOPA conjugate.

Embodiment 8

Rheometry

Carry out the ftheoloqical measurements of gelation process with Bohlin VOR flow graph (Bohlin Rheologi, Cranbury, New Jersey).All mensuration is all used the rustless steel cone-plate geometry (coneand plate geometry) of 30mm diameter, and cone angle is 2.5 °.Control temperature with circulator bath.Before the liquid solution of 0.5mL transferred in the instrument with sample but in refrigerator and cooled.At 0.1Hz, stress is that () measures storage and loss modulus G ' and G under 0.45% the oscillation mode ".The rate of heat addition is 0.5 ℃/minute, but the rate of heat addition is reduced to 0.1 ℃/minute during contiguous gelling temperature.Detected the dependency of the viscoelasticity data counter stress amplitude of several samples.Only measure in the range of linearity, wherein modulus does not rely on stress amplitude.Circle at the sample room external surface peripheral has used mineral oil to prevent the dehydration in the mensuration process.

Embodiment 9

Differential scanning calorimetry (DSC)

(TA Instruments, New Castle DE) carry out DSC and measure on the calorimeter at TA Instruments DSC-2920.In the heating and cooling circulation, obtain the heat spectrum of 3 samples of each concentration.Having used the sample volume of 20ul in the aluminum evaporation ware of sealing, is 3 ℃ of/minute following writing scan data in heating and cooling speed, and empty evaporating dish in contrast.

Embodiment 10a

With amino-end capped methoxyl group-PEG, mPEG-NH ₂(2.0g, 0.40 mM, M _w=2,000 or 5,000, Sun-Bio PEGShop), N-Boc-L-DOPA dicyclohexyl ammonium salt (0.80 mM), HOBt (1.3 mM) and Et ₃N (1.3 mM) is dissolved in the mixture of 50: 50 dichloromethane of 20mL (DCM) and DMF.Add the HBTU (0.80 mM) that is dissolved in 10mL DCM then, reaction is 30 minutes under room temperature and ar gas environment.Reaction solution is used saturated nacl aqueous solution, 5%NaHCO successively then ₃, rare HCl solution and distilled water wash.The concentrating under reduced pressure crude product is used column chromatography purification on Sephadex  LH-20, methanol is made mobile phase.Precipitation is further purified product mPEG-DOPA 3 times in cold methanol, and the room temperature vacuum drying is stored under the nitrogen at-20 ℃. ¹H NMR(500MHz，CDCl ₃/TMS)：δ6.81-6.60(m，3H，C ₆H ₃(OH) ₂-)，6.01(br，s，1H，OH-)，5.32(br，s，1H，OH-)，4.22(br，s，1H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，3.73-3.38(m，PEO)，3.07(m，2H，PEO-CH2-NH-C(O)-)，2.73(t，2H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，1.44(s，9H，(CH ₃) ₃C-)，1.25(s，3H，CH ₃CH ₂O-)。

Embodiment 10b

By analogizing, contain the peptide of DOPA and the synthetic and correlation technique that oligopeptide (natural or synthetic source) can be expanded previous embodiment with other.According to concrete composition sequence, can randomly adopt N-end blocking group.As mentioned above, also can use multiple other the sticky ingredient of similar DOPA, these be know of the present invention well known to those skilled in the art.For example, the glycine DOPA analog that can use B-aminoacid and N-to replace.

According to above-mentioned synthetic technology and method, no matter concrete DHPD sticky ingredient can use the multiple polymers component.The molecular weight of polymers compositions can change, and it is subjected to the restriction of corresponding dissolubility requirement.As mentioned above, much other polymer can be used for surperficial anti-soil dirt and/or stable particle, and these polymer include but not limited to, hyaluronic acid, glucosan etc.According to the requirement of dissolubility and required surface action, polymers compositions can be branching, high branching or dendroid (dendrimeric), these components be commercially available maybe can be by known synthetic technology preparation.

Though the compositions of embodiment 10a is the amidated products of above-mentioned parent material, should understands end group, main chain or the side chain that the N-terminal of DHPD component can be coupled to suitable functionalized natural or synthetic polymer (comprising above-mentioned polymer) and prepare similar polymer-DHPD conjugate.Such as but not limited to, as mentioned above, available end is the N-terminal reaction of the suitable polymers component and the required DHPD component of carbonate functionalities, and required conjugate is provided.

Embodiment 11a

With the solid phase method of peptide synthesis; on the Rink resin (0.6mMol/g) with aminoacid, BOP, HOBt and the DIEA of Fmoc protection as activator; and be solvent with NMP; total (consensus) decapeptide repetitive sequence (sea-mussel mucin decapeptide of synthetic mytilus edulis Mytilus edulis foot albumen 1 (Mefp 1); MAPd, NH ₂-Ala-Lys-Pro-Ser-Tyr-Hyp-Thr-DOPA-Lys-CO ₂H).The deprotection that uses the nmp solution of 25% piperidines Fmoc to be carried out 20 minutes.The pre-activation step through initial 10 minutes, use 2 normal 1: 1: 1: 1 Fmoc-aminoacid: amino acid whose coupling was carried out in BOP: HOBt: DIEA reaction in 20 minutes.After finishing decapeptide, use the carbodiimides chemical method, with the free ammonia cardinal extremity and the activatory methoxyl group-PEG-CO of decapeptide ₂H (mPEG-SPA, M _w=2k or 5k, Shearwater Polymers) coupling.Under 0 ℃, use TFA solution and EDT, thioanisole and the m-cresol of 1M TMSBr that PEG-decapeptide conjugate (mPEG-MAPd, 2k or 5k) was carried out cracking 2 hours.Under 0 ℃, in ether, precipitate thick mPEG-MAPd product, and use Vydac 218TP reversed-phase column (220 * 22mm * 10 μ m) to carry out purification with preparation HPLC.Degree of purity of production by analytical type HPLC be determined as＞90%, and determine structure with PerSeptive BiosystemMALDI-TOF-MS.

Embodiment 11b

Synthetic expanding to process among the embodiment 11a can be similar to embodiment 10b or consistent with the described variation of embodiment 10b.In addition, available other conjugate for preparing the polymer that contains DOPA by the contained tyrosine residue of enzymatic conversion.Also other technology of knowing in the synthetic field of peptide can be effective to provide other desirable protein sequence, peptide conjugate and obtain adhesion/anti-soil dirt effect.

Embodiment 12a

The modification of gold surface is DCM or the phosphate buffered saline (PBS) (PBS by being 0.1-75mg/ml from polymer concentration; PH=3,7.4 and 11) absorption mPEG-DOPA or mPEG-MAPd (2k, 5k) carry out in the solution.Base material placed bottle and immerse mPEG-DOPA or mPEG-MAPd solution leaves standstill and reaches 24 hours.After from solution, taking out, with The suitable solvent (DCM or DI H ₂O) clean base material removing unconjugated polymer, and vacuum drying.As a comparison, with PEG-monomethyl ether (mPEG-OH, average M _w=5000) carry out same finishing.Perhaps, under 37 ℃, a solution that contains mPEG-DOPA or mPEG-MAPd (the PBS solution of 10mM, PEG molecular weight=2000) (was hatched 30 minutes on the Au thickness～10nm), cleaned the surface of (3x) this coverslip then with PBS at the glass cover slide that applies with Au.The analysis announcement of modified surface being carried out by forward/backward contact angle (advancing/receding contact angle), XPS and TOF-SIMS has formed mPEG-DOPA or mPEG-MAPd chemisorbed layer.

That Fig. 7 A-C is not depicted as is modified, the XPS collection of illustrative plates on surface that modify with mPEG-OH and that modify with mPEG-DOPA.As desired, the ether peak of handling 286.5eV place, back with mPEG-OH is maximum only minimumly, then observes significant increase behind the absorption mPEG-DOPA, and this shows a large amount of existence of ether carbon.Reported the ether peak of the pure PEG with identical combination energy in the document.The 285.0eV place is to be caused by the aliphatic in PEG and the DOPA headgroup (headgroup) and aromatic series carbon and some hydrocarbon pollutants of introducing in preparation/vacuum than small peak among Fig. 7.

Flight time SIMS data acknowledgement the discovery among the XPS.Carry out the TOF-SIMS analysis to unmodified with Au base material and mPEG-DOPA powder that mPEG-DOPA-modifies with the auri material that is exposed to mPEG-OH.Each base material is carried out about 4 minutes data collection.

The cation spectrum of not modified Au demonstrates the (C that has typically represented hydrocarbon pollutant _nH _2n+1) ⁺(C _nH _2n-1) ⁺Peak (data not shown goes out).There is other less pollution thing, comprises NH ₄ ⁺, Na ⁺C with relatively small amount _aH _bO _c ⁺Class.Owing to be used for the method for deposit Au thin film,, also observe the Cr peak at m/z～52 places except the Au peak of m/z～196.9.Gold surface is exposed to the only feasible C that represents of mPEG-OH _aH _bO _c ⁺Medium increase takes place in the segmental peak of PEG, and this peak may be that the non-specific adsorption by pollutant or mPEG-OH causes.This point can be by m/z～225 (AuOC ⁺) and 254 (AuOCCO ⁺) the peak confirmation, when comparing with the base material of modifying with mPEG-DOPA, these peaks do not demonstrate significant increase.(Fig. 8 A-C).

Accounting for leading in the cation spectrum on the Au surface of modifying with mPEG-DOPA is the C that representative is adsorbed molecule _aH _bO _c ⁺The existence at peak.As shown in Figure 9, with respect to surface not modified and that modify with mPEG-OH, C ₂H ₃O ⁺And C ₂H ₅O ⁺Relative abundance improve.C ₃H ₇ ⁺(m/z～43) and C ₄H ₅ ⁺The relative abundance of (m/z～53) also shows a marked increase, and these peaks may be to have the tert-butyl group in hydrocarbon pollutant or the Boc protecting group partly to cause.

Notable attribute may be that graphical triplet (triplet) in the high-quality scope repeats (Figure 10) in the Au base material cation of the PEGization spectrum.These triplets bunch each corresponding to an Au-DOPA-(CH ₂CH ₂O) _nFragment.When further differentiating, the inferior bunch representative of each in the triplet has added CH ₂, CH ₂CH ₂Or CH ₂CH ₂O, these peaks separate about 14-16amu separately.This repetitive pattern can recognize that when n=0-15 this signal then is lower than detectability when exceeding this scope.

In originally the anion on Au surface is composed, the O during except n=1-3 ^-, HO ^-And Au _n ^-Outside the strong detectable peak, only observe the record (data not shown goes out) of a little.At m/z～13 (CH ^-), 24 (C ₂H ₂ ^-) and 37 (C ₃H ^-) there is a spot of hydrocarbon pollutant.Accounting for leading in the anion on the Au surface of the PEGization spectrum is the C of m/z～126.893 ₇H ₁₁O ₂ ⁺The peak.This peak exists with moderate strength and shows that it has represented bigger ethylene glycol fragment in the Au spectrum of modifying with mPEG-OH.The most interesting peak position in the high-quality scope (＞200m/z), represented the catechol oxygen that is coupled to Au.This collection of illustrative plates shows that an Au atom can be in conjunction with nearly 6 oxygen atoms corresponding to 3 DOPA.

The contact angle data show when modifying gold thin film with mPEG-DOPA the strong dependency (data not shown goes out) of the characteristic of used adsorption solvent.The surface of modifying in DCM demonstrates the θ a that significantly is lower than without modification of surfaces (p＜0.001) and the surface (p＜0.05) modified in all aqueous solutions.Usually, pH raising along with aqueous solution, the hydrophilic of treated surface reduces, this ability that shows the PEGization surface reduces, may be because DOPA tends to be oxidized to the lower quinone form of its viscosity when pH improves, the explanation of being supported by research formerly shows that the not oxidation catechol form of DOPA plays main effect to viscosity.

Embodiment 12b

Below to protein adsorption and cell adhesion/be diffused on the unprocessed and treated coverslip and assess.Surface plasmon resonance (surface plasmon resonance, SPR) test shows that the polymer that contains DOPA is incorporated into gold surface rapidly, and the modified surface of gained has enhanced resistance (Figure 11) to protein adsorption.Be adsorbed in the protein on the gold of mPEG-MAPd (5k) modification than being adsorbed in lacking of not modified gold surface about 70%.To being incubated at fibroblastic mPEG-DOPA concentration (Figure 12), adsorption solvent and modification time used when the analysis showed that cell adhesion depends on preparation PEG strongly and modifies base material on the modified base material.With the mPEG-DOPA of＞25mg/ml or mPEG-MAPd 24 hours being modified at being carried out on the surface demonstrates in cell adhesion and the diffusion and reduces (Figure 12-14) on the statistics significantly.The gold surface of modifying with mPEG-MAPd (5k) shows that its always outstanding cell area (total projected cellulararea) has reduced by 97%, and the cell density that adheres to the surface has then reduced by 91%.

Embodiment 12c

Modification shown in the embodiment 12a can randomly change with reference to embodiment 10b and 11b, and can expand to other noble metal, includes but not limited to: silver and platinum surface.As described herein, these are used also can expand to and comprise any the have bulk metal of passivation or oxidized surface or the finishing of alloy.For example, can be as described herein, the bulk metal oxide is modified with relevant ceramic surface.Also can for example be used for those surfaces that integrated circuit and MEMS device are made with this technological expansion to semiconductor surface, these are hereinafter about also there being explanation in the context of nano-particle stabilisation.

Embodiment 13

With the described method of embodiment 12a,, silicate glass surface (glass cover slide) modified by from the 10mM aqueous solution, adsorbing mPEG-MAPd (2k).As described above the cell density that adheres to the NIH 3T3 cell on the modified and not modified glass surface is assessed.Modify cell density on 24 hours the glass surface compares with not modified glass surface and demonstrates reduction (cell density (individual cell/mm of 43% with mPEG-MAPd ²): not modified on glass be 75.5+/-6.5; Be 42.7+/-9.8 with the on glass of mPEG-MAPd modification).

Embodiment 14a

For the stabilisation of metal-oxide (and being specially metal oxide nanoparticles) is described, with 50mgmPEG-DOPA (5k) soluble in water (18M Ω-cm, Millipore) and add 1 mg magnetic powder (Fe ₃O ₄).Be used as the mPEG-NH of contrast ₂(5k) (Fluka) and mPEG-OH (2k) (Sigma) prepare similar prepared product.Carry out 1 hour ultrasonic with Branson Ultrasonics 450 probe type ultrasonic instrument to being immersed in each aqueous solution in 25 ℃ of baths.The frequency of probe is 20kHz, and length is 160mm, and end diameter is 4.5mm.Take out sample then, make its at room temperature standing over night so that any not modified magnetite from solution, be settled out.With comparison polymer (mPEG-NH ₂And mPEG-OH) Zhi Bei suspension is separated out rapidly, obtains brown solid and the colourless supernatant of clarification.In the sample of use with the preparation of nanoparticles of PEG-DOPA stabilisation, sample is that clarification is brown.The brown supernatant of separating clarifying is used Spectra/Por in water ^Film pipe fitting (MWCO:15,000) dialysis 3 days.After the dialysis, sample is carried out lyophilization and it at room temperature is stored in the vacuum until use.

Embodiment 14b

By transmission electron microscopy (TEM), thermogravimetric analysis (TGA), fourier transform infrared spectrometry method (FTIR) and UV/ visible light method the nano-particle with the mPEG-DOPA stabilisation is characterized.The result of TEM shows that the diameter of most of nano-particle is 5-20nm (data not shown goes out).0.4mg be the analysis showed that with the TGA of the magnetite of mPEG-DOPA stabilisation described granule contains the mPEG-DOPA of 17 weight % (data not shown goes out).The FTIR that undressed magnetite is carried out is presented at 4000-400cm ^-1Wave-length coverage in less absorption is arranged, the FTIR of the nano-particle of handling with mPEG-DOPA then is presented at 800-1600cm ^-1And 2600-3200cm ^-1Absorption bands, this point has just confirmed the existence of mPEG-DOPA.

Embodiment 14c

Exsiccant magnetite nano-particle with the PEG-DOPA stabilisation easily is scattered in the clarification brown suspension that (for example, dichloromethane) do not have obvious sediment formation to obtain to stablize the several months in aqueous and the polar organic solvent.Be scattered in 1ml water by the magnetite that 1mg is handled with mPEG-DOPA and (use Millex ^AP 0.22 μ m filter carries out 18M Ω-cm and filters (Millipore)), in DCM or the toluene, prepared the suspension of nano-particle in different solvents with the mPEG-DOPA stabilisation.Place bath formula ultrasonoscope 10 minutes with the dispersing nanometer granule suspension.At room temperature, three kinds of solution have all been stablized 6 months at least, and undressed magnetite and with mPEG-OH or mPEG-NH ₂The contrast suspension of the magnetite of stabilisation in each solvent is being less than in 24 hours and precipitating.

Embodiment 14d

In the presence of physiological concentration salt, discovery also is stable with the suspension of the nano-particle of mPEG-DOPA stabilisation.Can suppress the inductive nanoparticle aggregate of salt in order to measure mPEG-DOPA, 0.3mg is placed quartz colorimetric utensil with the magnetite that mPEG-DOPA handles, add 0.7ml water (carrying out 18M Ω-cm with 0.25 μ filter filters).Saturated NaCl solution (5 μ l, 10 μ l, 20 μ l, 50 μ l, 100 μ l) equal portions (aliquot) are added in the cuvette continuously, before carrying out the UV-VIS spectrum, leave standstill 10 minutes (Figure 15).Be suspended in the solution that contains cumulative NaCl concentration with the absorption spectrum of the nano-particle of mPEG-DOPA stabilisation much at one, this just shows that mPEG-DOPA has effectively stablized nano-particle and prevented gathering.The peak that concentrates on the 280nm place is the indication of the catechol side chain of DOPA.

Embodiment 14e

Known the step that illustrates among the embodiment of the it will be understood by those skilled in the art that 14a-14d of the present invention and can expand to other different metal-oxides or ceramic nano granule with technology.Equally, these application of the present invention can further comprise and are used to be similar to or consistent polymer-DHPD conjugate in the broad range of those compositionss described in embodiment 10b and the 11b and variant.As hereinafter explanation, in the preparation of semiconductor composition, in the presence of polymer of the present invention-DHPD conjugate, can in formation, carry out the original position stabilisation to metal-oxide or ceramic nano granule.

Embodiment 15a

In order to confirm the stability of metal nanoparticle, with commercially available gold colloid suspension (Sigma, particle diameter be 5 or 10nm) place that (molecular cut off that 5nm is used is 8000 in the Dialysis tubing, employed to 10nm then is 15000), and dialysis 2-3 days is present in Hydrazoic acid,sodium salt in the commercial articles with removal in ultra-pure water.To place vial through the suspension of dialysis then, and add mPEG-DOPA (10mg/ml).Sample was left standstill about 2 days at room temperature, then sample is dialysed once more to remove excessive mPEG-DOPA.Undressed 10nm Au nano-particle is unstable in the presence of NaCl also assembles (Figure 16), and treated Au nano-particle is at next maintenance stable suspersion of existing of NaCl aqueous solution (Figure 17).

Embodiment 15b

Can stablize various other metal nanoparticles by embodiment as described above is described, these metals include but not limited to: silver, platinum etc.Though the stability of having used the representational coupling compositions display of the present invention also can be by preparing various other compositionss with the similar or consistent mode of the replacement embodiment described in embodiment 10b and the 11b.Can obtain comparable result by the nano-particle that original position forms stabilisation, the nano-particle of described stabilisation is synthetic by the corresponding metal precursor in the presence of suitable viscosity coupling polymer of the present invention.

Embodiment 16a

The data show of present embodiment the stability of semiconductor nanoparticle.Employing is based on Cd (NO ₃) ₂Weak solution and Na ₂The slow blended standard method of S weak solution prepares CdS nano-particle (quantum dot).With nanoscale pure water preparation Cd (NO ₃) ₂And Na ₂The fresh storing solution (2mM) of S.Use gastight syringe, with 20 μ l s ^-1Speed with Na ₂S solution slowly injects 50ml Cd (NO ₃) ₂In the solution.Along with Na ₂The adding of S, 2mL Na is being injected in the solution flavescence ₂Behind the S, because yellow mercury oxide has appearred in the gathering of CdS nano-particle.Separate this CdS precipitation and be dried standby.Use the above-mentioned method that is used for magnetite, exsiccant CdS powder is scattered in the mPEG-DOPA solution to obtain clarifying yellow solution by ultrasonic.Above-mentioned yellow waterborne suspension is in the dark stored the several months under room temperature, do not observe sedimentary formation.There are not mPEG-OH or mPEG-NH not containing polymer ₂Condition under carry out controlled trial, obtained yellow mercury oxide and the colourless supernatant of clarification.In the presence of the NaCl aqueous solution, the CdS nano-particle stable with mPEG-DOPA keeps stable suspersion (Figure 18).

Embodiment 16b

The result of present embodiment has shown that the original position of stabilisation semiconductor nanoparticle forms.In the presence of mPEG-DOPA, by with Cd (NO ₃) ₂And Na ₂The dilute methanol solution of S slowly is mixed with CdS nano-particle (quantum dot).In methanol, prepare Cd (NO ₃) ₂And Na ₂The fresh storing solution (2mM) of S.25mgmPEG-DOPA (PEG molecular weight=2000) is dissolved in the Cd (NO of 5ml 2mM ₃) ₂In the methanol solution, use syringe then with 20 μ l s ^-1Speed slowly add the Na of 5ml 2mM ₂S solution.Solution turned yellow in adition process.Do not observe xanchromatic precipitation, dynamic light scattering method shows that particulate average diameter is 2.5nm.Carry out controlled trial not containing polymer or exist under the condition of mPEG-OH, obtained yellow mercury oxide and the colourless supernatant of clarification.Person of skill in the art will appreciate that according to selected material and corresponding ionic replacement or exchange reaction, can be in the presence of the cementitious compositions of type described herein various other inorganic particle base materials of preparation.

Embodiment 16c

The stabilisation that also polymerization coupling compositions of the present invention can be used for multiple other semi-conducting material.For example, can as described hereinly carry out surface-stable to core-shell nanoparticles.

Embodiment 17

The test of optimizing among the embodiment 17-20 is carried out with mPEG-DOPA-5K.Measured several parameters and be optimized so that mPEG-DOPA is adsorbed in gold from solution, these parameters comprise type and pH, adsorption time and the mPEG-DOPA solution concentration of solvent.Use adsorption solvent not make cell adhesion and diffusion that very big difference takes place.The total outstanding area of the cell number on the base material and they there is no significant difference between DCM and three kinds of different aqueous solutions.Compare with not modified base material (p＜0.01), the base material that adsorbs in neutral, alkaline and organic mPEG-DOPA solution all has remarkable enhanced anti-soil characteristic.Though do not observe difference in cell adhesion between solution and the diffusion, the contact angle data be supported in optimize to modify in the strategy can be with an organic solvent as a kind of method of minimizing catechol oxidation.In addition, the finishing of only carrying out in DCM demonstrates and has obviously less cell and lower total outstanding cell area from the teeth outwards.

Embodiment 18

Cell adhesion and diffusion demonstrate the strong dependency (Figure 12) to the mPEG-DOPA solution concentration.Compare with the surface of modifying with 10mg/ml solution (p＜0.05) with original gold surface (p＜0.001), the adhesion of cell and being diffused in is used to surpass on the base material that 25mg/ml mPEG-DOPA modifies and is significantly reduced.Compare cell adhesion and diffusion indifference when adopting the concentration that is lower than 10mg/ml with not modified base material.Difference is not to each other observed on the surface of modifying in the mPEG-DOPA of 25-75mg/ml solution in cell adhesion and diffusion.

Embodiment 19

The adsorption time section that prolongs mPEG-DOPA is also only observed less fibroblast adhesion and diffusion.As if though the base material that is as short as 5 minutes is modified and reduced cell adhesion and diffusion, 24 hours adsorption time makes the cell on the PEGization base material adsorb and spreads the base material (p＜0.05) that obviously is less than not modified base material (p＜0.001) and handles through the short time.

Embodiment 20

Be incubated at not modified surface and the lip-deep fibroblast form of modifying by Electronic Speculum (Hitachi 3500 SEM) observation through PEG.Not modified Au and the fibroblast on the Au that mPEG-OH-modifies are launched usually and spread apart well, and the diffusion that is incubated at those cells on the Au that mPEG-DOPA modifies then will reduce (Figure 14 A-C) far away.It should be noted that observed cell projection quantity is less than other surface on the mPEG-DOPA surface, this raised structures is working by integrating in plain cell adhesion and local the adhesion.Fig. 13 be depicted as fibroblast exposed Au, on Au that mPEG-OH handles and (50mg/ml handled 24 hours) handles with mPEG-DOPA 5K, mPEG-MAPd 2K or mPEG-MAPd 5K under optimal conditions Au adhesion and spread difference.The cell adhesion on the surface of modifying with the conjugate that contains DOPA and diffusion significantly are less than any one in other two kinds of surfaces.Make total protrusion cell area reduce 97% and make lip-deep cell density reduce 91% though mPEG-MAP5K modifies, this reduces to be far longer than reducing that mPEG-DOPA 2K modification obtains.

In cell adhesion and diffusion, there are differences between the surface of modifying with DOPA-and the link coupled PEG of MAPd-among Figure 13, can ascribe the physical property of associating (associated) PEG adhesion layer to.The analysis showed that with MAPd-PEG of SPR result formed the thicker and more blocky adhesion layer that has higher PEG concentration than with the fixed equimolecular quantity PEG of DOPA.Thereby the thicker adhesion layer that the PEGization that mediates with MAPd-makes the more successfully absorption of Profilin matter also more can successfully suppress cell adhesion.

Embodiment 21

Boc-DOPA (TBDMS) ₂-Osu's is synthetic

(0.110g 0.95mmol) joins Boc-DOPA (TBDMS) with N-hydroxy-succinamide (NHS) ₂(0.500g, dry methylene chloride 0.95mmol) (DCM) is (8.0mL) in the solution.This solution placed on the ice bath stir, and under nitrogen atmosphere, add 1, and 3-dicyclohexylcarbodiimide (DCC) (0.197g, 0.95mmol).Under 0 ℃, reactant was stirred 20 minutes, be warming to room temperature then, restir 4 hours.Filter reaction mixture is evaporated to it 1/5 of original volume subsequently to remove the urea by-product.Solution is cooled to 4 ℃, makes it leave standstill 2 hours to precipitate residual urea by-product, filtration and evaporation obtain the Boc-DOPA (TBDMS) of white foam shape ₂-OSu (0.567g, productive rate are 96%).

Embodiment 22

Boc-DOPA ₂(TBDMS) ₄Synthetic

With Boc-DOPA (TBDMS) ₂-OSu (0.567g, 0.91mmol) be dissolved in dry dimethyl formamide (DMF) (2.5mL) in, add DOPA (TBDMS) at nitrogen atmosphere then next time ₂(0.405g, 0.95mmol).This mixture placed on the ice bath stirs, and with syringe dropwise add diisopropylethylamine (DIEA) (158 μ L, 0,91mmol).After 20 minutes, reaction is warming to room temperature, was stirring 17 hours, filter (if necessary), (40mL) dilute, be transferred in the separatory funnel, with the flushing of 5%HCl aqueous solution with ethyl acetate (EtOAc).Extract from water layer with EtOAc.Merge organic layer and use 5%HCl aqueous solution (3x), H ₂MgSO is used in O (1x) washing ₄Drying, and evaporation obtains the Boc-DOPA of white foam shape ₂(TBDMS) ₄(0.83g, productive rate 98%).

Embodiment 23

Boc-DOPA ₂(TBDMS) ₄-Osu's is synthetic

Use Boc-DOPA ₂(TBDMS) ₄Repeat the process of embodiment 21, to obtain Boc-DOPA ₂(TBDMS) ₄-Osu.

Embodiment 24

Boc-DOPA ₃(TBDMS) ₆Synthetic

Use Boc-DOPA ₂(TBDMS) ₄-OSu repeats the process of embodiment 22, to obtain Boc-DOPA ₃(TBDMS) ₆

Embodiment 25

DOPA ₂Synthetic

With Boc-DOPA ₂(TBDMS) ₄(0.5g 0.54mmol) is dissolved among the saturated HCl/EtOAc (3mL), stirs this solution then under nitrogen.After 5 hours, with other HCl gas bubbling 25 minutes gently in this solution.With the reactant standing over night, subsequently it is concentrated to the  of original volume.By centrifugal collection gained precipitation, with cold EtOAc (3x) washing, and the dry DOPA that obtains white powder ₂(0.15g, productive rate 74%).RP-HPLC is further purified product with the preparation type, and characterizes with ESI-MS.

Embodiment 26

DOPA ₃Synthetic

With Boc-DOPA ₃(TBDMS) ₆(1.06g 0.79mmol) is dissolved among the saturated HCl/EtOAc (3mL), and stirs this solution under nitrogen.After 12 hours,, continue reaction 40 hours then with other HCl gas bubbling 30 minutes gently in this solution.Use more HCl gas bubbling 30 minutes in solution once more, stop to stir.By the precipitation of centrifugal collection gained, with cold EtOAc (3x) washing, and the dry DOPA that obtains white powder ₃(0.424g, productive rate are 96%).RP-HPLC is further purified product with the preparation type, and characterizes with ESI-MS.

Embodiment 27

MPEG-DOPA _L-3Synthetic

With argon to the borate buffer solution of 0.1M (50mL pH8.5) carries out 20 minutes the degassing, and add L-DOPA (0.197g, 1.0mmol).With solution stirring after 15 minutes, add in batches the end capped PEG-SPA of methoxyl group (mPEG-SPA) 5K (0.5g, 0.1mmol), and reaction stirred 3 hours.With the HCl aqueous solution settled solution of gained being acidified to pH then is 1-2, with DCM (3x) extraction.Organic layer with 0.1M HCl washing merges passes through MgSO ₄Drying, and concentrate.Last residue is dissolved among the DCM, precipitates 3 times, obtain the rmPEG-DOPA (0.420g, productive rate are 84%) of white powder with ethyl acetate.With MALDI-MS and ¹H NMR spectrographic method characterizes product.

Embodiment 28

Finishing

(Al, 316L rustless steel and NiTi) grinds and polishes to the solid metal base material, use at last be the 0.04m silica sol (Syton, DuPont).Adopt Edwards FL400 electron-beam evaporator, with＜10 ^-6Holder is with 20nm TiO ₂Or 10nm TiO ₂/ 40nm Au evaporation deposition is cut to the sheet of 8mm * 8mm then to the Si wafer.In following each material, all base materials are carried out 20 minutes ultrasonic clean: 5%Contrad70 (Fisher Scientific), ultrapure H ₂O, acetone and petroleum ether.Then, by under 150 millitorrs and 100W, being exposed to O ₂(Harrick Scientific) further cleaned the surface in 5 minutes in the plasma.In order to prevent to form golden oxide (Au ₂O ₃) layer, some Au base material is not exposed to O ₂In the plasma.In order to form and the similar surface of biopolymer, (Fisher Scientific) carried out aforesaid cleaning to the glass cover slide, and it was soaked in 0.01% PLL (Sigma) solution 5 minutes, uses ultrapure H ₂O cleans, and dry under nitrogen.

In order to study various modification conditions with minimum sample, used the Luo Basite design method (Robust Design approach) of 9 key elements.Under the cloud temperature condition, by under 50 ℃, being soaked in mPEG-DOPA _1-3With 0.1M MOPS buffered 0.6M K ₂SO ₄In the solution base material is modified.PH to buffer as shown in table 1, modification time and mPEG-DOPA concentration improve.Use ultrapure H then ₂O cleans modified base material, and dry under nitrogen current.

Table 1

Test	Anchoring group	The pH of buffer	Adsorption time	Polymer concentration
					1	-DOPA	3.0	1h	0.5mg/mL
2	-DOPA	6.0	4h	1.0mg/mL
					3	-DOPA	9.0	24h	3.0mg/mL
4	-DOPA 2	3.0	4h	3.0mg/mL
					5	-DOPA 2	6.0	24h	0.5mg/mL
6	-DOPA 2	9.0	1h	1.0mg/mL
					7	-DOPA 3	3.0	24h	1.0mg/mL
8	-DOPA 3	6.0	1h	3.0mg/mL
					9	-DOPA 3	9.0	4h	0.5mg/mL

Embodiment 28a

TiO ₂Base material

Under the cloud temperature condition, by the following method to TiO ₂Base material is modified: under 50 ℃, base material is soaked in mPEG-DOPA _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28b

Under the cloud temperature condition, with the following method 316L rustless steel (Goodfellow, Devon PA) is modified: under 50 ℃, base material is soaked in mPEG-DOPA _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28c

Under the cloud temperature condition, with the following method to Al ₂O ₃(Goodfellow, Devon PA) modifies: under 50 ℃, base material is soaked in mPEG-DOPA _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28d

Under the cloud temperature condition, with the following method to SiO ₂(South Boston MA) modifies: under 50 ℃, base material is soaked in mPEG-DOPA for the thermal oxide of 1500 , University Wafer _1-3With 0.1MN-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28e

The NiTi alloy (10mm * 10mm * 1mm) available from Nitinol Devices﹠amp; (Fremont CA), and under the cloud temperature condition, modifies it: under 50 ℃, base material is soaked in mPEG-DOPA Components with the following method _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28f

Under the cloud temperature condition, with the following method Au (the deposited by electron beam evaporation method is deposited on the Si wafer available from UniversityWafer) is modified: under 50 ℃, base material is soaked in mPEG-DOPA _1-3With 0.1MN-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28g

Under the cloud temperature condition, with the following method to Au ₂O ₃(the Au sample described in the embodiment 28f is exposed in the oxygen plasma to form Au ₂O ₃) modify: under 50 ℃, base material is soaked in mPEG-DOPA _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28h

Under the cloud temperature condition, (University Wafer, South Boston MA) modify: under 50 ℃, base material is soaked in mPEG-DOPA to GaAs with the following method _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 28i

Prepare the p-L-Lys surface with the following method: with glass cover slide (Fisher Scientific) be soaked in 0.01% PLL (p-L-Lys, Sigma) in 5 minutes, use ultrapure H ₂O cleans, and at N ₂Following dry.Then, under the cloud temperature condition, with the following method they are modified: under 50 ℃, base material is soaked in mPEG-DOPA _1-3With 0.1M N-3-morpholinyl propane sulfonic acid (MOPS) buffered 0.6M K ₂SO ₄In the solution 24 hours.Use ultrapure H ₂O cleans modified base material, and dry under nitrogen current.

Embodiment 29

Cell adhesion

At 37 ℃ and 5%CO ₂Under cultivate 12-16 for 3T3 Switzerland albino fibroblast (ATCC, Manassas, VA), culture medium is for being supplemented with 10% hyclone (FBS) (Cellgro, Herndon, VA), Da Erbaike (family name) improvement Iger (family name) culture medium (DMEM) of 100g/mL penicillin and 100U/mL streptomycin (steptomycin) (Cellgro, Herndon, VA).Before carrying out the cell adhesion analysis, the trypsin-EDTA with 0.25% is gathered into fibrocyte, is resuspended in the growth medium, and counts with blood counting chamber.

The general steps of analyzing in 4 hours

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) (epifluorescent microscope) obtains 9-16 image (depending on the base material size) and obtains quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular Devices Corporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29a

TiO ₂Base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, fixing recovery cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29b

TiO ₂Base material (studying for a long period of time)

For TiO ₂Study for a long period of time, with analyzed identical density in 4 hours and on base material, carry out semiweekly renewed vaccination.In periodic interval, remove not adherent cell by the culture medium of drawing in each hole.

Embodiment 29c

316L stainless steel substrate (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mLDMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29d

Al ₂O ₃Base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29e

SiO ₂Base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29f

NiTi base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29g

Au base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29h

Au ₂O ₃Base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 29i

GaAs base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).Meansigma methods and the standard deviation measured have been reported.

Embodiment 29j

P-L-Lys base material (analyzing in 4 hours)

At 37 ℃ and 5%CO ₂Condition under, the setup test base material is 30 minutes in the 12 hole tissue culturing polystyrene plates that the 1.0mL DMEM that contains FBS is housed.With 2.9 * 10 ³Individual cell/cm ²Density with the cell kind on base material, and at 37 ℃ and 5%CO ₂Condition under, in containing the DMEM of 10%FBS, kept 4 hours.Adhere to analysis in order to carry out 4 hour cells, the secure attachment cell is 5 minutes in 3.7% paraformaldehyde, use 5 μ M 1 then, 1 '-two octadecyls-3,3,3 ', 3 '-tetramethyl indole carbocyanine perchlorate (DiI) (MolecularProbes, Eugene, DMSO solution OR) dyeed 45 minutes down at 37 ℃.

With having disposed SPOT RT digital camera (Diagnostic Instruments, Sterling Heights, Leica TIRF MI) obtain 9-16 image (depending on the base material size) and obtain quantitative cell adhesion data on the optional position on every plate substrate.Adopt the threshold value among the MetaMorph, quantitatively the total outstanding cell area of gained image.(Universal Imaging Corporation ^TM, it is the subsidiary of Molecular DevicesCorporation, Downington, PA).The meansigma methods and the standard deviation of measurement result have been reported.

Embodiment 30

Modified and analyzed in 4 hours in 24 hours of base material

With the pH value shown in Figure 24, under 50 ℃ at the mPEG-DOPA of 1.0mg/mL ₃Modified each surface in (or mPEG-OH in contrast) 24 hours.As above carrying out 4 hour cells described in the embodiment 9 adheres to and diffusion analysis.The results are shown among Figure 24.Compared and used mPEG-DOPA ₃The cell adhesion resistance of all base materials of handling.Cell adhesion and the diffusion and not modified surperficial as broad as long (data not shown goes out) of the base material of handling through mPEG-OH.

Embodiment 31

Prepare on surface and surface

By the physical vapor deposition method, (PSI, Villigen is Switzerland) with TiO with reactive magnetron sputtering ₂(20nm) be coated on the silicon wafer (WaferNet GmbH, Germany).The sheet that will cut into 1cm * 1cm then with the wafer of metal oxide-coated is to be used to (ex-situ) ellipsometry (ellipsometry) mensuration of offing normal.Be used for optical waveguide sheet that OWLS measures available from Microvacuum Ltd. (Budapest, Hungary), and by AF45 glass baseplate (8 * 12 * 0.5mm) and the thick Si of 200nm _0.25Ti _0.75O ₂The waveguide surface layer is formed.With with the identical condition that is used for silicon wafer as mentioned above, at the TiO of the deposited on top 8nm of ducting layer ₂Layer.Before polymer-modified, in the 2-propanol to using TiO ₂The supersound process that silicon wafer that applies and waveguide sheet carry out 10 minutes is cleaned in ultra-pure water, and dry under nitrogen current, is exposed to O then ₂In the plasma (Harrick Scientific, Ossining, the U.S.) 3 minutes to remove all organic principles from the surface.After OWLS measures, as described below the waveguide sheet is regenerated with recycling: the waveguide sheet is placed cleaning solution (300mM HCl, 1% detergent; Roche Diagnostics, Switzerland) middle supersound process, clean to remove adsorbate with ultra-pure water then.

Finishing.

Use cloud temperature buffer (CP buffer: the 0.6M K that is buffered to pH=6.0 with 0.1M MOPS ₂SO ₄), in 25-50 ℃ temperature range, use mPEG-DOPA according to embodiment 27 preparations _1-324 hours modification is carried out on the surface, and used polymer concentration is 1.0mg/ml.Modify the back water and clean base material, at N ₂Dry in the stream, as described below immediately then the analysis.

X-ray photoelectron spectroscopy (XPS) is measured

Use is at standard (polyenergeticization) AlK that flies away from work under the angle (take-off angle) of 325W (13kV, 25 mA) and 0 ° _αX-ray source, and on SAGE 100 (SPECS, Berlin Germany) gather measurement result and high-resolution spectroscopy,, the described angle of flying away from is defined as angle between optoelectronic detector and surface normal.Be used to measure and the passing through and (pass energy) to be respectively 50eV and 14eV of high-resolution spectroscopy.In data acquisition, the pressure in the analysis room is remained below 2 * 10 ^-8Pa.All XPS spectrums are reference with the aliphatic hydrocarbyl composition in the Cls signal under the 287.4eV all.Use CasaXPS software, adopt Shirley background rejection and 90% Gaussian function and 10% Lao Lunsi function sum to carry out curve fitting.With the atom sensitivity factor intensity of measuring (peak area) is converted into standardized intensity, and can calculates the atom composition on surface thus.Three meansigma methodss that repeat base material have been reported among the table 7-8.Standard deviation is usually less than the l0% of meansigma methods, for the sake of clarity with its omission.

Table 7

For the TiO that modifies through mPEG-DOPA ₂The quantitative analysis of the XPS data on surface

The surface	Atomic concentration (atom %)
								Ti	O			C
	Ols A TiO 2	Ols B TiOH	Ols C C-O，H 2O	Cls A C-C，C-H	Cls B C-O	Cls C NHC(＝O)
							The TiO of cleaning 2 mPEG-DOPA 1 mPEG-DOPA 2 mPEG-DOPA 3		24.3 17.9 11.1 7.4	50.9 36.8 22.5 15.9	14.1 5.2 3.2 0.5	4.1 13.1 20.2 25.3	4.5 2.8 3.7 4.0	1.2 23.1 37.3 43.7	0.9 1.2 2.1 3.2

Table 8

From the TiO that modifies through mPEG-DOPA ₂The atomic ratio that the XPS data computation on surface obtains

The surface	Atomic ratio a
							C/Ti	O A/Ti	O B/O A	C B/C A	C C/C B	C C/C A
	The TiO of cleaning 2mPEG-DOPA mPEG-DOPA 2mPEG-DOPA 3	0.27 1.52 3.89 6.87	2.10 2.06 2.03 2.14	0.28 0.14 0.14 0.03	0.33 8.42 10.07 11.00	0.42 0.052 0.055 0.074							0.20 0.42 0.56 0.80

^aBe defined in the table 7 for amount (Contribution) A, B and C.

Beam split ellipsometry (Spectroscopic Ellipsometry)

Measure for ELM, as mentioned above to using TiO ₂The modification of offing normal of the Si base material of sputter, the temperature of modifying solution changes between 25-50 ℃.After the modification, use H ₂O cleans base material, and at room temperature (pH=7.4) hatched 48 hours in 10mM HEPES buffer, used H once more ₂O cleans, and uses N ₂Dry.In order to detect protein-resistant, modified and not modified base material is exposed in the pure human serum 15 minutes, water cleans, and at N ₂Dry in the stream.Before modification and just modified back, HEPES and hatched after back and serum exposes, under 65 °, 70 ° and 75 °, (Lincoln USA) carries out ELM and measures for J.A.Woollam Co., Inc., and used wavelength is 193-1000nm on the oval photometer of M-2000D beam split.In the WVASE32 analysis software, utilize generalization Cauchy polymeric layer (Cauchy polymer layer) (A _n=1.45, B _n=0.01, C _n=0) optical property is with multilayered schema match ELM collection of illustrative plates, to obtain " doing " thickness of adsorbed PEG and serum adhesion layer.(" doing " or dehydration thickness are meant and are using N ₂The thickness that dry back is measured under environmental condition).Reported the average thickness of three replications among the table 9-10.

Table 9

Adsorption temp is to TiO ₂ ^aThe influence of last PEG adhesion layer thickness

Adsorption temp (℃)	Thickness ()
		25 28 31 34 37 40 45 50	10.6±1.8 13.9±0.3 16.4±1.1 19.4±1.9 19.6±3.5 22.5±2.0 24.4±2.0 33.8±4.6

^aTiO ₂The surface is exposed to mPEG-DOPA ₃(1mg/ml) 2 hours, in HEPES, cleaned each surface 48 hours then.

Table 10

TiO by beam split ellipsometry mensuration ₂On organic adhesion layer apparent thickness ()

Handle a	mPEG-DOPA 3Adsorption time (minute)
							0	1	30	60	240	1080
	Be exposed to be exposed to serum before the serum after	- 60.2	5.1 23.4	24.2 24.7	27.6 ＜28.1 b	31.8 ＜32.3 b							35.0 ＜35.5 b

^aWith TiO ₂The surface is exposed to mPEG-DOPA ₃(1mg/ml) 0-1080 minute, water cleaned, and hatches in HEPES 48 hours, was exposed in the serum 15 minutes then.

^bIt is long less than 0.5  to be exposed to having a net increase of of adsorbent layer thickness behind the serum, and this numerical value approaches the resolution of ELM technology.

Optical waveguide optical mode spectrographic method (Optical Waveguide Lightmode Spectroscopy, OWLS).

As mentioned above, with 2-propanol and O ₂Plasma cleaning TiO ₂The wave conductor that applies.With the cleaning wave conductor be fixed on the mensuration head of OWLS110 (Microvacuum Ltd.), and under room temperature at cloud temperature buffer (CP buffer: the 0.6M K that is buffered to pH=6.0 with 0.1M MOPS ₂SO ₄) in stabilisation at least 48 hours.The described stabilisation stage makes TiO ₂Lip-deep ion exchange reaches balance, and obtains stable baseline.Be the absorption of monitoring polymer, be infused in the mPEG-DOPA in the CP buffer to arrhea pattern (stop-flow mode), inject the CP buffer then and remove unconjugated PEG, signal is able to stabilisation after this.Write down not coupling angle (incoupling angle)---α _TMAnd α _TE, the software that provides by manufacturer is translated into refractive index (N _TM, N _TE).Use de Feijter formula that effective refractive index real-time change of pick off is converted into the absorption quality.Township's feelings of the reference that need include in [de Feijter, 1978#14] are with the 0.13cm of pure PEG ³The 0.18cm of/g and pure amino acids ³Linear interpolation between/g is rised in value to the refractive index of each mPEG-DOPA polymer---and dn/dc calculates.For protein adsorption test, the hygral equilibrium of measuring head at 37 ℃, until signal stabilization, was injected serum 15 minutes then, injecting buffer.The absorption quality does not have tangible difference with the prolongation of serum open-assembly time.

Embodiment 32

N-methacryl 3,4-dihydroxy-L-phenylalanine synthetic

With 1.15g (5.69mmo1) Na ₂B ₄O ₇Be dissolved in the 30ml water.With Ar solution is carried out 30 minutes the degassing, add 0.592g (3.0mmol) L-DOPA then, stirred 15 minutes.Add 0.317g (3.0mmol) Na then ₂CO ₃, solution is cooled to 0 ℃, stir down slowly adding 0.3ml (3.0mmol) methacrylic chloride.In course of reaction, use Na ₂CO ₃The pH of solution is remained on 9.After at room temperature stirring 1 hour, with dense HCl solution being acidified to pH is 2.With this mixture of ethyl acetate extraction 3 times.After the 0.1NHCl washing, use anhydrous MgSO ₄Drying, vacuum is removed solvent, obtains the thick solid of light brown.By from silicagel column, product being further purified with dichloromethane (DCM) and methanol (95: 5) eluting.Behind the evaporative removal solvent, the productive rate with 35% obtains white sticky solid. ¹H NMR (500MHz, acetone-d ₆):

7.1d (1H ,-NH-); 6.6-6.8 (3H, C ₆H ₃(OH) ₂-); 5.68s (1H, CHH=); 5.632s (unknownpeak); 5.33s (1H, CHH=); 4.67m (1H ,-CH-); 2.93-3.1m (2H, CH ₂-); 1.877s (3H ,-CH ₃).

Embodiment 33

3, two (tert-butyl group dimethyl methyl siloxy)-L-phenylalanine of 4-synthetic

3.60g (24.0mmol) TBDMS-Cl is dissolved in the 18ml anhydrous acetonitrile.1.60g (8.0mmol) L-DOPA is added in the solution, stir this suspension and be cooled to 0 ℃, add 3.6mlDBU (24.0mmol) then.Stirred reaction mixture 24 hours at room temperature then.Cold acetonitrile is added in the reaction solution, obtain colourless precipitation.Filter this precipitation and, then it is carried out vacuum drying with cold acetonitrile washing several.Productive rate with 78% obtains white powder. ¹H NMR (500MHz, methanol-d); 6.7-6.9 (eH, C ₆H ₃(O-Si-) ₂-); 3.72 (m, 1H ,-CH-); 2.82-3.2 (m, 2H ,-CH ₂-); 1.0 (d, 18H ,-C (CH ₃)); 0.2 (d, 12H, Si-CH ₃).

Embodiment 34

3, two (tert-butyl group dimethyl methyl siloxy)-N-tertbutyloxycarbonyl-L-phenylalanine of 4-synthetic

With 1.60g (3.77mmol) 3, two (tert-butyl group dimethyl methyl siloxy)-L-phenylalanine of 4-add 10ml and contain 0.34g (4.05mmol) NaHCO ₃Water in.Add 0.96g (4.30mmol) Bis(tert-butoxycarbonyl)oxide (solution of di-t-butyl dicarbonatel in the 10ml oxolane, and stirred reaction mixture 24 hours at room temperature.Behind the evaporative removal oxolane, in residue, add 10ml water.With rare HCl solution being acidified to pH is 5, uses ethyl acetate extraction 3 times.Use anhydrous MgSO ₄After the drying, vacuum is removed solvent.By column chromatography purification crude product (silica gel; Eluent; The DCM solution of 10% methanol).Productive rate with 70% behind the evaporative removal eluting solvent obtains white solid. ¹H NMR (500MHz, methanol-d);

6.68-6.81 (3H, C ₆H ₃(O-Si-) ₂-); 4.28 (m, 1H ,-CH-); 2.78-3.08 (m, 2H ,-CH ₃-); 1.4 (s, 9H ,-O-C (CH ₃) ₃); 1.0 (d, 18H ,-Si-C (CH ₃) ₃); 0.2 (d, 12H, Si-(CH ₃) ₂).

Embodiment 35

3, two (tert-butyl group dimethyl methyl siloxy)-N-tertbutyloxycarbonyls of 4--L-phenylalanine pentafluorophenyl group ester synthetic

With 1g (1.90mmol) 3, two (tert-butyl group dimethyl methyl siloxy)-N-tertbutyloxycarbonyl-L-phenylalanine of 4-and 0.351g (1.90mmol) Pentafluorophenol (pentafluorophernol) are dissolved in the solvent mixture of being made up of 24ml diox and 1ml DMF, add 0.432g (2.10mmol) DCC down at 0 ℃.0 ℃ of following agitating solution 1 hour, at room temperature stirred then 1 hour, stir the back solution is filtered to remove 1,3-Dicyclohexylurea, vacuum evaporation.By column chromatography product 4 is carried out purification (silica gel; Eluent; Hexane/ethyl acetate=11.2).After removing eluent, the productive rate with 55% obtains lily sticky solid. ¹H NMR(500MHz，CDCl ₃)；

6.65-6.81(3H，C ₆H ₃(O-Si-) ₂-)；4.85(m，1H，-CH-)；3.05-3.2(m，2H，-CH ₃-)；1.41(s，9H，-O-C(CH ₃) ₃)；1.0(d，18H，-Si-C(CH ₃) ₃)；0.2(d，12H，Si-(CH ₃) ₂)。

Embodiment 36

N-(13 '-amino-4 ', 7 ', 10 '-trioxa, three decyls)-tertbutyloxycarbonyl-3 ', 4 '-two (tert-butyl group dimethyl methyl siloxy)-L-hydrocinnamamides synthetic

Under 0 ℃, with 0.869g (1.26mmol) 3, the 10ml DCM solution of two (tert-butyl group dimethyl methyl siloxy)-N-tertbutyloxycarbonyls of 4--L-phenylalanine pentafluorophenyl group ester, in 30 minutes time, dropwise join by 2.07ml (9.44mmol) 4,7,10-trioxa-1,13-three decane diamidogen and 1.32ml (9.44mmol) Et ₃In the mixture that N forms in 1mlDMF.At room temperature, go down to desolventize in vacuum then with solution restir 2 hours.Crude product is stated from the silica gel, with DCM, the DCM solution of 5% methanol, the DCM solution of 10% methanol and the DCM eluant solution of 15% methanol.Vacuum goes down to desolventize the chemical compound 5 that obtains white solid.Productive rate is 63%. ¹H NMR (500MHz, acetone-d ₆);

7.38 (m, 1H ,-CONH-); 6.60-6.80 (3H, C ₆H ₃(O-Si-) ₂-); 5.26 (m, 1H ,-CONH-); 4.30 (m, 1H ,-CH-); 3.4-3.8 (m, 12H ,-CH ₂O-; 3.03-3.4 (m, 4H ,-CH ₂-NH-,-CH ₂-NH ₂); 2.78-3.02 (m, 2H ,-CH ₂-); 2.0 (m, 2H ,-CH ₂-); 1.7 (m, 2H ,-CH ₂-); 1.39 (s, 9H ,-O-C (CH ₃) ₃); 1.0 (d, 18H, Si-C (CH ₃) ₃); 0.2 (d, 12H, Si-C (CH ₃) ₂).

Embodiment 37

N-(13-(N '-tertbutyloxycarbonyl-L-amino-3 ', 4 '-two (tert-butyl group dimethyl methyl siloxies)-4,7,10-trioxa three decyls)-MAAm synthetic

With 0.57g (0.79mmol) N-(13 '-amino-4 ', 7 ', 10 '-trioxa, three decyls)-tertbutyloxycarbonyl-3 ', 4 '-two (tert-butyl group dimethyl methyl siloxy)-L-hydrocinnamamides and 0.166ml (1.18mmol) Et ₃N is dissolved in the anhydrous chloroform of 5ml, and adds 0.176ml (1.18mmol) methacrylic anhydride therein.At room temperature agitating solution is 3 hours, goes down to desolventize in vacuum then.Obtain the pure compound 6 (silica gel of white sticky solid shape with 61% productive rate by column chromatography; Eluent: ethyl acetate). ¹H NMR(500MHz，CDCl ₃)；

6.60-6.80(3H，C ₆H ₃(O-Si-) ₂-)；6.40(m，1H，-CONH-)；5.71s(1H，CHH＝)；5.30s(1H，CHH＝)；5.096(m，1H，-CONH-)；4.21(m，1H，-CH-)；3.2-3.65(m，16H，-CH ₂O，-CH ₂-NH--CH ₂-NH ₂)；2.80-2.99(m，2H，-CH ₂-)；1.96s(3H，-CH ₃)；1.81(m，2H，-CH ₂-)；1.68(m，2H，-CH ₂-)；1.40(s，9H，-O-C(CH ₃) ₃)；1.0(d，18H，Si-C(CH ₃) ₃)；0.2(d，12H，Si-C(CH ₃) ₂)。

Embodiment 38

N-(13-(N '-t-Boc-L-3 ', 4 ' dihydroxy benzenes alanyl amido)-4,7, l0-trioxa three decyls)-MAAm synthetic

In the 10ml round-bottomed flask, add 0.344g (0.433mmol) N-(13-(N '-tertbutyloxycarbonyl-L-amino-3 ', 4 '-two (tert-butyl group dimethyl methyl siloxies)-4,7,10-trioxa three decyls)-MAAm, 3mL THF and 0.137g (0.433mmol) TBAF.At room temperature stir this solution 5 minutes, and added 3ml 0.1NHCl then.With DCM extraction solution 3 times, vacuum evaporating solvent then.By column chromatography (silica gel; Eluent: the DCM solution of 7% methanol) obtain white solid chemical compound 7 with 63% productive rate. ¹H NMR (500MHz, acetone-d ₆);

7.90 (m, 1H ,-CONH-); 7.23-7.40 (d 2H, C ₆HH ₂(OH) ₂-); 6.56-6.76 (3H, C ₆HH ₂(OH) ₂-; 5.930 (m, 1H ,-CONH-); 5.71s (1H, CHH=); 5.30s (1H, CHH=); 4.20 (m, 1H ,-CH-); 3.1-3.60 (m, 16H ,-CH ₂O ,-CH ₂-NH-,-CH ₂-NH ₂); 2.70-2.95 (m, 2H ,-CH ₂-); 1.96s (3H ,-CH ₃); 1.78 (m, 2H ,-CH ₂-); 1.65 (m, 2H ,-CH ₂-); 1.39 (s, 9H ,-O-C (CH ₃) ₃).

Embodiment 39

Synthesizing of PEG-diacrylate (PEG-DA)

Come dry 40g (5mmol) PEG by azeotropic vaporization in benzene, then it is dissolved among the 150mL DCM.With 4.18mL (30mmol) Et ₃N and 3.6mL (40mmol) acryloyl chloride adds in this polymer solution.Under agitation reflux this mixture 5 hours makes its cool overnight at room temperature.Ether is added in this mixture to form luteotestaceous precipitation.Crude product is dissolved in the saturated NaCl solution then, and be heated 60 ℃ two-layer to form.In the upper strata, add DCM, and add MgSO ₄To remove moisture.Filter and remove MgSO ₄After, reducing solvent volume under the vacuum, sample is deposited in the ether.The vacuum drying end-product, and it is stored in-15 ℃.Productive rate is 75%. ¹H NMR(500MHz，D ₂O)：δ6.47(d，1H，CHH＝C-)；6.23(m，1H，C＝CH-C(＝O)-O-)；6.02(d，1H，CHH＝C-)；4.35(m，2H，-CH ₂-O-C(＝O)-C＝C)；3.23-3.86(PEG CH ₂)

The PEG-DA photopolymerization

Prepare PEG-DA, chemical compound 1, chemical compound 7 and light trigger initial soln (precusor solution), before photopolymerization, mix immediately.The storing solution of PEG-DA (200mg/mL) and chemical compound 1 (40mg/mL) is dissolved in uses N ₂The phosphate buffered saline (PBS) that purged (PBS, pH7.4) in, use N in advance and chemical compound 7 (60mg/mL) is dissolved in ₂In 50: 50 PBS/95% ethanol that purge.In order to prepare final polymerizable compound, with chemical compound 1 or 7 and PEG-DA to merge to obtain final concentration be PEG-DA and the DHPD derivant of 150mg/mL.Then 100 μ L mixture are added (100 μ L, diameter=9mm, the degree of depth=2.3mm, Secure Seal in the plate-like mould ^SA8R-2.0, Grace Bio Lab, Inc., OR), with UV lamp (Black Ray ^Lamp, 365nm, model UVL-56, UVP, CA) or blue-ray light (VIP ^, 400-500nm, BISCO Inc., IL) irradiation reaches 20 minutes.Photocuring for UV causes adds in the polymeric solution DMPA (the VP solution of 600mg/mL) to obtain the final concentration of 34mM.In the curing of using visible light-inducing, can use and contain DMAB (the VP solution of 30mg/mL, (the VP solution of 100mg/mL, final concentration=150mM) or use contain AA (the PBS solution of 100mg/mL, the FNa of final concentration=17mM) to the CQ of final concentration=151mM) ₂(the PBS solution of 188mg/mL, final concentration=2mM) as light trigger.The VP final concentration is adjusted to 135-300mM.

After the irradiation, blot gel with removal liquid surface layer, and it is weighed with filter paper.Then gel weight is drawn the gel conversion percentages divided by the weight of 100 μ L starting solns.

Embodiment 41

The bonded mensuration of DOPA

Adopted the amount that is incorporated into the DOPA in the photopolymerization gel by the improvement colorimetric DOPA assay of Waite and Benedict exploitation.The gel of photocrosslinking is stirred to extract the DOPA monomer that is not incorporated into gel network in 3mL 0.5N HCl.0.9mL nitrite reagent (1.45M sodium nitrite and 0.41M two molybdic acid hydrate sodium) and 1.2mL 1M NaOH are joined in the 0.9mL extraction solution, in 2-4 minute of adding NaOH, with the absorbance (500nm) of Hitachi U-2010 UV-Vis spectrophotometric determination mixture.Use the concentration of compound known 1-7 to make up standard curve.

Embodiment 42

Mechanical test

By being stated from, 25 μ L polyblends use 1H, 1H, and 2H on the sheet glass that 2H-perfluoro octyl group trichlorosilane was handled, forms hemispherical hydrogel.Irradiation gel 10 minutes, in 0.15M HCl dialysis at least 24 hours to extract unconjugated DOPA monomer, then before test in PBS balance more than 15 minutes.Be to measure the modulus of gel, with super binding agent with hemispherical gel cover and steel graduated cylinder (diameter=6mm, the end connection of length=30mm).The other end and piezoelectricity stepper motor (IW-701-00 with this graduated cylinder, BurleighInstruments NY) connects, and this motor resolution of having connected is about 50g load transducer (the loadtransducer) (FTD-G-50 of 0.1mN, Schaevitz Sensors, VA).(Inc. MD) measures steel column moving in the axial direction for RC100-GM2OV, Philtec with the fibre optics displacement transducer.To use TiO ₂The Si wafer that applies places under the hydrogel, with PBS submergence Tio ₂The surface is to keep the hydration status of gel.Pressure head advances with 5 μ m/s, until the maximum compression load that is measured to 4mN.

By concrete condition is assumed to can not the compression elasticity hemisphere with rigid planar between do not have the Hertzian mechanism that contact that adheres to, the calculating elastic modulus, (P in this case loads _h) and displacement (δ _h) between Hertzian relation become:

$P_h=\frac{16{\mathrm{<figure-callout\; id="1"\; label="R"\; filenames="A20058000618000811.png,A20058000618000862.png"\; state="\{\{state\}\}">R</figure-callout>}}^{1/2}\mathrm{<figure-callout\; id="9"\; label="E"\; filenames="A20058000618000891.png,A20058000618001051.png"\; state="\{\{state\}\}">E</figure-callout>}}{9}{{\mathrm{\&delta;}}_{\mathrm{<figure-callout\; id="3"\; label="h"\; filenames="A20058000618000811.png,A20058000618000861.png"\; state="\{\{state\}\}">h</figure-callout>}}}^{3/2}$

Wherein R and E are respectively the radius of curvature and the elastic modelling quantity of hemispherical gel.The radius of curvature of determining this gel by the height measured from the photo of gel and width.

Embodiment 43

With the PEG-DOPA chemical oxidation is hydrogel

4-arm-PEG-amine (PEG-(NH ₂) ₄, M _w=10,000) available from SunBio, Inc. (WalnutCreek, CAv), and straight chain PEG-couple-amine (PEG-(NH ₂) ₂, M _w=3,400) and methoxyl group-PEG-amine (mPEG-NH ₂, M _w=5,000) then available from Shearwater Polymers, and Inc. (Huntsville, AL).Sephadex ^LH-20 available from Fluka (Milwaukee, WI).N-Boc-L-DOPA two hexamethylene ammonium salts, sodium metaperiodate (NaIO ₄), Mushroom Tyrosinase (MT, EC 1.14.18.1) and horseradish peroxidase (HRP, EC1.11.1.17) available from Sigma Chemical Company (St.Louis, MO).Triethylamine (Et ₃N), hydrogen peroxide (30wt%, H ₂O ₂), two molybdic acid hydrate sodium and sodium nitrite available from Aldrich ChemicalCompany (Milwaukee, WI).L-Dopa available from Lancaster (Windham, NH).I-hydroxybenzotriazole (HOBt) available from Novabiochem Corp. (La Jolla, CA), and hexafluorophosphoric acid O-(benzotriazole-1-yl)-N, N, N ', N '-tetramethylurea (HBTU) available from Advanced ChemTech (Louisville, KY).

The PEG's that modifies with DOPA is synthetic

Use the synthetic nearly PEG with straight chain and side chain DOPA modification of 4 DOPA end groups that comprises of standard carbodiimides coupling chemical method as described below.The structure of the PEG that modifies with 4 DOPA is shown among Fig. 1.

PEG-(N-Boc-DOPA) ₄, I's is synthetic with PEG-(NH ₂) ₄(6.0g, 0.60mmol) with at 60mL50: N-Boc-L-DOPA two hexamethylene ammonium salts (4.8mmol), HOBt (8.0mmol) and Et in 50 dichloromethane (DCM) and dimethyl formamide (DMF) mixture ₃N (8.0mmol) reaction.Add the solution of HBTU (4.8mmol) in 30mL DCM then, under room temperature and argon, carry out 1 hour coupling reaction.With saturated nacl aqueous solution, 5%NaHCO ₃, rare HCl solution and distilled water wash this solution successively.The concentrating under reduced pressure crude product, and by column chromatography at Sephadex ^Be that mobile phase is carried out purification with methanol on the LH-20 post.3 times this product is further purified by precipitation in cold methanol, vacuum drying at room temperature ,-20 ℃ are stored in the nitrogen. ¹H NMR(500MHz，CDCl ₃/TMS)：δ6.81-6.77(m，2H，C ₆HH ₂(OH) ₂-)，6.6(d，1H，C ₆H ₂H(OH) ₂-)，6.05(br，s，1H)，5.33(br，s，1H)，4.22(br，s，1H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，3.73-3.41(m，PEO)，3.06(m，2H，PEO-CH ₂-N-C(O)-)，2.73(t，2H，C ₆H ₃(OH) ₂-CH ₂-CH-)，1.44(s，9H，(CH ₃) ₃C-)。GPC-MALLS： M _w＝11,900， M _w/ M _n＝1.01。

PEG-(DOPA) ₄, under the synthetic room temperature of II, (0.25mmol) is dissolved among the 15mLDCM with the 3.0g Compound I.15mL TFA was joined in the mixture under argon reaction 30 minutes.In Rotary Evaporators behind the evaporating solvent, with cold methanol precipitated product 3 times, vacuum drying at room temperature, and be stored in the nitrogen in-20 ℃. ¹H NMR(500MHz，D ₂O)：δ6.79(d，1H，C ₆H ₂H(OH) ₂-)，6.66(s，1H，C ₆H ₂H(OH) ₂-)，6.59(d，1H，C ₆H ₂H(OH) ₂-)，4.00(t，1H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，3.70-3.34(M，PEO)，3.24(m，2H，PEG-CH ₂-N-C(O)-)，3.01-2.88(m，2H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)。GPC-MALLS： M _w＝11,400， M _w/ M _n＝1.02。

PEG-(N-Boc-DOPA) ₂, III's is synthetic. and with PEG-(NH ₂) ₂(5.0g, 1.5mmol), N-Boc-L-DOPA two hexamethylene ammonium salts (5.9mmol), HOBt (9.8mmol) and Et ₃N (9.8mmol) is dissolved among 50: 50 the DCM and DMF mixture of 50mL.Add the solution of HBTU (5.9mmol) in 25mL DCM then, under room temperature and argon, carry out 30 minutes reaction.As product being reclaimed and purification as described in to Compound I. ¹H NMR(500MHz，CDCl ₃/TMS)：δ6.81-6.77(m，2H，C ₆HH ₂(OH) ₂-)，6.59(d，1H，C ₆H ₂H(OH) ₂-)，6.05(br，s，1H)，5.33(br，s，1H)，4.22(br，s，1H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，3.73-3.42(M，PEO)，3.06(m，2H，PEO-CH ₂-N-C(O)-)，2.74(t，2H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，1.44(s，9H，(CH ₃) ₃CO-)。GPC-MALLS： M _w＝4,600， M _w/ M _n＝1.02。

Methoxyl group-PEG-(N-Boc-DOPA), IV's is synthetic with mPEG-NH ₂(2.0g, 0.40mmol), N-Boc-L-DOPA two hexamethylene ammonium salts (0.80mmol), HOBt (1.3mmol) and Et ₃N (1.3mmol) is dissolved in the mixture of the 50:50 DCM of 20mL and DMF.Add the solution of HBTU (0.80mmol) in 10mLDCM then, under room temperature and argon, carry out reaction in 30 minutes.As product being reclaimed and purification as described in to Compound I. ¹H NMR(500MHz，CDCl ₃/TMS)：δ6.81-6.60(m，3H，C ₆H ₃(OH) ₂-)，6.01(br，s，1H，OH-)，5.32(br，s，1H，OH-)，4.22(br，s，1H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，3.73-3.38(m，PEO)，3.07(m，2H，PEO-CH ₂-NH-C(O)-)，2.73(t，2H，C ₆H ₃(OH) ₂-CH ₂-CH(N-)-C(O)N-)，1.44(s，9H，(CH ₃) ₃C-)，1.25(s，3H，CH ₃CH ₂O-)。GPC-MALLS： M _w＝6,100， M _w/ M _n＝1.02。

The DOPA assay

By the integration at relevant peak in the 1H NMR spectrum with by colorimetric DOPA analytic process, to measuring with the DOPA content among the PEG of DOPA modification.In the NMR method, by the Boc methyl proton integrated value of δ=1.44 and the PEG methene proton integrated value of δ=3.73-3.38 are relatively measured DOPA content.The DOPA analytic process is based on the method for aforementioned Waite and Benedict.In brief, handle the PEG-DOPA aqueous solution, add excessive NaOH solution then with nitrite reagent (1.45M sodium nitrite and 0.41M two molybdic acid hydrate sodium).In adding NaOH 2-4 minute, with the absorbance (500nm) of Hitachi U-2010UV/vis spectrophotometer record mixture.Use the solution of known DOPA concentration to make up standard curve.

The formation of PEG-DOPA hydrogel

In order to form the PEG-DOPA hydrogel, with sodium metaperiodate (NaIO ₄), horseradish peroxidase and hydrogen peroxide (HRP/H ₂O ₂) or Mushroom Tyrosinase and oxygen (MT/O ₂) (PBS is pH7.4) in the solution to add the phosphate buffered saline (PBS) of PEG-DOPA (200mg/mL).For by the inductive gelling of MT, before adding MT, use air douche PBS 20 minutes.The qualitative mixture of measuring for the bottle that contains mixture liquid by upset of gelling time is stopped the mobile time.

Vibration-rheological is measured

Vibration-rheological is measured the mechanical property that is used to monitor gelatinization and is used to measure hydrogel.Cross-linking agent is added the PEG-DOPA aqueous solution, and well-mixed solution is loaded on the Bohlin VOR flow graph.The condition that analysis is carried out is: frequency is that 0.1Hz, strain (strain) are 1%, diameter is the cone of 30mm and with 2.5 ° of fixed plates of coning angle.

The beam split assessment of DOPA oxidation

To be dissolved in the PEG that DOPA modifies in the 10mM PBS solution (for HRP/H ₂O ₂And NaIO ₄Use the argon bubbling, the air bubbling is then used in test for MT).After adding oxidant, under the wavelength of 200-700nm, with the time-dependent UV/vis spectrum of 800nm/ minute sweep speed monitoring solution.To all samples all earlier with the PBS buffer as blank, then at room temperature with Hitachi U-2010 UV/vis spectrophotometer record.

Molecular weight analyse

Use following condition determining molecular weight: use GPC-MALLS, on DAWN EOS (WyattTechnology), use Shodex-OH Pak post, (50mM PBS, 0.1MNaCl, 0.05%NaN in aqueous mobile phase ₃And use Optilab DSP (Wyatt Technology refractive index detector pH=6.0).For estimating of molecular weight, used dn/dc value (0.136) through the compound IV of test determination.

Embodiment 44

Material and method

Most advanced and sophisticated modify (Tip modification)

To silicon nitride (Si ₃N ₄) before the tip carries out finishing, use O earlier ₂Plasma instrument (a kind of title of machine) carries out 3 minutes cleaning, then they is transferred to (sulphuric acid: H in the piranha solution ₂O ₂=8: 2) 30 minutes.Use H ₂After O cleans, their were moved in the toluene solution of 20% (v/v) 3-aminopropyl trimethoxy monosilane 30-60 minute, with by amine-functionalized.Selected 2 kinds of Polyethylene Glycol (PEG) derivant to be used on the AFM tip, carry out PEGization: mPEG-N-N-Hydroxysuccinimide (NHS) (Mw 2000) and Fmoc-PEG-NHS (Mw 3400) (Nektar Inc.).At the 0.6M of 50mM sodium phosphate buffer, pH7.8 K ₂SO ₄With preparation (Fmoc-PEG-NHS: mPEG NHS=1: 5-10, the mixture of PEG 5mM) in the mixture of chloroform.As described belowly carry out the PEGization reaction continuously: at first carry out at 40 ℃ sodium phosphate buffer, react in chloroform then, each step was all carried out 3 hours.Using the reason of PEG mixture is in order to prevent that a plurality of DOPA are incorporated into TiO ₂On.Fmoc-PEG-NHS provides amine in Fmoc fracture back for the Boc-DOPA combination.With piperidines (nmp solution of 20%v/v) Fmoc was carried out deprotection 5 minutes, then cantilever is transferred in BOP/HOBt/DOPA (mol ratio is 1: 1: 1, the nmp solution of the final 8mM) solution that contains 10 μ L DIPEA.Same step also is used for tyrosine and modifies.

The AFM test

All data all gather AFM instrument from the Nikon inverted microscope top that places (AsylumResearch, Santa Barbara, CA).By equiparition theorem (equipartition theorem) is applied to thermal noise frequency spectrum (S1), calculate each cantilever spring constant (information that provides according to producer be 45,100 and 300pN/nm about).With 1 drip put on through cleaning in advance (in organic solvent for ultrasonic with use O ₂Plasma) TiO ₂The surface.The moment curve (Force-distancecurve) that selection comprises PEG elasticity and profile length is used for further statistical analysis.For the test of DOPA quinone, all tests are all carried out in the 20mM of pH9.8 Tris.

Dynamic test

Load up speed (loading rate) dependency power is measured and has been disclosed the bonded energy view of DOPA (energy landscape) (17).The slope of the linear graph of [1n on the force rate (load up speed)] (=kBT/xb) determined that energy barrier xb edge applies the distance of the axle of power.By changing in the logarithm intercept power at zero load up speed place and come the energy barrier of calculations incorporated available from the xb of slope available from change the power takes place by pulling speed.Used silicon nitride AFM cantilever (Bio-Levers, Olympus, Japan), this be since they have less spring constant (～5pN/nm and～28pN/nm).In this research minimum load up speed of 2nN/ second be pulling speed by using 400nm/ second and cantilever (～5pN/mm) obtain.Promotion load rate (1500nN/ second) is by the work of 5 μ m/ seconds of piezoelectric device and uses hard cantilever (300pN/nm Veeco) produces.The surperficial sign with x-ray photoelectron spectroscopy (XPS) (Omicron, Taunusstein Germany) the analysis surface that is equipped with monochromatic Al K α (1486.8eV) 300W x-ray source and is used to eliminate the electron gun of electric charge accumulation (build-up).With the identical step described in modifying with AFM is most advanced and sophisticated, to the silicon nitride surface (0.7 * 0.7cm of manufacturing in hot room (Keun Ho customization) ²) clean and modify.Photosignal from carbon 1s track relates to Si ₃N ₄All are rich in the main indication of the finishing of kind Si, O and N on the surface.

Though combined the specific embodiment principle of the present invention is described, should knows that the adding that is understood that these descriptions only is as embodiment, rather than in order to limit the scope of the invention by any way.For example, the present invention can strengthen the adhesion characteristics of broad variety polymeric compositions, no matter whether they can the water-setting gels.Equally, the present invention can use various other synthetic technology well known to those skilled in the art to be used for the concrete polymers compositions of follow-up coupling and preparation corresponding D OPA conjugate with functional modification.By the claims by the appended claims herein and by the determined scope of their reasonable equivalents, other advantage, feature and benefit will become apparent, and this point should be able to be understood by those skilled in the art.

Claims (25)

1. dihydroxy phenyl (DHPD) viscous compound of a formula (I),

In the formula, R ₁And R ₂Can be identical or different, be selected from independently of one another hydrogen, saturated and undersaturated, side chain and straight chain, replacement and unsubstituted C _1-4Alkyl;

P separately and be independently selected from-NH ₂,-COOH ,-OH ,-SH,

R in the formula ₁And R ₂As defined above,

Singly-bound, halogen,

A in the formula ₁And A ₂Separately and be independently selected from H, singly-bound,

Blocking group,

Poly-(alkylene oxide) roughly,

N is 1 to about 3 in the formula,

A ₃Be

R ₄Be H, C _1-6Low alkyl group, or polyalkylene oxide

R ₃As defined above, D is suc as formula shown in (I).

2. chemical compound as claimed in claim 1 is characterized in that, described poly-(alkylene oxide) has following structure:

In the formula, R ₃And R ₄Separately and be independently selected from H or CH ₃, the value of m between 1 to about 250, A ₄Be NH ₂, COOH ,-OH ,-SH ,-H, DHPD or blocking group.

3. chemical compound as claimed in claim 1 is characterized in that, described DHPD has following structure:

4. chemical compound as claimed in claim 1 is characterized in that, described DHPD has following structure:

5. chemical compound as claimed in claim 1 is characterized in that, described DHPD has following structure:

A in the formula ₂Be-OH A ₁Roughly be poly-(alkylene oxide) of following structure

R ₃, R ₄With m such as claim 2 qualification.

6. DHPD as claimed in claim 5 is characterized in that, described poly-(alkylene oxide) is the block copolymer of oxirane and expoxy propane.

7. one kind adheres to the method for another base material with base material, and this method may further comprise the steps: the DHPD of following structure is provided,

R wherein ₁And R ₂As defined above;

With the DHPD of said structure put on want on adherent one or another base material or two base materials on;

Will adherent base material contact so that described base material is adhering to each other with the DHPD of said structure between them;

Thereby randomly by base material separately with use the DHPD of the said structure between base material that base material is contacted with each other once more to make relative to each other transposition of base material.

8. method as claimed in claim 7 is characterized in that R ₁And R ₂Be hydrogen.

9. method as claimed in claim 7 is characterized in that, one of DHPD part or another have following structure:

10. method as claimed in claim 7 is characterized in that, two DHPD have following structure:

11. binding agent that contains chemical compound shown in the formula (II):

In the formula, R ₁, R ₂With P such as claim 1 definition.

12. binding agent as claimed in claim 11 is characterized in that, described binding agent is bonding in aqueous environments.

13. binding agent as claimed in claim 11 is characterized in that, the chemical compound of described formula (II) is DOPA.

14. one kind contains formula (III) compound compositions:

Wherein, for each chemical compound of formula (1a), R ₁And R ₂Separately and independently such as claim 1 definition;

For each chemical compound of formula (1a), P ₁And P ₂Define as the P in the claim 1 separately and independently, n and m independently are 0 to about 5, and condition is that among m or the n is at least 1.

15. compositions as claimed in claim 14 is characterized in that, R ₁And R ₂All be hydrogen.

16. compositions as claimed in claim 14 is characterized in that, P ₁Or P ₂In at least one contain roughly poly-(alkylene oxide).

17. compositions as claimed in claim 14 is characterized in that, P ₁Or P ₂In at least one contain the unsaturated site of at least one thiazolinyl (ethlynic).

18. compositions as claimed in claim 14 is characterized in that, P ₁Or P ₂In at least one contain PEG.

19. compositions as claimed in claim 14 is characterized in that, (1a) at least one is three-DOPA, and P is PEG.

20. compositions as claimed in claim 14 is characterized in that, P ₁And P ₂Coupling each other.

21. the medical apparatus of a coating, it comprises:

The device and

Coating, described coating contain the described compositions of claim 14.

22. the medical apparatus of coating as claimed in claim 20 is characterized in that, described device is selected from support and pacemaker.

23. the medical apparatus of coating as claimed in claim 14 is characterized in that, described coating is biodegradable.

24. one kind prevents that cell or protein adherence to the method for operation cutting part, comprising the step that applies described position with the described compositions of claim 14.

25. one kind at the active force between hydrogen bond and covalent bond on the intensity, described active force is reversibly to form basically, rupture and form.

Images