微纳米多孔不锈钢表面高效吸附活性生物大分子

[Article]物理化学学报(Wuli Huaxue Xuebao )Acta Phys.⁃Chim.Sin .2013,29(7),1595-1602July Received:March 4,2013;Revised:May 8,2013;Published on Web:May 8,2013.∗Corresponding author.Email:xyang@;Tel:+86-10-82801539;Fax:+86-10-82802724.The project was supported by the National Natural Science Foundation of China (30770581,20971008)and Research Fund for the Doctoral Program of Higher Education,China (20090001110068).国家自然科学基金(30770581,20971008)和高等学校博士学科点专项科研基金(20090001110068)资助项目ⒸEditorial office of Acta Physico ⁃Chimica Sinicadoi:10.3866/PKU.WHXB201305082微纳米多孔不锈钢表面高效吸附活性生物大分子余占江1陈永强2杨晓达1,*(1北京大学药学院化学生物学系,天然和仿生药物国家重点实验室,北京100191;2乐普(北京)医疗器械股份有限公司,北京100022)摘要:不锈钢(AISI 316L)是目前在医药器械中应用最为广泛的商业化材料.下一代的不锈钢智能材料将特殊功能的生物活性分子(或纳米粒子)修饰在金属表面以模拟组织功能、提高生物/细胞相容性,这是目前材料科学研究的热点领域之一.本文研究了具有微纳米多孔表面结构的316L 不锈钢对抗体和生物酶分子的吸附作用,并与这些生物分子在光滑表面以及镀金表面的吸附进行了比较.研究发现不锈钢可通过简单的电化学腐蚀方法在表面产生微纳米多孔结构.微纳米孔不锈钢表面可稳定地吸附抗体或辣根过氧化物酶分子,其吸附量与喷镀金表面相当或更好.用表面活性剂(10%牛血清白蛋白(BSA)或0.2%Tween-20)洗涤不能除去吸附的蛋白.用5%Tween-20预处理金属表面,则可减少一半的抗体吸附量;但表面活性剂预处理对辣根过氧化物酶的吸附没有影响.吸附蛋白质后的金属表面湿润度大大增加;蛋白质修饰的微纳米孔不锈钢表面表现出了很好的亲水性(水接触角小于50°),指示了很好的生物相容性.而金属表面的湿润度则主要取决于蛋白质物种,并与蛋白质的吸附量正相关.吸附于不锈钢微纳米孔表面的抗体仍保持了良好的生物活性;用此种方式制备的抗CD34抗体修饰的不锈钢血管支架可以高密度并高选择性地吸附其目标细胞(如KG-1细胞).本文工作为未来制备新型的无高聚物涂层的不锈钢智能医学生物材料提供了基础.关键词:微纳米结构;不锈钢;抗体;辣根过氧化物酶;表面吸附中图分类号:O647Effective Adsorption of Functional Biological Macromolecules onStainless Steel Surface with Micro/Nanoporous TextureYU Zhan-Jiang 1CHEN Yong-Qiang 2YANG Xiao-Da 1,*(1State Key Laboratory of Natural and Biomimetic Drugs,Department of Chemical Biology,School of Pharmaceutical Sciences,Peking University,Beijing 100191,P .R.China ;2Lepu Medical Technology (Beijing)Co.,Ltd.,Beijing 100022,P .R.China )Abstract:Stainless steel (AISI 316L)is commonly used as a material in medical devices.Modification of the metal surface with bioactive molecules and/or nanoparticles to develop next-generation smart biomaterial, e.g.,cardiovascular stents,has recently attracted great attention.The present work investigated adsorption of antibodies and enzymes on micro/nanoporous 316L stainless steel compared with that on smooth and gold-coated stainless steel surfaces.The experimental results showed that the micro/nanoporous stainless steel surface produced by electrochemical erosion can adsorb a large amount of proteins (antibodies or horse radish peroxidase (HRP)),with comparable or better results than the sputtered-gold surface.Washes with surfactants (e.g.,10%bull serum albumin (BSA)or 0.2%Tween 20solution)did not remove the enzymes/antibodies.In contrast,pretreatment of the metal plates with 5%Tween 20halved antibody adsorption but did not affect adsorption of HRP.The wettability of the porous metal surface coated with proteins depended on the protein species and amount of protein adsorbed.The1595Acta Phys.⁃Chim.Sin.2013V ol.29protein-coated porous surface was hydrophilic(water contact angle<50°),which should make it biocompatible.The proteins on the micro/nanoporous stainless steel surface retained their high biological activity;in particular,micro/nanoporous stainless steel stents modified with an anti-CD34antibody usingthe present method can effectively and selectively capture KG-1cells.Our work provides a basis for developing novel polymer-free,smart,economic biomaterials with stainless steel for biomedical applications.Key Words:Micro/nano texture;Stainless steel;Antibody;Horse radish peroxidase;Surface adsorption1IntroductionIn the efforts for discovery of novel biomaterials in medical devices and implants,improving the biocompatibility and me-chanical performance has always been the two primary issues. The next generation of biomaterials has been proposed to be smart or biomimetic materials.A key challenge in designing smart biomaterials is to modify material surface with function-al biological or synthetic molecules/nanoparticals to mimic the extracellular matrix(ECM)of natural tissue.1Among biopolymers,alloys,and ceramics,stainless steel (AISI316L)is one of the most prominent available commer-cial materials for medical devices,2-4e.g.,cardiovascular stents, bone,and dental implants.The316L stainless steel exhibits long-standing performance and good biocompatibility,2which make stainless steel implants safe and efficient treatment op-tion over the much more expensive anecdotal superior titanium alloys.5One major limitation for stainless steel is the lack of chemi-cally active groups on the metal surface for covalently immobi-lization of functional molecules.A great deal of efforts have been made to engineer the metal surface with a variety of or-ganic and inorganic coatings,for instance,heparin hydrogel,6 carbohydrates,7polydopamine,8,9poly(ethylene glycol)and vari-ous hydrophilic polymers,10-12peptides and peptide nanofiber,13-16 polyelectrolyte micelles,17alkanethiol layers,18doped dia-mond-like carbon,19sputtered TiN/TiO2,20,21hydroxylapatite,22 hydrothermal calcium nanocomposites,23and S-phase layers,24 etc.On these bases,antibodies,vascular endothelial growth factor(VEGF),VE-cadherin,thrombin inhibitor,and lipo-somes were covalently attached to the surface of metal im-plants.These works improved greatly the cytocompatibility of the materials,for instance,the pro-healing approach immobi-lized antibodies capturing endothelial progenitor cells(EPC) from circulation on the blood contact surface of the stents;25the stents were shown to significantly reduce the thrombosis by fa-cilitating stent endothelialization.26,27However,the use of syn-thetic polymer matrixes was suggested negative for in-stent re-stenosis by some studies.25,28Surface modification at the nanoscale was suggested to pro-mote protein adsorption and cell adhesion.12,29-31Grafting the surface roughness and topography by electrochemical erosion29 or ultrafast laser irradiation32has been investigated to improve cytocompatibility.A functionalized TiO2nanonodule-in-micro-pit smart titanium surfaces was shown to enhance osteoblast proliferation and differentiation while the micropitted surface actually inhibited osteoblast growth.20Comparing with mir-ror-polished stainless steel surfaces,nanostructured surfaces showed better adhesion and differentiation for osteoblastic cells.29It was found that cells responsed to surface energy and three dimensional(3D)patterns.31The40-75nm nanopores on 316L stainless steel enhanced fibroblast cell proliferation and signal transduction while~200nm nanopore surfaces greatly attenuated.30However,the effects of micro/nano surface on ad-sorption of functional biological molecules for smart biomateri-al have so far not been well investigated.It has long been recognized that stainless steel surface can ir-reversibly adsorb proteins33-35but far less effective for function-al protein immobilization than some noble metals,e.g.,gold that is most frequently used for immobilization of biomole-cules.36,37Providing that micro/nano-structured surface of stain-less steel can effectively adsorb active biomacromolecules,a novel polymer-free smart metal platform for developing new biomaterials,e.g.,stents,would be achieved.For this purpose, the present work investigated adsorption of antibodies and en-zymes on micro/nanoporous316L stainless steel in comparison with smooth and gold-coating stainless steel surfaces.2Experimental2.1Materials316L stainless steel plates and stents were from Lepu Medi-cal Technology Co.,Ltd.(Beijing).Mouse monoclonal anti-body against human CD34was from Biolegend(USA),FITC-labeled goat anti-mouse monoclonal antibody(FITC-IgG)and horseradish peroxidase conjugated goat anti-mouse immuno-globulins(HRP-IgG)from BD Biosciences(USA).4ʹ,6-Di-amidino-2-phenylindole(DAPI,the purity≥99%),RPIM1640, 4ʹ,6-diamidino-2-phenylindole,diaminobenzidine(DAB,the purity≥99%)and tetramethylbenzidine(TMB,the purity≥99%) from Amresco(USA),L929fibroblast and CD34positive KG-1a cells were from American Type Culture Collection (ATCC,USA).Horse radish peroxidase(HRP,EC.1.11.1.7) and all other reagents of analytical grade were from Sigma-Aldrich(USA).2.2Preparation of316L stainless steel surface1596YU Zhan-Jiang et al.:Effective Adsorption of Functional Biological Macromolecules on Stainless Steel Surface No.7The polished316L stainless steel plates(1.0cm×1.0cm) were cleansed with20%hydrochloride acid for10h at room temperature and75%ethanol for15min at100kHz ultrasoni-cator,the plates were dried in a stream of filtrated air.To pro-duce a porous surface,the plates were acid-etched with10% hydrochloride acid for10min in the assistance of0.2A,500 Hz electric current.For gold coating,the plates were either sputter-coated with gold or incubated with5%H2AuCl4solu-tion for17h at37°C.All the treated metal plates were cleaned finally by75%ethanol as described above.Then the products were observed and analyzed with a S-4800scanning electron microscope(SEM,Hitachi,Japan).2.3Adsorption of anti-CD34antibodies or HRP onmetal surfaceThe metal plates were incubated for30min at37°C with anti-human CD34monoclonal antibody(0-200μg·mL-1)or HRP (0-0.5mg·mL-1)in0.1mmol·L-1of carbonate sodium buffer, pH9.6.Then the plates were washed three times with10 mmol·L-1phosphate buffered saline(PBS,pH7.4)containing 0.2%Tween-20.To investigate the effect of surfactant on protein adsorption, the cleaned plates were pre-incubated with0.5%-5% Tween-20before incubation with200μg·mL-1of monoclonal antibody against human CD34or0.5mg·mL-1of HRP as de-scribed above.2.4Analysis of protein adsorption on metal surface For analysis of the amount of HRP adsorbed on the metal surface,38the treated plate was put into a12-well cultural plate and then0.8mL of colorization solution containing0.1mmol·L-1of tetramethylbenzidine(TMB)was added and incubated for15min at37°C.The reaction was terminated with0.5mL of1mol·L-1H2SO4and the absorbance at450nm was mea-sured with a microplate reader(ASCENT,Labsystems Oy,Fin-land).For analysis of the amount of anti-CD34antibodies on the metal surface with an ELISA assay,the plates were first blocked with10%bovine serum albumin(BSA)in10mmol·L-1phosphate buffered saline(pH7.4)for24h at4°C,then in-cubated with HRP-conjugated goat anti-mouse antibody(BD Biosciences,USA)diluted(1:500)in10mmol·L-1PBS,pH 7.4for1h at37°C.After three-times washing,colorization with TMB substrate was conducted as described above.2.5Wettability assessmentFor comparison of the wettability,the contact angles for met-al plates were measured with an optical contact angle instru-ment(DSA100,Kruss Inc.,Germany).2.6In vitro cell capturing activity of anti-CD34antibodies-coated316L stainless steel stentsTo determine the specific activity of antibody coated on stainless steel surface,the in vitro cell capturing activity of mi-cro/nanoporous stents with or without antibody coating were incubated at37°C with cell suspension(1×106mL-1,either CD34+KG-1a cells or fibroblast L929cells)for1h.These cells were previously stained with50μg·mL-1DAPI fluores-cent dye as described in literature.39After rinsing three times with PBS,the stents were photographed and analyzed on a Flu-orescence Microscope equipped with an image analyzing pro-gram(BX41,OLYMPUS,Japan).2.7Statistical analysisAll results were expressed as mean±standard error of each sample.Each experiment was repeated independently three times.One-way ANOV A was conducted using an Origin TM8.0 program(Microcal,USA)for data comparison.A value of p< 0.05was considered significant.3Results3.1SEM observation on316L stainless steel surface The surface of various316L stainless steel plates were ob-served under a microscopy(Fig.1).Although sputter-gold plates showed yellow color,these plates exhibited a similar shining smooth surface to that of polished metal plate(Fig.1 (A,B)).Chemically deposited-gold plates(Fig.1(C))showed a tarnished but plainer surface with a lightly golden color when compared with the sputter-gold plates.In contrast,the porous plates by anodization treatment(Fig.1(D))showed a rough sur-face.When taking a closer look on SEM(Fig.2),the plates showed a microtexture that is full of irregular pores with an av-erage size of(400±160)nm.The surface roughness was esti-mated and the contour arithmetic mean deviations(R a)were 0.007,0.005,0.013,and0.033μm for polished stainless steel plate,sputter-gold plate,chemically deposited-gold plate and porous plate,respectively.3.2Protein adsorption on surface of metal plates For assessment of protein adsorption,an enzyme(HRP)and a mouse monoclonal antibody against human CD34were used as the representative of functional biologicalmacromolecules.Fig.1Microscopic images(30×)of surfaces of polished stainless steel plate(A),sputter-gold plate(B),chemically deposited-gold plate(C),and porous plate by anodization treatment(D)1597Acta Phys.⁃Chim.Sin.2013V ol.29The physical data of HRP and antibody are listed in Table1. Adsorption of HRP on various stainless steel plates were shown in Fig.3(A).The amount of HRP,expressed as enzymat-ic activity,increased with enzyme concentrations in solution. Polished plates and gold-coated plates exhibited similar extents of enzyme adsorption while porous plate adsorbed most amount of HRP than the other three plates.For antibody(monoclonal anti-CD34antibodies)adsorption (Fig.3(B)),polished stainless steel plates hardly adsorb antibod-ies.Chemically deposited-gold plates adsorbed a few with in-crease of antibody concentration in solution.The sputter-gold plates could most effectively adsorb antibody in a concentra-tion-dependent manner.The porous plates exhibited a similar capacity of antibody adsorption as the sputter-gold plate,how-ever,with a saturation concentration.The maximal amount of antibody adsorption on porous plates was calculated to be~1200ng·cm-2according to a calibration method described previously.383.3Effect of surfactant on protein adsorptionAs shown in Fig.4,pretreatment of metal plates by surfac-tant Tween-20could significantly reduce antibody adsorption almost by half;Herein,the porous plates exhibited a similar ef-fect with the sputter-gold plates.However,Tween-20did not affect adsorption of HRP on both metal surfaces.3.4Wettability of metal plates upon proteinadsorptionSurface wettability was thought to be one important factor tuning cell adhesion and protein adsorption31,40,41and also close-ly related with the adsorbed amount of proteins.42The results of water contact angles of the plates before and after protein ad-sorption were shown in Fig.4.It is noted that porous treatment and gold coating barely reduced the contact angles.However, adsorption of protein can significantly increase the wettability of the metal surface.Although on the porous plate,protein ad-sorption produced the best wetness surface,nevertheless,the difference between the plates was far less than that between the protein species.This indicated that change of surface wettabili-ty for metal plates is more dependent on the adsorbed protein.3.5Cell capture capacity of antibodies adsorbed tomicro/nanoporous316L stainless steel stents In smart biomaterials,antibodies are used to selectively at-tach target cells(e.g.,stem cell or progenitor cells)to form mi-metic tissue on the implants in situ.To test the efficiency and the selectivity of the antibodies immobilized,we tested thecell Fig.2Scanning electron microscope(SEM)images of the surface of porous stainless steel plate by anodization treatmentTable1Physical data of tested proteinsProteinhorse radish peroxidase(HRP)(EC.1.11.1.7) mouse monoclonal antibody(IgG)Molecular weight/kDa40(Ф~6nm)150(Ф~15nm)Isoelectric point(pI)7.28.0NotepI3-9for HRP isomersagainst CD34in presentworkFig.3Adsorption curves for HRP(A)and mouse monoclonal antibodies against CD34(B)on316L stainless steel platesvariously treated as described aboveData are average of triplicate independent measurements.p<0.01vs polished plate.OD:optical density1598YU Zhan-Jiang et al .:Effective Adsorption of Functional Biological Macromolecules on Stainless Steel SurfaceNo.7capture capacity of porous metal stents coated with anti-CD34mouse monoclonal antibodies.The results are shown in Fig.5and Fig.6.For CD34negative L929fibroblast cells,bare po-rous metal surface caught a few cells (~9844cells ·cm -2);while the antibody-coated metal surface held a little more (~11593cells ·cm -2),probably due to better wettability after antibody coating.For CD34positive KG-1a cells,bare porous metal sur-face caught much fewer amount (~3929cells ·cm -2)than L929cells.This is conceivable because fibroblasts usually have higher adhesive capacity.Remarkably,the antibody-coated metal surface caught almost ten folds of cells (~36256cells ·cm -2).These results indicated the antibody can remain high efficiency and specificity on the micro/nanoporoussur-4Effect of surfactant on antibody (A)and horse radish peroxidase (B)adsorption on porous andsputter-gold stainless steel platesFig.5Contact angles of various 316L stainless steel plates before and after adsorption of proteins (HRP or mouse monoclonalantibodies)Data are average of triplicate independentmeasurements.Fig.6Fluorescence microscopic images (40×)of CD34positive or negative cells adhesive to micro/nanoporous 316L stainless steel stents(A)CD34-L929fibroblast cells on bare surface;(B)CD34-L929fibroblast cells on antibody-coated surface;(C)CD34+KG-1a cells on bare surface;(D)CD34+KG-1a cells bare on antibody-coatedsurfaceActa Phys.⁃Chim.Sin.2013V ol.29face of stainless steel.4DiscussionFor effective immobilization of functional biological mole-cules on surface of biomaterials,the amount of biomolecules, the stability of immobilization,and the residue activity are the key concerns.Therefore,most methods use covalent bonds to attach biomolecules10,43,44and in this way modification of metal surface with organic polymers or inorganic particles with ac-tive groups(e.g.,―OH,―CHO,―COOH,―NH2,etc.) would be necessary.In the present work,we tested the efficiency of direct physi-cal adsorption of antibodies and enzymes on stainless steel sur-face by making use of micro/nano structures with an aim to de-velop novel smart metal implant,e.g.,prohealing stents.The experimental results indicate that the stainless steel with micro/ nano texture can high-efficiently adsorb biomacromolecules with desired biological activity.First,the micro/nanoporous stainless steel surface adsorbed high amount of proteins(Fig.3),which is close to(for antibod-ies)or even more(for HRP)than those attached to sputter-gold surface.Gold can form coordination bond with―SH of pro-teins or adsorb protein via strong van der Waals interactions.36 Gold surface is well-known in biomedical and bioanalytical ap-plications for immobilization of protein or even protein parti-cles.37Herein,the protein adsorption capacity of the porous stainless steel surface is shown to be at least comparable with the gold surface that is much more expensive.The reasons for high protein adsorption capacity for the po-rous stainless steel surface may lie on the followings:(i)the po-rous plates exhibited much rougher surface.The surface rough-ness is one key factor for molecular adsorption because the rough surface has bigger external surface area and pocket ef-fect supports more protein loading.45,46In fact,the amount of HRP adsorption was by and large with the surface roughness (Fig.3(A));(ii)formation of protein multilayers by surface-in-duced aggregation as observed previously on stainless steel mi-croparticles;34,47(iii)the monoclonal antibody showed different adsorption profile from that of HRP.The molecular size of monoclonal antibody(~15nm)is larger than that of HRP(~6 nm);however,considering the pore size(~400nm)of the met-al plate,this size difference is too small to explain the adsorp-tion profile.The mechanism of interaction between protein molecules with porous surface is worthwhile to be investigated further.Second,the protein attached on the porous stainless steel sur-face is stable.The experimental results showed that adsorption treatment with10%BSA or0.2%Tween-20solution could not remove the enzymes/antibodies from the metal plate,which agrees with that stainless steel-protein interaction is strong48 and protein adsorption to stainless steel could be irreversible.34,47 While pre-treatment with Tween-20buffer could reduce anti-body adsorption by half(Fig.4(A))but had no effects on HRP adsorption,possibly because of the interaction between the an-tibody and surfactant.The wettability of the stainless steel has been proposed to be a predominant mechanism governing both protein adsorption and cell adhesion.40,41As shown in Fig.5,there was a great re-duce of water contact angles upon antibody or HRP adsorption, indicating significant reduce of surface Gibbs free energy and suggesting a highly spontaneous and strong adsorption of anti-body/HRP protein onto the surface like fibrinogen.49Several points are worthwhile to note here:(1)the wettability of HRP coated surfaces are much higher than that of antibody-coated surface,indicating that the wettability of protein-modified met-al surface is primarily dependent on the properties of the pro-tein rather than the nature of metal.Similar adsorption behav-ior of proteins at stainless steel-liquid interfaces has been ob-served previously;33(2)although the surface of gold-modified plates adsorbed more proteins than the stainless steel plates, however,the wettability of gold surface is apparently less. Since surface wettability is correlated to the transition of sur-face cytocompatibility from cell-phobic to cell-philic,31,41this result may suggest that the protein-engineered stainless steel surface could be better than gold for medical implants;(3)for the same type of metal surfaces,higher amount of protein ad-sorption gave higher wettability,which is consistent with previ-ous observation.42Third,many works have shown that due to the strong sur-face interaction,adsorption of protein(e.g.,fibrinogen and BSA)on316L stainless steel could result in partial unfolding of proteins and significant changes in the secondary structure that occur predominantly within the first minute of adsorp-tion.46,49This raises a question of whether the proteins directly on the metal surface can keep their biological activity and spec-ificity.Fortunately,we observed that both HRP enzyme and anti-bodies retained high activity on the porous metal surface.Espe-cially,as shown in Figs.6and7,stainless steel stents with micro/Fig.7Quantatification of adhesion of CD34positive(KG-1a)or negative(L-929)cells to bare or antibody-coated micro/nanoporous316L stainless steel stentsEach counting averaged five randomly selected visual fields.Data are average of triplicate independentmeasurements.YU Zhan-Jiang et al.:Effective Adsorption of Functional Biological Macromolecules on Stainless Steel Surface No.7nanoporous surface coated with an anti-CD34antibody can capture the target cells with both high efficiency and high spec-ificity,which shall allow the development of novel poly-mer-free and economic smart biomaterials with stainless steel by direct protein adsorption on micro/nanoporous surface.The reasons for antibodies and HRP to keep their specific ac-tivity remain further investigated.However,several possibili-ties may include:(1)unlike fibrinogen and BSA,HRP and anti-bodies are rigid global proteins and thus resist to conformation-al change;(2)the surface pocket of the porous metal may ac-commodate the enzyme/antibody molecules in favorable states similarly to the case of protease on microporous zeolite MCM-22;45(3)the proteins may form multilayers on the metal surface.Then the proteins in the upper layers may be less influ-enced by surface forces and then keep a full activity.Nonethe-less,in the future studies,systematic exploration to the roles of 3D micro/nano morphology of metal surface on immobiliza-tion of a variety of biological macromolecules and the effects on their structure and biological functions are envisaged.5ConclusionsIn summary,the present work investigated adsorption of two important functional biomolecules,i.e.,monoclonal antibodies and HRP enzymes,on micro/nanoporous316L stainless steel in comparison with smooth and gold-coating stainless steel sur-faces.Our results indicate that antibodies and enzymes can be loaded firmly on the micro/nanoporous surface in a large amount and these proteins retained high biological activity.In addition,the porous 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