Preparation of functional coating on magnesium alloy with hydrophilic polymers and bioactive peptides for improved corrosion resistance and biocompatibility

Lingchuang Bai,Yahui Wang,Lan Chen,Jun Wang,Jingan Li,Shijie Zhu,Liguo Wang,Shaokang Guan,c,*

a School of Materials Science and Engineering,Zhengzhou University,Zhengzhou 450001,China

b Henan Key Laboratory of Advanced Magnesium Alloys,Zhengzhou 450002,China

c Key Laboratory of Materials Processing and Mold Technology(Ministry of Education),Zhengzhou 450002,China

Abstract Biodegradable magnesium alloy stents(MAS)have great potential in the treatment of cardiovascular diseases.However,too fast degradation and the poor biocompatibility are still two key problems for the clinical utility of MAS.In the present work,a functional coating composed of hydrophilic polymers and bioactive peptides was constructed on magnesium alloy to improve its corrosion resistance and biocompatibility in vitro and in vivo.Mg-Zn-Y-Nd(ZE21B)alloy modified with the functional coating exhibited moderate surface hydrophilicity and enhanced corrosion resistance.The favourable hemocompatibility of ZE21B alloy with the functional coating was confirmed by the in vitro blood experiments.Moreover,the modified ZE21B alloy could selectively promote the adhesion,proliferation,and migration of endothelial cells(ECs),but suppress these behaviors of smooth muscle cells(SMCs).Furthermore,the modified ZE21B alloy wires could alleviate intimal hyperplasia,enhance corrosion resistance and re-endothelialization in vivo transplantation experiment.These results collectively demonstrated that the functional coating improved the corrosion resistance and biocompatibility of ZE21B alloy.This functional coating provides new insight into the design and development of novel biodegradable stents for biomedical engineering.

Keywords:Magnesium alloy stent;Functional coating;Corrosion resistance;Biocompatibility;Hemocompatibility;Endothelialization.

1.Introduction

Cardiovascular diseases seriously threaten human life and health.Vascular stent implantation is an important method for the clinical treatment of cardiovascular diseases[1].The currently used vascular stents are mostly permanent metal stents,for example,316 L stainless steel,cobalt-chromium alloy,titanium alloy[2].After completing the supporting role,these non-degradable stents remain in the human body for a lifetime,and this may cause vascular dysfunction,local chronic inflammation,neointimal hyperplasia,and eventually in-stent restenosis.New-type fully biodegradable vascular stents(e.g.,magnesium alloy stents(MAS))will degrade and disappear after completing the treatment process and can overcome the abovementioned problems for non-degradable stents[3].However,the clinical use of MAS still faces two critical challenges:too fast degradation to achieve the treatment purpose and too poor biocompatibility to promote rapid endothelialization.Given the challenges,surface coating/modification is regarded as an effective strategy to simultaneously improve the corrosion resistance and biocompatibility of MAS.

Surface coating/modification can protect magnesium alloy from fast corrosion and endow it with diverse biological functions[4].Micro arc oxidation coating,degradable polymer coating,bio-ceramic coating,and chemical conversion layer were often applied to strengthen the corrosion resistance of magnesium alloy[5–8].Among these coatings,the chemical conversion layer,especially the MgF2conversion layer,is easier to form a dense and uniform coating structure,and has a strong bonding force with the substrate[9].Therefore,the chemical conversion layer is more suitable to enhance the anti-corrosion performance of MAS due to its convenience and efficiency.

In addition to improving the corrosion resistance,the coatings on MAS also need some necessary biological functions,such as improving hemocompatibility,inhibiting neointimal hyperplasia,and promoting rapid endothelialization.However,a single chemical conversion layer cannot provide the corresponding biological functions and lacks functional groups for further modification.Recently,it was reported that dopamine can be copolymerized with hexamethylenediamine to form an amine-rich coating[10],which provided a method for further modification of Mg alloy stents.Nevertheless,this amine-rich coating can readily interact with blood components and induce thrombosis because of its electropositivity and adhesive capacity.Heparin,hyaluronic acid,albumin,and hydrophilic polymers can be used to neutralize and shield the excessive positive charges in the amine-rich coating[10–13].Polyethylene glycol(PEG)as a kind of hydrophilic polymer with low immunogenicity and low toxicity was widely used in surface modification and drug delivery systems to enhance hemocompatibility and long circulation[14,15].Biomaterials modified with PEG usually possess too hydrophilic surface to profitably support vascular cells adhesion and growth,thus likely leading to delayed endothelialization.Besides improving hemocompatibility,achieving rapid endothelialization is of great significance for vascular stents[15].

The continuous functional endothelium on vascular grafts can not only suppress thrombus formation but also regulate the behaviors of smooth muscle cells(SMCs)and avoid intimal hyperplasia.Surface modification with proendothelialization biomolecules is beneficial for the formation of functional endothelium on vascular stents.Antibodies,aptamers,genes,and bioactive peptides were widely studied to promote the rapid endothelialization of vascular stents[16–20].Among these biomolecules,bioactive peptides draw more and more attention due to their high efficiency and stable bioactivity.Arg-Glu-Asp-Val(REDV)peptides derived from fibronectin in the extracellular matrix have been proven to specifically recognize and adhere endothelial cells(ECs)viathe ligand-integrin interaction,and simultaneously suppress SMCs adhesion[21,22].In addition,MAS also need to have anticoagulant ability to avoid the use of antiplatelet or anticoagulant drugs.ACH11peptides with LTFPRIVFVLG amino acid sequence have been confirmed with the potent inhibition effect on the activation of coagulation factor X(FXa)in the coagulation cascade and can be used as an anticoagulant agent to impede blood coagulation as well as enhance blood compatibility of vascular grafts[23,24].Therefore,combining the MgF2conversion layer,hydrophilic polymer coating,ECsselective REDV peptides,and anticoagulant ACH11peptides into one functional coating is a promising approach to simultaneously improve the corrosion resistance and biocompatibility of MAS.To our knowledge,such a functional coating developed on MAS has never been reported.It is expected that the functional coating can systematically enhance the anticorrosion capacity,hemocompatibility,anti-hyperplasia ability,and rapid endothelialization of MASin vitroandin vivo.

To solve the problems of too fast degradation and too poor biocompatibility for MAS,a novel functional coating was developed with MgF2conversion layer,hydrophilic polymers coating,REDV peptides,and ACH11peptides.In this functional coating,the MgF2conversion layer and hydrophilic polymers coating can synergistically protect Mg alloy from fast corrosion.Meanwhile,REDV peptides can promote rapid endothelialization by regulating the behaviors of ECs and SMCs,and ACH11peptides can strengthen the anticoagulant ability by inhibiting the FXa activation.In the present study,Mg-Zn-Y-Nd(ZE21B)alloy was firstly passivated with hydrofluoric acid to form a protective MgF2layer and then deposited in the mixture solution of dopamine hydrochloride and hexamethylenediamine to obtain the amine-rich coating,followed by PEG grafting and bioactive peptides immobilization.Herein,surface chemical structure and composition,micromorphology,wettability,and corrosion resistance were characterized to assess the performance of the functional coating.Hemolysis,fibrinogen adsorption/denaturation,and activated FXa assays were performed to evaluate the hemocompatibility of modified ZE21B alloy.The growth behaviors of ECs and SMCs on modified ZE21B alloy were investigated to verify the cytocompatibility.Rabbit carotid artery implantation experiment was finally performed to study thein vivocorrosion resistance,anti-hyperplasia,and pro-endothelialization of ZE21B alloy with the functional coating.

2.Materials and methods

2.1.Materials

The as-extruded Mg-Zn-Y-Nd(ZE21B,2.00wt% Zn,0.46wt% Y,0.50wt% Nd)alloy was specially designed for vascular stent application and produced in our lab(Henan Key Laboratory of Advanced Magnesium Alloy,Zhengzhou,China)[25–29].Briefly,the as-cast magnesium alloy was first fabricated through the alloy melting technology,and then it was hot extruded under specific extrusion temperature(300 °C)and extrusion ratio(17.4)conditions to obtain the as-extruded ZE21B alloy.Hydrofluoric acid(HF)was bought from Luoyang Haohua Reagent Co.,Ltd.,China.Anhydrous ethanol,methanol,and dimethyl sulfoxide(DMSO)were purchased from Concord Technology Co.,Ltd.(Tianjin,China).Dopamine hydrochloride(DA)and hexamethylenediamine(HDA)were obtained from Sigma-Aldrich.Orthopyridyl disulfide-PEG-N-hydroxysuccinimide(OPSS-PEG-NHS,Mn=2294 Da,99%)was obtained from JemKem Technology Co.,Ltd.(Beijing,China).Cys-Arg-Glu-Asp-Val-Trp(CREDVW)and Cys-Gly-Gly-Leu-Thr-Phe-Pro-Arg-Ile-Val-Phe-Val-Leu-Gly(CGG-LTFPRIVFVLG,CGG-ACH11)peptides were supplied by GL Biochem Ltd.(Shanghai,China).Human umbilical vein endothelial cells(HUVECs)and Human umbilical artery smooth muscle cells(HASMCs)were kindly provided by Cell Bank,Chinese Academy of Sciences,Shanghai,China.Nitric oxide(NO)detection kit,cell counting kit-8(CCK-8),and hemotoxylin-eosin(HE)staining kit were bought from Beyotime Biotechnology Ltd.(Shanghai,China).FITC-phalloidine and 4′,6-diamidino-2-phenylindole(DAPI)were purchased from Solarbio Co.,Ltd.(Shanghai,China).CD31 antibody was obtained from Abcam(HK)Ltd.

2.2.Preparation of ZE21B alloy with functional coatings

ZE21B alloy discs(Φ10 mm×H 3 mm)were first polished with metallographic sandpaper(200–1000 meshes).Afterward,the ZE21B alloy discs were thoroughly washed with acetone and anhydrous ethanol.The polished ZE21B alloy discs were immersed in HF solution(40%,v/v)at room temperature for 48 h to obtain a protective MgF2layer.The HF-treated samples were denoted as ZF.The ZF samples were rinsed with deionized water(dH2O)and dried for further use.To prepare amine-rich coating through the copolymerization of DA and HDA,both DA(2 mg/mL)and HDA(2.5 mg/mL)were dissolved in Tris–HCl buffer solution(10 mM,pH=8.5)and then the ZF samples were deposited in this solution at 25 °C for 24 h.The DA/HDA-pretreated ZF samples were named ZFD.For polyethylene glycol(PEG)grafting,the OPSS-PEG-NHS(150 μM)was first dissolved in phosphate buffer solution(PBS,pH=7.4)and DMSO mixed solution(97.5/2.5,v/v),and then the ZFD samples were immersed in this solution at 40 °C for 6 h.PEG was covalently grafted onto the ZFD surface through the reaction between the NHS group on OPSS-PEG-NHS and the amine group on ZFD.The PEG-grafted ZFD samples were marked as ZFDP.

For peptide immobilization,CREDVW(30 μM)and CGGACH11(30 μM)peptides were dissolved in Tris–HCl buffer solution(10 mM,pH=8.5)and methanol,respectively,and then the ZFDP samples were immersed into 3 mL of the corresponding peptide solution at 40 °C for 4 h.The bioactive peptides were chemically immobilized onto the ZFDP surfaceviathe reaction between thiol in the peptides and OPSS group in OPSS-PEG-NHS.The tryptophan(W)residue in CREDVW and the phenylalanine(F)residue in CGG-ACH11were used as fluorophores to determine the grafting densities of CREDVW and CGG-ACH11peptides on the sample surfaceviaa fluorescence spectrophotometer(FLS1000,Edinburgh,UK).The CREDVW,CGG-ACH11,and CREDVW/CGG-ACH11grafted ZFDP samples were labeled as ZFDPR,ZFDPA and ZFDPRA,respectively.

2.3.Characterization of modified ZE21B alloy with functional coatings

The surface chemical composition and structure of the modified ZE21B alloy samples were investigated by X-ray photoelectron spectroscopy(XPS,SHIMADZU AXIS Supra,UK)and attenuated total reflection Fourier transform infrared spectroscopy(ATR-FTIR,NICOLET iS10,USA).The XPS instrument was equipped with a Al KαX-ray source and operated at 5×10-10Torr.The survey scans and high-resolution scans were performed in steps of 1.0 eV and 0.1 eV,respectively.The FTIR spectra were recorded in the range of 4000–600 cm-1with a scan resolution of 2 cm-1by taking 32 scans.The micromorphology of the sample surface after the gold spray treatment was observed at the accelerating voltage of 5 kV by a focused ion beam scanning electron microscope(FIB-SEM,Zeiss Auriga,DE).The wettability of the sample surface was measured using a Krüss Easy Drop goniometer(Hamburg,DE).The droplet volume was set as 3 μL and five measurements were conducted to obtain the average water contact angle value.The corrosion resistance of the modified sample surface in Hank’s solution was studied using an electrochemical method through a three-electrode electrochemical system(RST5000,China).The auxiliary electrode,reference electrode and working electrode were platinum electrode,saturated calomel electrode(SCE)and the sample,respectively.The potentiodynamic polarization curves were conducted from-2.0 V to-1.0 V(vs.SCE)at a scanning rate of 0.001 V/s.The corrosion current density was fitted from the polarization curve detected by the three electrochemical systems.

2.4.In vitro hemocompatibility

The hemocompatibility of unmodified and modified samples,namely,ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA,and ZFDPRA,was evaluated by hemolysis tests,fibrinogen adsorption/denaturation measurements,and activated coagulation factor Xa(FXa)quantitative tests.The detailed experimental procedures were given in supporting information(SI).Fresh human whole blood was supplied by the Affiliated Hospital of Zhengzhou University.Platelet-rich plasma and platelet-poor plasma were prepared from fresh blood at 1000 rpm and 3000 rpm for 15 min,respectively.

2.5.In vitro cell experiments

Thein vitrocell behaviors of HUVECs and HASMCs,i.e.,the release of nitric oxide(NO),cell proliferation and migration,ECs/SMCs competitive adhesion,on biofunctionalized sample surfaces were systematically studied.The detailed experimental processes were given in SI.HUVECs and HASMCs were cultured with DMEM supplemented with 10%FBS at 37 °C in a humidified atmosphere with 5% CO2.The cultured HUVECs and HASMCs between the third and sixth passage after 80% confluence were chosen for cell experiments.

2.6.In vivo implantation

The animal experiments were conducted following the protocol approved by the Ethics Review Committee of Life Science of Zhengzhou University(Zhengzhou,China)and con-formed to the“Guide for the Protection and Use of Experimental Animals”of the American National Institutes of Health.Thein vivocorrosion resistance,neointimal hyperplasia,and endothelialization of ZE21B and ZFDPRA wires(Φ0.5 mm×H 10 mm)were evaluated byin vivoimplantation.Eight male New Zealand white rabbits(weight,2–2.5 kg)were used forin vivoimplantation.After the isolation of blood vessels under anesthetized conditions,the ZE21B and ZFDPRA wires were implanted into rabbits’right carotid arteries.After implantation for 1 month,the sample with surrounding vessels was isolated and fixed in 4% formaldehyde for further analyses.The fixed vessel tissues were dehydrated,embedded,sectioned,and stained with HE and CD31 antibody.

2.7.Statistical analysis

In this study,all assays were performed at least three samples,and all data were expressed as mean±SD(standard deviation).Statistical analysis was performed using one-way analysis of variance(ANOVA)through SPSS software.Pvalue less than 0.05 indicated statistically significant differences.

3.Results

3.1.Preparation of functional coating on ZE21B alloy surface

To especially improve the corrosion resistance and biocompatibility of ZE21B alloyin vitroandin vivo,the multifunctional coating on the ZE21B alloy surface was prepared from the MgF2conversion layer,hydrophilic polymer layer,and two bioactive peptides(REDV and ACH11).The abbreviations used in this study were listed in Table 1.The preparation process of multifunctional coating on the ZE21B alloy surface was presented in Scheme 1.The anti-corrosion property and biological performance of modified ZE21B alloy samples were systemically evaluatedvia in vitroandin vivoexperiments.

Table 1List of the abbreviations used in this paper.

Scheme 1.(A)Preparation process of the functional coating on ZE21B alloy surface.(B)ZFDPRA wires with functional coating used in rabbit carotid artery implantation.

The chemical structures of modified ZE21B surfaces were characterized by ATR-FTIR.ATR-FTIR spectra of ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA,and ZFDPRA were shown in Fig.1.No obvious characteristic peaks were found on ZE21B and ZF sample surfaces.This was because there were no organic compounds on the sample surfaces.To prepare the amine-rich coating,DA and HDA were deposited on the sample surfaceviathe copolymerization reaction.Compared to ZE21B and ZF samples,the new characteristic peaks occurred on the ZFD surface.For the ZFD sample,the adsorption signal at 3342 cm-1belonged to the N–H bonds(-NH-,-NH2),and correspondingly,the signal at 712 cm-1resulted from the deformation vibration of N–H bonds.The adsorption signal at 1327 cm-1resulted from the C–N(-CNH2)stretching vibrations.The signals at 2930 cm-1and 2860 cm-1were attributed to the stretching vibration of saturated C–H bonds in the methyl(-CH3)and methylene(-CH2-),respectively.The characteristic peaks at 1637 cm-1and 1398 cm-1were ascribed to carbonyl(C=O)and the skeleton vibration of benzene,respectively.After PEG grafting,the signals at 1080 cm-1became strong and broad because many C–O—C bonds were introduced onto the ZFDP sample surface.For peptide-immobilized groups,the peptide bonds(-CO–NH-)were the main characterstic peaks.But because these signals produced by REDV and ACH11peptides were overlapped by the precoating on ZFDP sample,it was difficult to distinguish the corresponding peaks belonging to the peptides.

The detailed chemical composition of functional coatings on modified ZE21B samples was analyzed by XPS.XPS survey spectra,S 2p high-resolution spectra,N 1s high-resolution spectra,and O 1s high-resolution spectra of ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA,and ZFDPRA were presented in Fig.2a,Fig.2b,Fig.2c,and Fig.2d.The major peaks of Mg 2s,F 1s,C 1s,N 1s,O 1s,and S 2p indicated Mg,F,C,N,O,and S elements,respectively,were the six main elements on the sample surfaces.Besides the peaks of the Mg element,there was no other obvious characteristic peak for the ZE21B sample.After HF treatment,the peak of F 1s at 683 eV was easily observed because of the MgF2conversion layer.Compared with the ZF sample,the newly appeared peak of N 1s at 396.5 eV indicated the successful preparation of amine-rich coating on the ZFD sample,because DA and HDA contained nitrogen element.Different from ZE21B,ZF,and ZFD samples,the peak of S 2p at 164.2 eV was found in the spectra of ZFDP,ZFDPR,ZFDPA,and ZFDPRA(Fig.2b),because both the heterobifunctional PEG and bioactive peptides contained sulfur element.Moreover,the C/N molar ratios(figures not shown)of ZFDP,ZFDPR,ZFDPA,and ZFDPRA were 2.06,1.59,1.71,and 1.86,respectively.After peptide conjugation,it was evident that the nitrogen content increased and the C/N molar ratios decreased.This can be attributed to the relatively high nitrogen content of REDV and ACH11peptides.The changes in C/N molar ratios confirmed the immobilization of REDV and ACH11peptides.Furthermore,the grafting densities of REDV,ACH11,and REDV/ACH11on ZFDPR,ZFDPA,and ZFDPRA were 27.1,24.6,and 12.5/10.8 nmol/cm2,respectively,determined by a fluorescence quantitative assay.Collectively,the functional coatings on ZE21B samples were successfully prepared with PEG,REDV,and ACH11peptides.

3.2.SEM observation

The micromorphology of materials influences the physicochemical property and cell behavior[30].The surface micromorphologies of ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA,and ZFDPRA samples were observed using SEM.The representative micrographs of each sample were shown in Fig.2e.Except for a few scratches caused by polishing,the ZE21B sample exhibited a smooth and even surface.For the ZE21B sample,there were obvious differences before and after HF treatment.It was visible that the ZE21B sample surface changed from silver-gray to black after fluorination treatment.A dense and grainy layer composed of MgF2could be observed under SEM with high magnification(Fig.2e).For the ZFD sample,lots of nano/microparticles were found on the surface due to the copolymerization of DA and HDA.Both the MgF2conversion layer and nano/microparticles layer could protect ZE21B samples from too fast corrosion.These findings confirmed the successful fabrication of the MgF2layer and amine-coating on ZE21B samples at a microscopic level.Moreover,there were no significant differences in size and morphology between ZFDP,ZFDPR,ZFDPA,ZFDPRA,and ZFD samples.The possible reason for this was that the PEG and peptides with too small size were directly covalently attached to the coating surface,and did not cause a change in the topography.

Fig.1.ATR-FTIR spectra of ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA and ZFDPRA.

3.3.Surface wettability

The surface wettability can influence the interaction between materials and blood/cells,especially for bloodcontacting materials[31].Compared to hydrophobic surfaces,hydrophilic surfaces can significantly reduce the friction between the cardiovascular device and the vessel wall,and benefit the adhesion,spreading and proliferation of vascular cells.The surface wettability of modified ZE21B samples was assessed by water contact angle measurements,and the results were shown in Fig.2f.ZE21B and ZF samples with a relatively low water contact angle showed good surface wettability.For other groups,the values of water contact angle ranged from 50° to 64°,resulting from the coatings composed of hydrophilic polymers and peptides.The values of water contact angle for ZFDPR,ZFDPA,and ZFDPRA groups were 50.3±4.0°,60.7±3.0°,and 56.4±3.7°,respectively.These groups with relatively higher water contact angles were possibly beneficial for further cell adhesion and migration.

3.4.Electrochemical test

Too fast degradation is a key problem for MAS in clinical application[32].The polarization curves(Fig.2g)of modified ZE21B with functional coating were measuredviaa three-electrochemical system,and the corrosion current density(Icorr)was determined from the polarization curves.It is generally considered that the lowIcorrvalue signifies high corrosion resistance.Compared with ZE21B group,all other groups exhibited relatively highIcorrvalues,indicating enhanced corrosion resistance.This was mainly because the functional coatings on the ZE21B samples could prevent the penetration of aggressive ion as a physical shielding.TheIcorrvalue of the ZFDPRA group(0.40 μA/cm2)was about 60%lower than that of ZE21B group(1.01 μA/cm2).According to theIcorr,the corrosion rates of ZE21B and ZFDPRA were 0.024 mm/year and 0.010 mm/year,respectively.The functional coatings can significantly reduce the corrosion rate of ZE21B alloy and benefit for the mechanical support of MAS during the follow-up period.The excellent corrosion resistance could ensure that the ZFDPRA used as vascular stents served well during the follow-up period.

3.5.Hemolysis rate

Hemolysis rate is an important indicator to judge the hemocompatibility of blood-contacting materials[33].For vascular stents,the hemolysis rate within an acceptable range is a prerequisite for clinical use.In this study,the hemolysis rates of ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA,and ZFDPRA were determined by hemolysis tests under static condition,and the results were presented in Fig.3a.The ZE21B,ZF,and ZFD groups could result in evident hemolysis,and their hemolysis rates were all above 8.0%.After PEG grafting,the hemolysis rate of the ZFDP group was reduced to 4.1±0.4%.It was likely due to the excellent blood compatibility of PEG and the shielding effect of PEG on abundant positive charges resulting from the amine-rich coating.Furthermore,no significant change in hemolysis rate was found after REDV and ACH11immobilization.The hemolysis rates of ZFDPR,ZFDPA,and ZFDPRA were 3.5±0.4%,4.5±0.5%,and 3.8±0.3%,respectively,which can meet the requirements of clinical use(below 5%).

3.6.Coagulation factor(FXa)generation

The coagulation factor Xa(FXa)is a key protease in the coagulation cascade,and plays an important role in the coagulation process[34].ACH11peptides possess the function of inhibiting the activated FXa,and their anticoagulant bioactivity in functional coatings was evaluated by an ELISA method.As shown in Fig.3b,compared with ZE21B,ZF,and ZFD groups,FXa concentrations for the other groups were significantly decreased at 5 min and 30 min.Especially,FXa concentrations for ZFDPA and ZFDPRA groups at 5 min and 30 min were the lowest among all groups,and reached 1.25±0.04 ng/mL and 1.37±0.06 ng/mL at 30 min,respectively.These findings verified that ACH11peptides restrained the FXa activation and exerted the anticoagulant bioactivity in functional coatings.

3.7.Fibrinogen adsorption and denaturation

Fig.2.(a)XPS survey spectra,(b)XPS S 2p high-resolution spectra,(c)XPS N 1s high-resolution spectra,(d)XPS O 1s high-resolution spectra,(e)representative SEM micrographs of surface morphology,(f)water contact angle,and(g)polarization curves of(A)ZE21B,(B)ZF,(C)ZFD,(D)ZFDP,(E)ZFDPR,(F)ZFDPA,and(G)ZFDPRA.(Error bars represent mean±SD.).

Fig.3.(a)Hemolysis rate,(b)activated FXa concentration,(c)fibrinogen adsorption,and(d)fibrinogen denaturation of(A)ZE21B,(B)ZF,(C)ZFD,(D)ZFDP,(E)ZFDPR,(F)ZFDPA,and(G)ZFDPRA.The hemolysis rates determined by using ultrapure water and physiological saline(0.9% w/w)groups as controls in the presence of samples after 60 min incubation at 37 °C.(mean±SD,*p<0.05 denotes statistically difference between pair.).

Plasma proteins are prone to adhere to the surface of a vascular stent,and the adsorbed proteins can further induce blood clotting and thrombosis[35].Fibrinogen was selected as the model protein to study the protein adsorption and denaturation of modified ZE21B surfaces,and the results were shown in Fig.3c and Fig.3d.The ZE21B,ZF,and ZFD groups exhibited a relatively high amount of fibrinogen adsorption.For the ZFD group,the high fibrinogen adsorption could be attributed to the adhesion effect of polydopamine.The ZFDP group showed the lowest fibrinogen adsorption among all groups because PEG could form a hydration layer to inhibit non-specific protein adsorption.The groups modified with REDV and ACH11peptides displayed a slight increase in fibrinogen adsorption,but the adsorption amount was still at a low level.Different from the fibrinogen adsorption,the amount of fibrinogen denaturation for the ZF and ZFD groups was higher than that of the ZE21B group.The fibrinogen denaturation of ZFDPR,ZFDPA,and ZFDPRA was still at a lower level,contributed by the PEG and bioactive peptides in functional coatings.

3.8.HUVECs adhesion and proliferation

The adhesion and proliferation behaviors of HUVECs on modified ZE21B surfaces after 1 and 3 days of culture were investigated using a FITC-phalloidine/DAPI staining assay.The cytoskeletons and nuclei of HUVECs after 1 and 3 days of culture were severally stained with green and blue,and the representative fluorescence micrographs were shown in Fig.4a.The number of HUVECs on each sample showed a gradual increase along with culture time.After 1 day of cell culture,almost all HUVECs on the modified ZE21B groups except for the ZFDPRA group displayed elliptical morphology,suggesting that the cells were not fully spread.In contrast,the cytoskeletons of fully spread HUVECs on the ZFDPRA sample can be intuitively and clearly observed.With the culture time extended to 3 days,HUVECs on all groups except for ZE21B group spread well,indicating the good interaction between the cells and the functional coatings on sample surfaces.Furthermore,it was found that the number of HUVECs on modified ZE21B surfaces showed a significant increase.It was noteworthy that HUVECs on the ZFDPRA surface can quickly spread,grow,migrate,and almost completely cover the sample surface after incubation for 3 days.

The proliferation of HUVECs on modified ZE21B surfaces was determined by CCK-8 assays,and the results were shown in Fig.4b.The number of HUVECs can be intuitively reflected by the optical density determined by CCK-8 assays.The cell proliferation results were consistent with those of the fluorescence staining.For ZFDPR,ZFDPA,and ZFDPRA groups,no significant differences in the number of HUVECs were found after 1 day of culture.The number of HUVECs on all modified ZE21B surfaces manifested an obvious increase,and the HUVECs number for each group was different after 3 days of culture.The number of HUVECs cultured on ZFDPR,ZFDPA,and ZFDPRA groups was distinctly higher than other groups.The possible reason for this was that the functional coating composed of PEG and bioactive peptides can provide a moderate wettability and abundant ECs binding site.These results demonstrated that the modified ZE21B surfaces with PEG,REDV,and ACH11can well support HUVECs adhesion and proliferation.

Fig.4.(a)Fluorescence micrographs of HUVECs stained with FITC-phalloidine and DAPI on different sample surfaces after 1 and 3 days of culture.(A)ZE21B,(B)ZF,(C)ZFD,(D)ZFDP,(E)ZFDPR,(F)ZFDPA and(G)ZFDPRA.(b)Proliferation of HUVECs cultured on different sample surfaces for 1 and 3 days(using CCK-8 kit).(mean±SD,*p<0.05 denotes statistically difference between pair.)(For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.).

Fig.5.NO release levels of HUVECs on ZE21B,ZF,ZFD,ZFDP,ZFDPR,ZFDPA and ZFDPRA samples after 1 and 3 days of culture(using NO detection assay kit).(mean±SD,*p<0.05 denotes statistically difference between pair.).

3.9.NO release

NO released from healthy ECs as a signal molecule can modulate the behaviors of vascular cells and accelerate endothelial repair[36].The amount of NO released from HUVECs on each sample surface was determined to evaluate ECs behaviors,and the results were shown in Fig.5.At the same time point(1 day and 3 days),the trend of NO release amount was consistent.Compared with other groups,the NO release amounts of ZE21B and ZF groups were low.Meanwhile,the ZFDPRA group exhibited the highest level of NO release at 1 day and 3 days(28.2±0.7 μmol/L and 34.1±0.8 μmol/L,respectively),the increase of NO release during the 48 h reached 5.9 μmol/L.This indicated that the functional coating on the modified ZE21B surface can provide a better microenvironment for the healthy growth of HUVECs.

Fig.6.(a)Optical micrographs of migration process of HUVECs cultured on different sample surfaces for 0,12 and 24 h(b)scratch width,(c)scratch closure and(d)scratch healing rate at the corresponding time points.The area between two parallel white lines in the micrographs represents the area uncovered by HUVECs.Yellow scale bar:200 μm.(mean±SD,*p<0.05 denotes statistically difference between pair.)(For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.).

3.10.HUVECs migration

The migration capacity of HUVECs is of significance to the rapid endothelialization of the vascular graft.The migration processes of HUVECs on the modified ZE21B surfaces were recorded and analyzed.As shown in Fig.6a,HUVECs can grow and migrate normally on the samples.The scratch widths for all groups showed a decreasing trend along with the migration time(Fig.6b).The scratch closure for the ZFD group was slightly higher than those of PEG and peptides modified groups after 12 h of cell migration,while the scratch closures of ZFDPR and ZFDPRA groups were 176.1±5.7 μm and 165.4±6.5 μm after 24 h of cell migration,significantly higher than those of other groups(Fig.6c).The scratch healing rates of ZFDPR and ZFDPRA groups were also at the highest level among all groups(Fig.6d).This was mainly because REDV peptides in the functional coating on ZFDPR and ZFDPRA surfaces can profitably adhere ECs and promote the ECs migration.

3.11.HASMCs adhesion and proliferation

The functional coating on vascular stents should regulate the behaviors of both HUVECs and HASMCs.The pathological proliferation of HASMCs may induce intimal hyperplasia and in-stent restenosis.The adhesion and proliferation of HASMCs were investigated by a cell fluorescence staining experiment and the corresponding result was similar to that of HUVECs.The cytoskeleton and nuclei of HASMCs were observed as shown in Fig.7a,and these HASMCs can adhere and grow on ZE21B and modified ZE21B surfaces after 1 and 3 days of culture.It was evident that the HASMCs growth on the ZE21B surface was not well,proved by the fewer cells and smaller cell spreading area in the fluorescence images,which may be caused by the over high metal ion concentration due to ZE21B degradation.Most of HASMCs on all sample surfaces displayed oblate morphologies after 1 day of culture,demonstrating the insufficient cell spreading,especially for the ZFDPRA group(Fig.7a).After 3 days of culture,HASMCs on modified ZE21B samples were further spread,and some HASMCs with slender actin filaments can be also observed on these samples.Except for cell adhesion and morphology,the number of HASMCs on these samples was also significantly different.It can be found that the numbers of HASMCs on the ZFDPR and ZFDPRA samples were lower than those of other groups.

The proliferation of HASMCs on ZE21B and modified ZE21B surfaces were further studied by CCK-8 assays(Fig.7b).The numbers of HASMCs on all groups were at a low level after 1 day of culture.It was found that more HASMCs could be detected on ZFDP and ZFDPA sample surfaces on day 1,suggesting that the coatings on these two samples were beneficial for HASMCs growth.After 3 days of culture,significant increases were found in the numbers of HASMCs on all samples except for ZE21B group.However,compared with ZFD,ZFDP,and ZFDPA groups,the numbers of HASMCs for ZFDPR and ZFDPRA groups were still relatively small,which was due to the inhibitory effect of functional coatings containing bioactive REDV peptides on HASMCs growth.

3.12.Competitive adhesion and proliferation of HUVECs and HASMCs

The ideal functional coatings for vascular stents can not only promote the adhesion,proliferation,and migration of ECs but also inhibit the excessive proliferation of SMCs.The competitive adhesion and proliferation of HUVECs and HASMCs on modified ZE21B surfaces were studied through a coculture experiment.The representative fluorescent images of HUVECs and HASMCs labeled with green and red separately after 6 h coculture and the corresponding statistical results were shown in Fig.8.For ZE21B and ZF groups,the adhesion amount of HUVECs were more than those of HASMCs,but the numbers of HUVECs on the sample surfaces were relatively lower because of their poor cytocompatibility.After modifying the sample surefaces with amine-rich coating and PEG,the HASMCs amouts were higher than the HUVECs amounts for the ZFD and ZFDP samples,which was not favorable to rapid endothelialization.For peptideimmobilized samples,more HASMCs on the ZFDPA surface suggested that this surface was more conducive to the adhesion and growth of HASMCs,and the opposite results were found in the ZFDPR group.Considering the anticoagulant function of ACH11peptides and ECs-selective ability of REDV peptides,the ZFDPRA sample was prepared.For ZFDPRA group,the significant changes were found in cell adhesion compared to the ZFDPA group,and HUVECs showed a clear competitive growth advantage over HASMCs on ZFDPRA surface.For example,the HUVECs and HASMCs adhesion amounts of ZFDPRA groups were 90±5 N/mm2and 55±3 N/mm2,respectively.Additionally,the ratios of HUVECs/HASMCs for ZFDPR and ZFDPRA groups were 2.5±0.2 and 1.6±0.2,respectively,contributed by the biological effect of REDV peptides in the functional coatings on ZFDPR and ZFDPRA surfaces.

3.13.Rabbit carotid artery implantation

Too fast degradation rate and too slow endothelialization are two major challenges for the clinical application of MAS.

Fig.8.Coculture of HUVECs and HASMCs on different sample surfaces after 6 h of incubation.(a)Fluorescence micrographs,(b)cell density,and(c)ratio of HUVECs/HASMCs varied with different samples.(A)ZE21B,(B)ZF,(C)ZFD,(D)ZFDP,(E)ZFDPR,(F)ZFDPA and(G)ZFDPRA.(mean±SD,*p<0.05 denotes statistically difference between pair.).

Thein vivocorrosion resistance,hyperplasia,and endothelialization of ZE21B and ZFDPRA wires were evaluated by 30-day rabbit carotid artery implantation experiments.The hyperplasia ratio and endothelial coverage ratio of each group were separately calculated from the HE staining images and CD31/DAPI immunofluorescence images through Image-Pro-Plus 6.0 software.The images of HE staining and CD31/DAPI immunofluorescence staining,and corresponding statistical results were shown in Fig.9.The mass residual ratios of ZE21B and ZFDPRA wires after 30-dayin vivoimplantation were also given in Fig.9b.The mass residual ratios for ZE21B and ZFDPRA groups were 65.7±5.2% and 82.4±1.2%,respectively,indicating that the ZFDPRA group had a slower degradation ratein vivo.This was mainly because the functional coating on the ZFDPRA surface composed of the MgF2conversion layer,amine-rich coating,PEG,and bioactive peptides,could synergistically protect the ZE21B matrix to avoid rapid corrosionin vivo.According to the images of HE staining,serious hyperplasia was found in the ZE21B group(Fig.9a),and the hyperplasia ratio even reached 77.4±2.9%,potentially caused by the poor hemocompatibility and cytocompatibility of the ZE21B sample.Though the biocompatibility of the ZFDPRA group had been dramatically improved by the multifunctional coating on the surface,the excessive size of ZFDPRA wire and surgical procedures can still inevitably cause endothelial damage and hyperplasia.The hyperplasia ratio of the ZFDPRA group was only 36.0±3.6%,indicating the hyperplasia around the modified sample was relatively mild.The anti-hyperplasia ability of the ZFDPRA group was significantly improved compared to the blank ZE21B group.CD31 is the most commonly used marker for vascular ECs.It can be seen from the CD31/DAPI immunofluorescence images(Fig.9a)that amounts of CD31-positive cells were found in the ZFDPRA group,whereas only a few were found in the ZE21B group.This indicated that the ZFDPRA group profitably supported the adhesion,proliferation,and migration of HUVECs on the sample surface,which was consistent within vitrocell experiments.The endothelial coverage ratios for ZE21B and ZFDPRA groups were 27.5±1.2% and 84.3±2.5%,respectively.Though the endothelial coverage of the ZFDPRA group was incomplete,its endothelialization rate had been enhanced compared with that of the ZE21B group.This can be owed to the synergy of hydrophilic PEG,ECs-selective REDV peptides,and antithrombotic ACH11peptides in the functional coating on the ZFDPRA surface.These results confirmed that thein vivocorrosion resistance,anti-hyperplasia,pro-endothelialization were systematically improved by the functional coating prepared with hydrophilic polymer and bioactive peptides.

Fig.9.(a)HE and CD31/DAPI staining of vessel tissue surrounding the ZE21B and ZFDPRA wires after 30 days of implantation in rabbits,and(b)corresponding quantitative analysis of mass residual ratio,hyperplasia ration and endothelial coverage ratio.(# denotes the implantation location of each wire;the white dotted lines represent the neointimal hyperplasia boundary;the white arrows mark the endothelial monolayer,which expressed positive CD31;mean±SD,*p<0.05 denotes statistical difference between pair).

4.Discussion

To improve the corrosion resistance and biocompatibility of MAS,a functional coating was constructed with MgF2conversion layer,hydrophilic polymers,and bioactive peptides.Generally,a coating can prevent the biodegradation of magnesium alloys.However,for a biodegradable vascular stent,it is expected that it can fully biodegrade in the human body to avoid the side effects.Our developed functional coating can not only biodegrade in the human body,but also make ZE21B alloy stents degrade at a slower rate,thus minimizing the mismatch between the protective coating and the biodegradation of ZE21B alloy.The MgF2conversion layer was prepared from the reaction between HF and Mg,and the obtained MgF2layer was tightly bonded to the ZE21B substrate and can effectively protect ZE21B alloy from fast degradation.More importantly,the MgF2conversion layer can effectively avoid the influence of substrate corrosion on the following preparation of the functional coating via inhibiting the rapid degradation of bare ZE21B alloy.To further enhance the biocompatibility of the ZE21B alloy,amine-rich coating and PEG were successively modified onto the ZF surfaces,and these hydrophilic polymer coating can enhance the corrosion resistance and hemocompatibility of the ZE21B samples.To endow ZE21B with the ECs-selective ability and anticoagulant function,two bioactive peptides(i.e.,REDV and ACH11peptides)were covently immobilized onto the ZE21B samples.The novel multifunctional coating was designed and developed to systematacially improve the corrosion resistance and biocompatibility of the ZE21B alloy.

The functional coating on the ZE21B surface was successfully prepared,which was confirmed by ATR-FTIR(Fig.1)and XPS(Fig.2a-d).The proper grafting densities of REDV and ACH11were beneficial for pro-endothelialization and anticoagulation.The changes in surface microtopography after modification indicated the existence of the MgF2layer and amine-rich coating(Fig.2e),which can collaboratively avoid the rapid corrosion of ZE21B alloy.The MgF2layer and amine-rich coating with a dense and uniform structure can act as a protective barrier against the erosion from body fluid and reduce the degradation rate of the ZE21B alloy.After grafting of PEG and bioactive peptides,the surface wettability had changed from super-hydrophilic to hydrophilic(water contact angles,～65°)(Fig.2f),which was more favorable for cell adhesion and growth.Icorrcan reflect the corrosion rate of samples from the aspect of corrosion kinetics.The anti-corrosion property of the ZFDPRA group in Hank’s solution was also significantly improved compared with the blank ZE21B group according to the electrochemical tests(Fig.2g).ZFDPRA group with proper peptide grafting density,moderate hydrophilicity,and enhanced corrosion resistance was suitable for biomedical use.

Ideal vascular stents should not induce hemolysis or coagulation when exposed to blood[37].The hemolysis rates of PEG,REDV,and ACH11modified samples were all below 5.0%(Fig.3a),in line with the standards for clinical use.This likely benefited from the shielding effect of PEG and the good hemocompatibility of bioactive peptides[38].Nonspecific protein adsorption and denaturation is important cause of thrombosis[39–41].Compared with the ZE21B group,ZFDPR,ZFDPA,and ZFDPRA groups showed obviously low levels of fibrinogen adsorption and denaturation(Fig.3c and Fig.3d),mainly contributed by the benign compatibility of fibrinogen with bioactive peptides consisting of amino acids.PEG in the functional coating can combine with water molecules to form a hydration layer,thus reducing hemolysis and inhibiting non-specific protein adsorption[42,43].Activation of coagulation factors is an important process in the coagulation cascade.ACH11peptides can inhibit the catalytic function of FXa towards its substrate and prevent platelet aggregation,thus exerting the potent antithrombotic activity[23,24].FXa concentrations of ZFDPA and ZFDPRA groups were much lower than other groups(Fig.3b),owing to the powerful inhibitory effect of ACH11peptides on FXa activation.PEG and ACH11peptides endowed the ZE21B alloy with the blood-compatibility surface and anti-coagulation function,togother improving the hemocompatibility of magnesium alloys.These findings demonstrated the desirable hemocompatibility of the ZFDPRA group with the functional coating.

Effective regulating the behaviors of vascular cells(HUVECs and HASMCs)is of great significance for inhibiting hyperplasia and accelerating endothelialization.REDV peptides have high affinity to integrinα4β1,which was highly expressed on HUVECs but not expressed on HASMCs.It was reported that REDV peptides can be recognized by HUVECs and regulate the migration behavior of HUVECs[21,22].According to the results ofin vitrocell experiments,the functional coating on the ZFDPRA surface can effectively promote the adhesion,proliferation,and migration of HUVECs(Fig.4 and Fig.5),benefited from the selective adhesion and growth promotion of REDV peptides to HUVECs,which was also verified in previous research[44,45].Whereas,the adhesion and proliferation of HASMCs were suppressed to a certain extent for the ZFDPRA group(Fig.7),which was different from the ZFDPA group.Moreover,because NO as a gas signal molecule can regulate the behaviors of vascular cells and avoid vascular dysfunction,NO release from HUVECs on ZFDPRA surface was much higher than other groups after 1 and 3 days of cell culture(Fig.6),suggesting that the functional coating provided a better microenvironment for HUVECs growth.The better microenvironment resulting from the profitable surface wettability and ECs-selective capacity can allow ECs to remain high growth and metabolic activity.The generated NO as signal molecules in turn inhibited the over-proliferation of SMCs.The co-culture experiment also confirmed this,and HUVECs on the ZFDPRA surface exhibited obvious advantages in cell adhesion and proliferation(Fig.8),which can help avoid intimal hyperplasia and promote rapid endothelialization.

For MAS,they need to provide radial support during the service life.Thus,too fastin vivodegradation should be avoided.The mass residual ratio of the ZFDPRA group was much higher than that of the ZE21B group(Fig.9b),showing a slowerin vivodegradation due to the protection of the functional coating.Furthermore,compared with the blank ZE21B group,the low hyperplasia ratio and high endothelial coverage ratio for the ZFDPRA group both confirmed that the functional coating can inhibit intimal hyperplasia and acceleratein situendothelialization simultaneously(Fig.9).Overall,these findings collectively demonstrated that the functional coating prepared with hydrophilic polymer and bioactive peptide can efficiently improve corrosion resistance and biocompatibility of ZE21B alloyin vitroandin vivo.Additionally,it was reasonable that the developed functional coating can be possibly applied on the other magnesium-based alloys,such as WE43,AZ31,and AZ91.It is valuable to further explore the effects of materials substrate on the properties of our developed coating in the near future.

Conclusions

In summary,a functional coating for magnesium alloy stents was successfully prepared with an MgF2conversion layer,hydrophilic polymer,and bioactive peptides.This functional coating endowed ZE21B alloy with better corrosion resistancein vitroandin vivo,compared to blank ZE21B alloy.The lower hemolysis rate,less fibrinogen adsorption/denaturation,and minor activated FXa suggested the improved hemocompatibility of modified ZE21B alloy owing to the functional coating.In vitrocell experiment results indicated that ZE21B alloy with the functional coating can provide a better microenvironment for the competitive growth of ECs over SMCs.In vivoimplantation further demonstrated that the functional coating can promote rapid endothelialization and suppress intimal hyperplasia,and has great potential in the development of biodegradable stents.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This project was supported by the National Natural Science Foundation of China(Grant No.52101291),China Postdoctoral Science Foundation(Grant No.2020TQ0273),the National Key Research and Development Program of China(Grant No.2018YFC1106703),and the Key Projects of the Joint Fund of the National Natural Science Foundation of China(Grant No.U1804251).

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2022.05.023.