Pseudopeptide polymer coating for improving biocompatibility and corrosion resistance of 316L stainless steel

Abstract

This article described for the first time using a pseudopeptide polymer, poly(2-methyl-2-oxazoline) (PMOXA), to form a bionic non-brush coating on a 316L stainless steel surface by electrochemical assembly for improving biocompatibility and corrosion resistance. In order to produce a stable coating, dopamine (DA) and partially hydrolysed PMOXA (H-PMOXA) were used to assemble on the surfaces of the 316L stainless steel via electrochemical oxidation and a Michael addition between the multi-imino groups of H-PMOXA and poly(dopamine). The effect of the molecular weights and hydrolysis degree of PMOXA on the formed coatings with respect to the biocompatibility and corrosion resistance of the modified stent materials were studied in detail. The results showed that the coating formed by PMOXA with moderate molecular weights and hydrolysis degree possessed excellent anti-fouling properties and biocompatibility. Moreover, the migration and proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) on this kind of coating were also greatly enhanced.

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Introduction

In the last two decades vascular stents have become the most important treatment for cardiovascular diseases. 316L stainless steel (316L SS), titanium alloy, and the shape memory alloy have been widely used for vascular stents. Nevertheless, the biocompatibility of these metallic materials is insufficient for the long-term anti-thrombogenic demand in blood vessels, because of protein adsorption and blood cell adhesion caused by the release of metal ions and the complex interaction between the metallic material surfaces and blood. Recently, surface modification of the metallic materials has become an effective strategy to control the release of metal ions from metallic materials and to improve the biocompatibility of metallic materials. Various surface modification strategies have been developed, among which the surface modification via thin polymer coatings represents an important method.^1,2 Corrosion resistance, resistance of nonspecific protein adsorption,³ and biocompatibility^4–7 can be achieved by polymer coating modification.

These days, polymers based on 2-oxazoline such as poly(2-methyl-2-oxazoline) (PMOXA), are considered to be a type of ‘pseudopeptide’ with the ability to form novel biomaterials.⁸ It is reported that PMOXA has good biocompatibility and stability in physiological environment⁹ for the chemical composition of PMOXA is similar to polypeptide. In our previous work, PMOXA brush coatings have been confirmed of possessing excellent protein-repellent property and biocompatibility by immobilizing on silicon¹⁰ and gold^11,12 surfaces. As we know, only improving biocompatibility of metallic stent is not enough for preventing in-stent restenosis, because the adhesion, differentiation, and proliferation of endothelial progenitor cells (EPCs) on vascular end epidermis or implanted vascular stents are also necessary to the repair of vascular endothelium and the inhibition of the proliferation of smooth muscle cells.¹³ However, the cells adhesion assay proved that the Human Umbilical Vein Endothelial Cells (HUVECs) can hardly adhere to the coating formed by PMOXA brush¹⁴ although PMOXA itself is able to improve the proliferation of HUVECs. It is likely because HUVECs are difficult to migrate and proliferate on the hydrophilic polymer brush coating, due to the elastic repulsive forces of the hydrophilic brush coating.^15,16 Therefore, in order to utilize the cell viability of PMOXA and produce a coating for endothelial cell migration and proliferation, a non-brush PMOXA coating was expected to immobilize on the metallic stent material surfaces to form a stable coating which will benefit the cell migration and proliferation.

A significant challenge in polymer coatings used for modifying metallic stent surface is that the coating needs to withstand the physiological effect of body in long term. Surface cracking, peeling, and flaking of the coating will result in thrombosis. Recently, inspired by mussel-adhesive proteins, Messersmith et al. have used an adhesive polydopamine (PDA) layer for modifying a wide range of materials.¹⁷ Moreover, the PDA layer is an active coating which can be served as the anchor for further immobilization of polymers¹⁸ or bioactive molecules with functional groups, such as amino and sulfydryl groups, by one-step or multi-steps strategies.^17,19 Tsai et al.²⁰ have synthesized poly(ethylene imino)-graft-poly(ethylene glycol) (PEI-g-PEG) copolymer, and then blended PEI-g-PEG with DA to prepare PEI-g-PEG/PDA mixed coatings by an one-step strategy, which could inhibit the adsorption of serum proteins as well as the attachment of fibroblast. Several groups have also studied the electrochemical oxidative polymerization of DA (ePDA) on metal material to form a functionalized surface,^21,22 because more smooth and more stable PDA coating can be produced by this method. For example, Wang et al.²² have reported that DA could form the ePDA coating on the surface of cardiovascular metallic stents through electropolymerization of DA, and this kind of ePDA coating could be functionalized further with vascular endothelial growth factor (VEGF) to enhance the proliferation of endothelial cells (ECs) and prevent the formation of neointima after stent implantation.

In this paper, we developed a novel strategy of immobilization of non-brush PMOXA coating on 316L SS surface to produce a long-term stable coating for improving biocompatibility, enhancing endothelial cell migration and proliferation. A series of hydrolysed PMOXAs (H-PMOXA) with different molecular weight and hydrolysis degree were prepared to produce multi-imino groups inserted PMOXA, i.e. poly(2-methyl-2-oxazoline-co-ethyleneimine) (P(MOXA-co-EI)) random copolymer, in which the EI repeating unit can be used as the anchors for immobilization of 2-methyl-2-oxazoline (MOXA) segments, as the imino groups inserted in back bone of P(MOXA-co-EI) can react with o-quinone produced by electrochemical oxidation of dopamine. Since the imino groups were randomly inserted in the main chains of H-PMOXA, the coating formed by PMOXA was a non-brush coating. After, the coatings were characterized by X-ray photoelectron spectroscopy (XPS) and water contact angle investigations. The corrosion resistance of the modified 316L SS was characterized by potentiodynamic polarization measurements, electrochemical impedance measurements (EIS), and cell viability assay. Furthermore, fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) and rich platelet serum were used to test the biocompatibility of non-brush PMOXA coating formed on 316L SS surface. The migration and proliferation of HUVECs on the non-brush PMOXA modified 316L SS surface were also investigated. Finally, this strategy was used to successfully modify the 316L SS vascular stent.

Materials and methods

Materials

Poly(2-methyl-2-oxazoline) with three kinds molecular weight (PMOXA-2.5k, PMOXA-5k, PMOXA-10k; M̄_n are 2.5 kD, 5 kD, and 10 kD, respectively) was synthesized according to previously reported procedures and shown in ESI.†²³ Sodium hydroxide (NaOH, AR), concentrated hydrochloric acid (HCl, AR), tris (hydroxymethyl) aminomethane (Tris, BR), ethyl alcohol (AR), acetone (AR), and methylbenzene (AR) were purchased from Sinoreagent (Shanghai, China). Dopamine hydrochloride (DA·HCl) was purchased from Sigma-Aldrich (St. Louis, MO, US). 316L SS slices were from Baosteel Co., Ltd (Shanghai, China) and 316L SS stents were purchased from Yinyi Co., Ltd (Dalian, China). Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was prepared according to previously reported procedures.²⁴ Platelet-rich plasma was obtained from fresh human blood by centrifugation at 200 × g for 10 min. High glucose Dulbecco's Modified Eagle Medium (DMEM) and 0.25% trypsin solution were obtained from Hyclone (Logan, Utah, US). Fetal bovine serum (FBS) was obtained from Tianhang Biotechnology Co., Ltd (Hangzhou, China). 6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotechnology (Shanghai, China). HUVECs were kindly provided by Prof. Longping Wen of the University of Science and Technology of China (Hefei, China). Deionized water was used throughout the whole experiment.

Preparation of partial hydrolysed PMOXA (H-PMOXA)

A series of H-PMOXA with different hydrolysis degree and molecular weight were prepared using procedures described in the literature.^25,26 Hydrolysis of PMOXA-5k was described as a representative procedure below. Briefly, 16.8 wt% HCl solution (100 mL) was added into a reaction flask and heated to 100 °C under reflux, and then PMOXA-5k (denoted as P₀, 4 g) was added. The hydrolysis reaction was performed for 15 min at 100 °C. After, the reaction flask was immersed into ice-water bath, and then 10 M NaOH aqueous solution was added into the flask to adjust the pH value of reaction mixture to about 9. Subsequently the reaction mixture was extracted with CHCl₃ (300 mL × 3). The combined oil phases were dried over MgSO₄, filtered, and then concentrated under vacuum. Finally, the raw products were precipitated in ice-cold ether, filtered, and dried in vacuum overnight, and then hydrolysed product of PMOXA-5k with 15 min hydrolysis denoted as P₁ was obtained. For change the hydrolysis degree of PMOXA, PMOXA-5k with 30 and 50 min hydrolysis time were also prepared, denoted as P₂ and P₃, respectively. In addition, the hydrolysis of PMOXA-2.5k and -10k under the same time as P₂ (30 min) were also performed, and the obtained products were defined as P₄ and P₅, respectively. The obtained polymers were characterized by ¹H NMR spectroscopy (AVANCE 300, Bruker Biospin, Switzerland).

Immobilization of H-PMOXA on 316L SS slice or vascular stents

A CHI600D electrochemical workstation (Chenhua, Shanghai, China) was used to immobilize PDA or H-PMOXA/dopamine coating on the surface of 316L SS slices or the surface of 316L SS vascular stents by electrochemical oxidation. The electrochemical deposition was carried out in a standard three-electrode cell. Pt and KCl-saturated Ag/AgCl electrodes were used as the counter and reference electrodes, respectively. 316L SS slices or stents were used as the working electrode (the illustration of the modification of slices was shown in Fig. S2 of ESI†). Electrochemical potentiostatic technology was employed to prepare H-PMOXA/dopamine coating on the working electrode surfaces. DA·HCl (20 mg) and H-PMOXA (30 mg) were added into 10 mL Tris·HCl buffer (50 mmol L⁻¹, pH 8.4) after being bubbled with high purified nitrogen for 10 min to remove oxygen. Immobilization of H-PMOXA was carried out by electrochemical potentiostatic sweep in a series of time at the potential 0.4 V. After co-deposition, the coating modified electrode was rinsed with deionized water and then immersed in deionized water thermostated at 60 °C for an hour, and then dried with a stream of purified nitrogen.

Characterization of the modified surfaces

The chemical compositions of modified surfaces were determined using a VG ESCALAB MK II XPS (VG Scientic Instruments, England) with an Al (Kα) X-ray source (1486.6 eV). The static water contact angles (WCA) of the modified surfaces were measured at room temperature by using an Optical Contact Angle & Interface Tension Meter SL200KS goniometer from KINO (US). The volume of the water drop was 2 μL. Measurements were performed 6 times for each sample. The results were reported as mean ± standard deviation.

Electrochemical impedance measurements (EIS) and potentiodynamic polarization measurements were employed to further confirm the surface modification.²⁷ Potentiodynamic polarization measurements were carried out in 5 wt% KCl solution at room temperature with modified 316L SS as the working electrode. The Tafel plots were recorded at a scan rate of 2 mV s⁻¹ within the range of −600 to 0 mV versus KCl-saturated Ag/AgCl electrodes, and analyzed to determine the corrosion potentials (E_corr), respectively. EIS were carried out in 0.1 M KCl solution containing 5 mM K₃[Fe(CN)₆/K₄[Fe(CN)₆] at open circuit potential and the frequency range was from 0.01 Hz to 100 kHz.

The corrosion resistance of the modified 316L SS was also characterized by anodized the samples in 5 wt% KCl solution at 1 V for 2 h. Cross-sectional images of the anodized samples were observed by a scanning electron microscope (SEM, SIRION 200, FEI, US).

Protein adsorption assay

Bare or modified 316L SS slices were immersed in PBS (0.01 M, pH 7.4) for 2 h, subsequently incubated in 0.25 mg mL⁻¹ FITC-BSA PBS solution for 2 h; and then the slices were rinsed with deionized water, dried under a stream of high purified nitrogen, and subsequently analyzed by laser scanning confocal microscope (LSCM, LSM510 from Zeiss, Germany).

Platelet adhesion assay

The platelet adhesion experiments on bare or modified 316L SS slices were performed according to previous work.²⁴ The treated slices were sputter-coated with gold and the morphology was observed by SEM.

Cell culture

HUVECs were cultured in DMEM medium supplemented with 10% FBS in thermostatic incubator (37 °C in a humidified atmosphere containing 5% CO₂). Culture medium was replenished every day. After growing to approximately 80% confluence, cells were trypsinized to prepare cell suspension solution. Then, the cell suspension solution was used for passage, cell viability assay or cell proliferation onto bare or modified 316L SS slices.

Cell viability assay: MTT

MTT tests have been used to assess the release of metal ions of bare or modified 316L SS slices. Bare or modified 316L SS slices (4 mm × 6 mm) were sterilized by immersing in absolute ethyl alcohol for 1 hour and washed by PBS buffer. Then, 100 μL of cell suspension solution (5 × 10⁴ cells per mL) was added into 96-well plates and kept in incubator for 3 h before the slices being placed in. After, the slices were respectively placed into the 96-well plates (leaned the slices against the well and the cells were on the bottom of the well) and the cells were cultured in incubator for 2 days, 4 days, or 6 days, respectively.

MTT tests also have been used to assess the cell viability of different PMOXAs and H-PMOXAs. Firstly, PMOXA and H-PMOXA were added into cell culture medium to prepare a series of polymer precursor solutions with different concentration (0.002, 0.02, 0.1, 0.2, 0.4, 1, 2 mg mL⁻¹) and the solutions were filtered through a 0.22 μm filter to remove bacteria. Secondly, 50 μL of cell suspension solution (1 × 10⁵ cells per mL) and 50 μL of polymer precursor solution were mixed and added into 96-well plates (the final polymer concentration was 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1 mg mL⁻¹, respectively), and the 96-well plates were cultured in incubator for 24 h. Non-treated cells were used as a control.

After treatment, the MTT solution (0.5 mg mL⁻¹) was added to plate and then incubated for 2 h. After 2 h, the reaction medium was removed from plates and DMSO was added. The absorbance was measured at 570 nm using a Microplate reader (ELX800, BIO-TEK Instruments Inc., US). At least three same samples were measured. All values corresponding to the cell viability assay were expressed as mean ± standard deviation. Statistical analysis was performed using Student's t-test for the calculation of significance level of the data. Differences were considered statistically significant at P < 0.05.

HUVEC proliferation assays

Bare or modified 316L SS slices (4 mm × 4 mm × 0.2 mm) were laid on the bottom of the 96-well plates, respectively. After, 100 μL of cell suspension solutions (5 × 10⁴ cells per mL) were added into each well of 96-well plates. The plates were cultured in incubator (37 °C, in a humidified atmosphere containing 5% CO₂) for 24 h. And then, the slices with adherent cells were carefully washed with PBS buffer and immersed in 2.5% glutaraldehyde for 2 h at room temperature. The slices were rinsed three times with PBS and then dehydrated using an ethanol series (25%, 50%, 75%, 90%, and 100%; 20 min each). After being dried with a stream of nitrogen, the slices were sputter-coated with gold for examination by SEM.

HUVEC migration assays

Bare or modified 316L SS slices (1 cm × 2 cm × 0.2 mm, allocated into two same groups) partly covered with a 316L SS ringy mask were laid on the bottom of the 6-well plates. After, 3 mL of cell suspension solutions (5 × 10⁵ cells per mL) were added into each well of 6-well plates. The plates were cultured in incubator (37 °C, in a humidified atmosphere containing 5% CO₂). After 6 h, the ringy masks were removed from all slices. One group of the slices was carefully washed with PBS buffer and immersed in 2.5% glutaraldehyde for 2 h at room temperature to fix the HUVECs. The slices were then rinsed three times with PBS and 1 mL of DAPI (2 μg mL⁻¹ in PBS) were carefully added to the plates to stain the nuclei of the fixed cells for 5 min.^28–30 Subsequently, the slices were rinsed three times with PBS and then observed using a Fluorescence Microscopy (Olympus, JPN) under 365 nm excitations for DAPI. The slices of the other group were cultured in incubator for another 48 h, and then the cells were stained and observed with the same procedure as the slices of the former group.

Results and discussion

The immobilization of H-PMOXA on 316L SS substrates

Fig. 1A shows the structure and model of PMOXA and H-PMOXA, respectively. After hydrolysis, part of amido bond of side groups in PMOXA could be broken and the imino groups could be produced in the main chain of PMOXA, and then multi-imino groups inserted PMOXA was obtained. The hydrolysis degree was used to describe the number of imino groups of the H-PMOXA, and it increases with increasing the hydrolysis time. The hydrolysis degree (η) was calculated according the ¹H NMR spectra (Fig. 1B) and obtained bywhere S_a is the integral quantity of peak a in the ¹H NMR spectroscopy of H-PMOXA, S_c is the integral quantity of peak c in the ¹H NMR spectroscopy of H-PMOXA (Fig. 1B). The results were presented in Table 1. The process of DA and H-PMOXA electrochemical assembly on 316L SS surface was described in Fig. 1C. During the process of oxidation and polymerization of DA on the working electrode surfaces, many o-quinone groups could be produced and assembled on the 316L SS surfaces. The produced o-quinone could react with the amino groups of DA and imino groups inserted in H-PMOXA, and then DA and H-PMOXA could be immobilized on the 316L SS surfaces. Thus, the PMOXA segments can be immobilized not only on the coating surfaces, but also in the inner of the coating with PDA via multi-imino group anchors. After being immersed in 60 °C deionized water for an hour, the coating could become more stable (date not shown) for the elevated temperature is of benefit to improve the reaction ratio of imino groups inserted in H-PMOXA with PDA, leading to the immobilization of PMOXA segment on the 316L SS surfaces with more anchors. Furthermore, after being treated with hot water, some PMOXA segments that had been embedded in PDA during electrochemical assembly process could migrate from inner coating to surface in aqueous environment due to the excellent hydrophilicity of PMOXA, thus, the modified surface treated with hot water will become more hydrophilic (Fig. 1C).

Fig. 1 (A) The preparation of H-PMOXA, (B) ¹H NMR spectra of hydrolyzed PMOXA-5k with different hydrolysis time, (C) schematic illustration of the electrochemical assembly process of DA and H-PMOXA on 316L SS surfaces, and the anti-fouling and HUVEC adhesive properties of H-PMOXA/dopamine modified surface.

Table 1 Molecular weight, hydrolysis time, and hydrolysis degree of PMOXA

Polymer	Molecular weight^a	Molecular weight^b	Hydrolysis time	Hydrolysis degree
a Theoretical number-average molecular weight of PMOXA before its hydrolysis.b The number-average molecular weight of PMOXA obtained from ^1H NMR spectra of PMOXA in Fig. S1 (ESI).
P0	5.0 kD	4.6 kD	0	0
P1	5.0 kD	4.6 kD	15 min	8.1%
P2	5.0 kD	4.6 kD	30 min	17.4%
P3	5.0 kD	4.6 kD	50 min	33.2%
P4	2.5 kD	2.4 kD	30 min	18.8%
P5	10.0 kD	7.5 kD	30 min	16.7%

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Characterization of the modified surfaces

Surface chemical composition of the bare or modified 316L SS slices has been measured by XPS. All modified slices were obtained with 2 h modification. The basic elements including carbon (C), oxygen (O), nitrogen (N), iron (Fe), and chromium (Cr) were surveyed by scanning bonding energy (Fig. 2). The main peaks at 284.8, 400.1, 531.7, 576.1, and 741.0 eV have been labeled in Fig. 2 and separately represent C1s, N1s, O1s, Fe2p, and Cr2p. The XPS results (Fig. 2A) showed that the PDA modified 316L SS slice surface was covered by the PDA coating completely, for the Fe and Cr signal existed on the surface of bare 316L SS slice cannot be detected. Fig. 2B shows the XPS spectra of the 316L SS surface modified with different H-PMOXA/dopamine deposited coating. The Fe2p and Cr2p signals of the bare 316L SS slice disappeared and the intensity of C1s, N1s, and O1s signals of all H-PMOXA/dopamine deposited coating modified 316L SS slices increased, which indicated that the surface of 316L SS slice could be covered by the H-PMOXA/dopamine deposited coating completely. Table 2 shows the surface relevant atomic ratios based on the intensity of the C1s, N1s, and O1s signals of PDA modified 316L SS slice and different H-PMOXA/dopamine deposited coating modified 316L SS slices. As shown in Table 2, the value of O/N and C/N of PDA coating was 2.53 and 8.21, respectively. Meanwhile, the corresponding values of all H-PMOXA/dopamine coatings decreased. The lower O/N and C/N ratios of H-PMOXA/dopamine coatings suggested that the PMOXA segments were involved in the coating forming process, for the O/N and C/N ratios of all H-PMOXA/dopamine coatings were between the theoretical value of PDA and PMOXA (theoretical values of O/N and C/N ratios of PMOXA are 1 and 4; theoretical values of O/N and C/N of PDA coating are 2 and 8, respectively). In order to characterize more clearly the H-PMOXA composition in the coating, the values of O/N and C/N in different modified 316L SS slices were amended by formula (2) and (3), respectively; the H-PMOXA content in different coating was calculated by formula (4) and (5), respectively:where α(P_x) and β(P_x) are the value of O/N and C/N of H-PMOXA/dopamine coating, for example α(P₀) is the value of O/N of P₀/dopamine coating; α(PDA) and β(PDA) represent the value of O/N and C/N of PDA coating, respectively. α*(P_x) and β*(P_x) are the amendatory value of O/N and C/N of H-PMOXA/dopamine coating. η(P_x) is the hydrolysis degree of P_x as shown in Table 1. γ₁(P_x) is the content of H-PMOXA in the P_x/dopamine coating calculated from the value of O/N. γ₂(P_x) is content of H-PMOXA in the P_x/dopamine coating calculated from the value of C/N. The results were displayed in Table 2.

Fig. 2 (A) The XPS spectra of bare and PDA modified 316L SS slices, (B) the XPS spectra of different H-PMOXA/dopamine coating modified 316L SS slices.

Table 2 Atomic ratios (%) of C1s, N1s, and O1s; the value of α, β, α*, β*, γ₁, and γ₂ of different coatings^a

	C1s	N1s	O1s	α	β	α*	β*	γ1	γ2
a α and β are the values of O/N and C/N, respectively; α* and β* are the amendatory values of O/N and C/N, respectively; γ1 and γ2 are the content of H-PMOXA in the coating which are calculated from the value of O/N and C/N, respectively.b The value in brackets is the theoretical value of O/N and C/N of PDA coating, respectively.
PDA coating	69.9	8.51	21.59	2.53(2)^b	8.21(8)^b	2	8	0	0
P0/dopamine coating	68.64	9.49	21.87	2.3	7.23	1.81	7.04	19.0%	24.0%
P1/dopamine coating	68.03	10.12	21.85	2.16	6.72	1.71	6.55	26.8%	30.8%
P2/dopamine coating	69.42	10.87	19.71	1.81	6.39	1.43	6.23	48.6%	40.7%
P3/dopamine coating	69.45	11.74	18.81	1.60	5.92	1.26	5.77	55.6%	47.8%
P4/dopamine coating	68.66	10.81	20.53	1.90	6.35	1.50	6.19	42.1%	41.4%
P5/dopamine coating	68.86	10.55	20.59	1.95	6.52	1.54	6.35	39.4%	38.1%

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As depicted in Table 2, there are two different values (γ₁ or γ₂) of H-PMOXA content in the H-PMOXA/dopamine coating based on the value of O/N and C/N, respectively. That of PMOXA-5k without hydrolysis (P₀/dopamine) coating was 19% (γ₁) and 24% (γ₂). Meanwhile H-PMOXA content (no matter γ₁ or γ₂) in H-PMOXA/dopamine coatings were all higher than that of P₀/dopamine coating, the H-PMOXA content of P₃/dopamine, in which the hydrolysis degree of PMOXA-5k is the highest, reached 55.6% (γ₁) and 47.8% (γ₂). These results suggested that a higher hydrolysis ratio can result in more imino groups inserted in one PMOXA chain, and increased imino groups can improve the reaction probability between H-PMOXA and PDA. In addition, among the H-PMOXA/dopamine coatings formed by the similar hydrolysis degree of PMOXA (all the hydrolysis time is 30 min, including P₂, P₄, and P₅/dopamine coating), the content of H-PMOXA in P₂/dopamine coating (PMOXA-5k) is the highest, since γ₁(P₁) value is the highest (γ₂(P₁) is little lower than γ₂(P₂) owing to system error). At the same time, the content of H-PMOXA in P₅/dopamine coating (PMOXA-10k) was the lowest, for the value of γ₁ and γ₂ of P₅/dopamine coating is the lowest compared with P₂ and P₄/dopamine coating. The electrochemical assembly of H-PMOXA and dopamine on the 316L SS surface is a coexisted process of cooperation and competition. On the one hand, the oxidation and assembly of dopamine on the surface is of benefit to the immobilization of H-PMOXA thanks to Michael addition reaction between dopamine and H-PMOXA; on the other hand, the existence of H-PMOXA can limit the assembly of dopamine on the surface of 316L SS because of the steric effect of macromolecules. The higher the molecular weight of H-PMOXA is, the more influence of the H-PMOXA exerts. Therefore, compared to P₂ and P₅, P₄ with the lowest molecular weight may result in the assembly of H-PMOXA and dopamine easier, but the short PMOXA segments of P₄ are difficult to limit the assembly of dopamine on the surfaces; furthermore, compared to P₂ and P₄, P₅ has the highest molecular weight, which could lower the reaction efficiency between the imino groups inserted in PMOXA and PDA due to the biggest steric effect of P₅. Consequently, the content of H-PMOXA of P₂/dopamine coating (PMOXA-5k) is the highest and that of P₅/dopamine coating (PMOXA-10k) is the lowest. Thus, hydrolysis degree and molecular weight of PMOXA are two important parameters for electrochemical assembly of H-PMOXA and dopamine, high hydrolysis degree and moderate molecular weight of PMOXA are advantageous to the H-PMOXA/dopamine coating immobilized on the surface of 316L SS slice.

The Nyquist plots of bare and modified 316L SS slices are shown in Fig. 3A. As displayed, the charge transfer resistance (R_ct) of K₃[Fe(CN)₆]/K₄[Fe(CN)₆] for slices could be obtained based on the diameter of the circular curve of Nyquist plot. The R_ct of bare slice was very small, while that of modified slices increased more than hundreds times, and R_ct of H-PMOXA/dopamine coating modified slices were higher than that of PDA and P₀/dopamine coating modified slices. Meanwhile, R_ct of modified slices were boosted while increasing the hydrolysis degree of PMOXA-5k as shown in Nyquist plots of P₀, P₁, P₂, and P₃/dopamine coating modified slices (Fig. 3A). These phenomena suggested that Michael addition reaction happened between imino groups inserted in H-PMOXA and PDA during the coating formed by electrochemical assembly process, and the coating should be reinforced with the increment of the hydrolysis degree of PMOXA, especially for the coating formed by dopamine and H-PMOXA (P₃) with the highest hydrolysis degree of PMOXA-5k. The reinforced coating can prevent the electrochemical redox of [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ on the electrode. These results are in line with the XPS results in which high hydrolysis ratio of H-PMOXA is beneficial to the immobilization of H-PMOXA. On the other hand, R_ct values of H-PMOXA/dopamine coating modified slices with H-PMOXA in different molecular weight (P₂, P₄, and P₅/dopamine coating) showed that H-PMOXA with the lowest molecular weight (P₄, PMOXA-2.5k) was easier to form a reinforced coating. Although the sequence of R_ct values is a little different with the sequence of H-PMOXA content obtained from XPS results, it further indicated that the assembly of P₄ with short chain and dopamine was becoming easier, and the short H-PMOXA segments of P₄ are difficult to limit the assembly of dopamine on the surfaces, therefore, the surface of P₄/coating include more PDA than other coatings, which were conducive to the formation of the reinforced coating due to the process of polymerization of DA.

Fig. 3 (A) The Nyquist plots of bare and modified 316L SS slices, (B) Tafel plots of bare and modified 316L SS slices, (C) cross-sectional SEM images of anodized bare (up) and P₂/dopamine coated (down) 316L SS (the samples were anodized in 5 wt% KCl solution at 1 V for 2 h), (D) illustration of cell viability assay, (E) HUVECs grown for 2, 4, and 6 days with bare and modified 316L SS slices immersed in cell culture well, the cell cultured without 316L SS slice as control, (n = 3, *P < 0.05, compared to the control group).

The Tafel plots of bare and modified 316L SS slices are shown in Fig. 3B, the corrosion potential, E_corr, undergoes a positive shift about 100 mV to 150 mV for PDA and H-PMOXA/dopamine coating modified slices comparing with that of bare slice. The positive shift of corrosion potential revealed that the electrochemical oxidation of 316L SS slice could be resisted by PDA and H-PMOXA/dopamine coating. In addition, the positive shift of H-PMOXA/dopamine coating is more than that of PDA coating, and the positive shift increased with increasing the hydrolysis degree of PMOXA-5k, which suggested that the coating formed by electrochemical assembly could be reinforced by imino groups inserted in PMOXA, the more the imino groups are, the more reinforced the coating is. Therefore, all the above results unambiguously demonstrated that the H-PMOXA/dopamine coating not only could be immobilized and cover fully on the surfaces, but also could seriously improve the corrosion resistance of 316L SS slice by forming reinforce coating via Michael addition reaction between imino groups inserted in PMOXA and PDA.

Cross-sectional SEM images of anodized bare and P₂/dopamine coated 316L SS slices were displayed in Fig. 3C, the thickness of oxidation layer on anodized bare 316L SS was reached 4 μm, while the oxidation layer could not be observed on P2/dopamine coated 316L SS slices, which also demonstrated that the coating on the 316L SS improved the corrosion resistance of 316L SS.

The cell viability assay was used to test the release of metal ions from bare or modified 316L SS slices. In this assay, the bare or modified 316L SS slices were immersed in cell culture well in which the HUVECs had been cultured for 3 h in advance (Fig. 3D). MTT was used to determine the cell viability, and the results are shown in Fig. 3E. The number of HUVECs in the culture well contained PDA or H-PMOXA coating modified slices was similar to that of the culture well without slice, and the number of HUVECs in culture well with bare slice was larger than the others, no matter for the 2 days, 4 days, or 6 days cell culture. The increment of cell number in culture well with bare slice is most likely caused by the trace amounts of iron ions released from 316L SS during the slice immersed in cell culture medium that could improve the cell proliferation.³¹ Zhu et al.³² have reported the similar phenomenon in their work in which lower iron concentration (<10 μg mL⁻¹) can produce the favorable effect on the metabolic activity of ECs. On the other hand, the number of HUVECs in the culture well with modified slice was similar to that in control well, indicating that the iron ions cannot release from PDA or H-PMOXA coating modified 316L SS slices.

The surface hydrophilicity of the modified 316L SS slice

PMOXA is a kind of super hydrophilic polymer, and therefore the changes of WCAs of bare or modified 316L SS slice surface can be used to study the effect of PMOXA on hydrophilicity of H-PMOXA/dopamine coating. As displayed in Fig. 4A, the WCAs of all of the modified slices were lower than that of bare slice, especially for the H-PMOXA/dopamine modified 316L SS slice, which indicated that PDA and H-PMOXA/dopamine coating can improve the hydrophilicity of 316L SS slice. The better hydrophilicity of H-PMOXA/dopamine coating should be attributed to the PMOXA segment on the outermost layer of the coating. The WCA of surface was decreased to 16° after 316L SS slice being treated with P₁/dopamine for 5 hours through electrochemical assembly, while the WCA of surface was decreased below 5° after being coated with P₂/dopamine coating using only 2 hours through electrochemical assembly, and the WCA of 3 hours and 5 hours modified slices change little. Obviously, higher hydrolysis degree is necessary for PMOXA immobilization on the 316L SS. However, the WCA of P₃/dopamine coating was a little higher than that of P₂/dopamine coating. As we know, the surface H-PMOXA content of P₃/dopamine coating modified 316L SS slice was higher than that of P₂/dopamine coating modified slice as shown in Table 2, nevertheless, the hydrophilicity of the coating is mainly related to the content of 2- MOXA repeating unit in the H-PMOXA. Hence, the surface PMOXA segment content of the H-PMOXA/dopamine coating was calculated, the results were displayed in Table S1 (ESI†). As displayed, the content of MOXA repeating unit in P₂/dopamine coating was higher than that of P₃/dopamine coating, which implied that while keeping the molecular weight of unhydrolysed PMOXA constant, the content of MOXA repeating unit in the H-PMOXA decreased with the increment of the hydrolysis degree of PMOXA, resulting in the P₂/dopamine coating with more MOXA repeating unit possessed better hydrophilicity. Fig. 4B–E shows the WCA photographs of bare 316L SS slice, PDA coating, P₂/dopamine coating modified 316L SS slices, and the P₂/dopamine coating modified slice after immersing in PBS for 30 days. It can be seen that the WCA of P₂/dopamine modified slice after immersing in PBS for 30 days is almost the same with that of fresh P₂/dopamine modified slice, which elucidated that the P₂/dopamine coating was very stable in physiological environment thanks to the excellent adhesion of DA and H-PMOXA segments immobilized with PDA through covalent bonding.

Fig. 4 (A) The WCA values of different modified 316L SS slices with different modification time, (B–E) the WCA photograph of bare 316L SS slice (B), PDA coating (C), P₂/dopamie coating (D) modified 316L SS slices, and the P₂/dopamine coating modified 316L SS slices immersed in PBS for 30 days (E) (n = 6, *P < 0.05, compared to the bare group (316L)).

The anti-fouling assay

To assess the anti-fouling properties of the modified surfaces, a fluorescence test was performed using FITC-BSA as a model protein. As depicted in Fig. 5, the protein adsorption was tested by LSCM image of the attachment tests of FITC-BSA on bare (Fig. 5A), PDA coating (Fig. 5B), and PMOXA or H-PMOXA/dopamine modified 316L SS slices (Fig. 5C–H). Intense fluorescence (appearing green) was observed on the bare slice (Fig. 5A), confirming that the BSA was efficiently adsorbed on the surface of bare 316L SS slice. By contrast, the adsorption of FITC-BSA on PDA (Fig. 5B) and P₀/dopamine coating (Fig. 5C) modified slices was similar and lower than that of bare slice. Obviously, P₀ cannot be stably immobilized on the slice with PDA due to its shortage of active groups, therefore the protein adsorption resistance property of P₀/dopamine coating is similar to that of the PDA coating. Whereas the attachment of FITC-BSA was hardly observed on the surface of H-PMOXA/dopamine coating modified slices (Fig. 5D–H). It can be concluded that the presence of H-PMOXA chains on the surface was responsible for the reduction in protein adsorption. The normalized fluorescence intensity values of FITC-BSA adsorption substrate, relative to the fluorescence intensity of Fig. 5A were shown in Fig. 5I. The fluorescence intensity of H-PMOXA/dopamine coating modified slices were extremely low, especially for P₂, P₃, P₄, and P₅/dopamine coating modified slices. As we discussed before, the WCA values of H-PMOXA/dopamine coatings were all below 10°. These results illustrated that the H-PMOXA/dopamine coating modified slices possess excellent protein adsorption resistant ability due to the excellent hydrophilicity of the H-PMOXA/dopamine coating.

Fig. 5 LSCM images of the attachment tests of FITC-BSA on bare 316L SS (A), PDA (B), P₀/dopamine coating (C), P₁/dopamine coating (D), P₂/dopamine coating (E), P₃/dopamine coating (F), P₄/dopamine coating (G), P₅/dopamine coating (H) modified slices. (I) The normalized fluorescence intensity values of FITC-BSA adsorption substrates, relative to the fluorescence intensity of A (n = 3, **P < 0.01, compared to the bare group (316L)).

Platelet adhesion in vitro

The biocompatibility of the bare and modified 316L SS slices was investigated by the attachment tests of platelet. As depicted in Fig. 6, there were many platelets aggregated and activated on the bare slice (Fig. 6A) and a fewer platelets were observed on the PDA coating modified slice (Fig. 6B). Obviously, the biocompatibility of PDA coating is better than that of bare 316L SS. Meanwhile, under the same molecular weight of PMOXA (PMOXA-5k), the number of platelets attached on the H-PMOXA/PDA coating modified slices decreased with increasing the hydrolysis degree of H-PMOXA as shown in Fig. 6C–E, and it is hard to see any platelets attached on the P₂/dopamine modified slice. But with increasing the hydrolysis degree further, the platelets seriously aggregated and activated on the P₃/dopamine coating (Fig. 6F), and even the number of platelets aggregated and activated on the P₃/dopamine coating modified slice was more than that on bare slice. As previously discussed, P₀ (PMOXA without hydrolysis i.e. hydrolysis degree is zero) cannot be stably immobilized on the slice in the long term due to its shortage of active groups, which is necessary for the interaction between PMOXA and PDA, resulting in the biocompatibility of P₀/dopamine coating is the same as that of PDA coating. Moreover, the slice couldn't be covered fully by P₁/dopamine coating because of the lower MOXA repeating unit existed in P₁/dopamine coating (Table S1, ESI†), leading to the biocompatibility of P₁/dopamine coating couldn't satisfied the requirements. Whereas, P₂/dopamine coating modified slice could display an excellent biocompatibility due to its hydrophilicity and higher content of MOXA repeating unit existed in the coating. As we know, the imino groups possess cell cytotoxicity, and P₃ is occupied by a large number of imino groups, which could be exposed out of the surface and bring about the platelets aggregated and activated seriously on P₃/dopamine coating modified slice. Although, as discussed before, the higher the hydrolysis degree is, the more H-PMOXA immobilized on the coating and the greater corrosion resistant ability of the modified 316L SS will be, a moderate hydrolysis degree is better for excellent biocompatibility of the coating. What's more, the moderate principle also can be observed from the comparison among the platelet attachment tests of H-PMOXA/dopamine coating formed by the H-PMOXA with similar hydrolysis degree and different molecular weight (P₂, P₄, P₅/dopamine coating). The number of attached platelets on the P₄ and P₅/dopamine coating (Fig. 6G and H) was higher than that on P₂/dopamine coating (Fig. 6E). The process of electrochemical assembly of H-PMOXA and dopamine is a combination of cooperation and competition process, the oxidation and assembly of dopamine on the surface are beneficial to the immobilization of H-PMOXA on the surface, and the H-PMOXA on the coating can limit the dopamine to assemble continually at the same time. Although P₄ with low molecular weight (M̄_n 2.5 kD) could assemble with dopamine easily, the short PMOXA segments of P₄ is difficult to limit the assembly of dopamine on the surfaces; meanwhile, P₅ would result in less PMOXA segments immobilizing on the surfaces due to its bigger steric effect, therefore, P₄ and P₅/dopamine coating exhibited poorer biocompatibility. Thus, appropriate molecular weight and hydrolysis ratio are necessary for H-PMOXA modifying 316L SS slice by this strategy to build a coating with excellent antifouling and biocompatibility.

Fig. 6 The SEM images of the attachment tests of platelet on bare 316L SS slice (A), PDA coating (B), P₀/dopamine coating (C), P₁/dopamine coating (D), P₂/dopamine coating (E), P₃/dopamine coating (F), P₄/dopamine coating (G), P₅/dopamine coating (H) modified 316L SS slices. The scale bar is 50 μm. The magnified images of the attached platelets were shown in Fig. S3 of ESI.†

Furthermore, the PDA coating and P₂/dopamine coating modified 316L stent were prepared through 2 h electrochemical assembly, the stability and the biocompatibility of the coating on the complex-3D stent surfaces were investigated, respectively (Fig. 7). Firstly, the modified 316L SS stents were immersed in PBS buffer and shaken in shaking table for 72 h and then the surface cracking, peeling, and flaking of the coating was studied by SEM, the images were displayed in Fig. 7A. Compared with the bare 316L SS stent, it can be seen clearly that the surface of PDA and P₂/dopamine coating modified stents are still homogeneous, continuous and smooth. Only tiny particles can be observed in amplifying images. Thus, after being immersed in PBS buffer and shaken for 72 h, either the PDA coating or the H-PMOXA/dopamine coating could resist the destruction and surface cracking, peeling and flaking of the coating were hardly observed (the long-term stable property of the coating was also investigated as shown in Fig. S4 of ESI†). These results revealed that the PDA coating or the H-PMOXA/dopamine coating obtained by electrochemical assembly strategy is very stable under physiological environment. Besides, a simulation experiment was made to study the mechanical stability of the coating, the result was displayed in Fig. S5 ESI.† There are not cracking, peeling, and flaking in the coating on the stent, after being bended. Secondly, the attachment tests of platelet were also used to study the biocompatibility of the coating on the complex-3D stent surfaces. As shown in Fig. 7B, many platelets have attached on the surfaces of bare stent and PDA modified stent. Meanwhile the quantities of platelets attached on the P₂/dopamine coating modified surface decreased remarkably. The enlarged graphs of platelet adsorption were inserted in the right bottom corner of images. As depicted in the enlarged graphs, the platelets attached on bare stent and PDA modified stent were activated and that on the P₂/dopamine coating modified stent were not activated. Therefore, the results above unambiguously demonstrated that the P₂/dopamine coating modified surface possess excellent biocompatibility, implying that this surface modification strategy can be used to modify the complex-3D stent.

Fig. 7 The photograph of bare, PDA coated, and P₂/dopamine coated 316L SS stent, the color of PDA coated stent is brown, and the color of P₂/dopamine coated stent is light brown. SEM micrographs of bare 316L SS stent, PDA coated stent and P₂/dopamine coated stent after being immersed and shaken on the shaking table in PBS buffer for 72 h (A). SEM micrographs of the attachment tests of platelet on bare 316L SS stent, PDA coated stent and P₂/dopamine coated stent (B) (red circles show the attached platelets), inset: partially magnified images of attached platelets.

The endothelial cell proliferation and migration

As shown in Fig. 8, SEM images were conducted to estimate the HUVECs proliferation on the bare (Fig. 8A), PDA (Fig. 8B) and P₂/dopamine coating modified (Fig. 8C) 316L SS slices after 24 h cell culture. Contrastingly, the cell density of Fig. 8C is obviously higher than that of Fig. 8A and B, implying that HUVECs can excellently proliferate on P₂/dopamine coating. As shown in Fig. 9, HUVECs seeded on P₂/dopamine coating modified 316L surfaces spread out rapidly and covered the gap more than that on bare and PDA modified 316L surfaces, implying that HUVECs can excellently migrate on P₂/dopamine coating. According to previous work, PMOXA possess excellent vitro cell viability, for the chemical composition of PMOXA is similar to polypeptide. The cell viability assay (Fig. S7 in ESI†) confirmed that the hydrolysed products of PMOXA-5k also keep the good cell viability. Previous work¹⁴ has reported that although PMOXA possess excellent vitro cell viability, HUVECs cannot be adsorbed on the brush coating formed by PMOXA due to the cell difficult adhesion and proliferation on a swing polymer brush coating. However, HUVECs can excellently migrate and proliferate on P₂/dopamine coating thanks to the non-brush coating formed by the co-deposition of multi-imino groups inserted PMOXA and PDA, which implied that this kind of coating could be potentially used to enhance the vascular endothelialization and prevent in-stent restenosis.

Fig. 8 SEM images of HUVECs proliferation on the bare (A), PDA coating modified (B), and P₂/dopamine coating modified (C) 316L SS slices after 24 h cell culture. Scale bar is 200 μm. The magnified images of the attached platelets were shown in Fig. S6 of ESI.†

Fig. 9 Migration of HUVECs was visualized using DAPI staining. After culture 6 h on the slices covered with a 316L ringy mask, cell on the bare (A), PDA coating modified (B), P₂/dopamine coating modified (C) 316L slices were fixed, stained, and observed with fluorescence microscope. The samples of the other group were continue cultured for another 48 h (after the ringy mask removed), and then the cell on the bare (D), PDA coating modified (E), P₂/dopamine coating modified (F) 316L slices were fixed, stained, and observed with fluorescence microscope. Scale bar is 500 μm.

Conclusion

In summary, we have presented a simple strategy to modify the 316L SS slice and stent using the co-deposition of partially hydrolysed PMOXA and DA through electrochemical oxidation assembly, in which the coating formed is non-brush due to the Michael addition reaction between dopamine and multi-imino groups inserted in main chains of PMOXA. The studies demonstrated here indicate that hydrolysis degree and molecular weight of PMOXA are two important parameters for electrochemical assembly of H-PMOXA and dopamine on 316L SS materials. The modified 316L SS slice with hydrolysed PMOXA-5k (M̄_n of PMOXA: 5 kD, hydrolysis time: 30 min, hydrolysis degree: 17.4%) and DA possessed not only excellent anti-fouling property and biocompatibility, but also corrosion resistant ability as well. Moreover, the migration and proliferation of HUVECs were greatly enhanced on the modified 316L SS slice. What's more, the successful modification of complex-3D vascular stent implied that the surface modification technique shows a great potential for the elimination of late stent thrombosis and in-stent restenosis.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 21374109) and the Ministry of Science and Technology of China (Grant No. 2012CB933802).

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Footnote

† Electronic supplementary information (ESI) available: Additional ¹H NMR; the content of MOXA repeating unit in different coating; cytotoxicity of polymers. See DOI: 10.1039/c5ra17802a

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