Enhanced biocompatibility of biostable poly(styrene-b-isobutylene-b-styrene) elastomer via poly(dopamine)-assisted chitosan/hyaluronic acid immobilization

Abstract

The biostable poly(styrene-b-isobutylene-b-styrene) (SIBS) elastomers are well-known for their large-scale in vivo application as drug-eluting coatings in coronary stents. In this study, the SIBS elastomers were modified with a poly(dopamine) (PDA) adherent layer, followed by integrating both chitosan (CS) and hyaluronic acid (HA) onto their surfaces. The as-prepared samples (SIBS-CS-g-HA) presented excellent cytocompatibility because CS facilitates cell attachment and HA enhances cell proliferation. The initial adhesion test of E. coli on SIBS-CS-g-HA showed effective antiadhesive properties. The in vitro antibacterial test confirmed that SIBS-CS-g-HA has good antibacterial activity.

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Introduction

Thermoplastic styrenic elastomers (TPSs) possess physical properties that overlap with those of polyurethanes and silicone rubbers. The popularly used TPSs consisting of elastomeric components, i.e., isoprene, butadiene, ethylene–butylene, or ethylene–propylene hold promise as biomaterials, but over the years have found only limited in vitro biomedical applications,^1–5 because the double bonds more or less exist in their elastomeric segments.⁶ Pioneered by Kennedy and Puskas et al., poly(styrene-b-isobutylene-b-styrene) (SIBS) has been developed via a cationic polymerization strategy.⁷ It's oxidatively, hydrolytically and enzymatically stable over their lifespan in the body owing to only containing stable alternating secondary-and-quaternary carbons in its elastomeric backbone, and equally stable primary carbons as pendant groups. Therefore, it has found some in vivo applications,^8,9 such as ophthalmic implants,¹⁰ urinary tract,¹¹ artificial heart valve,¹² cartilage tissue,¹³ and drug-eluting stents (DES) coating.¹⁴ Notably, SIBS has been commercially used as the polymer platform in the Taxus™ and Firebird™ stents on a large scale.^8,15 SIBS is one of the most attractive DES coating options, but presents much less than the optimal biocompatibility because of its nature hydrophobicity. At present, just a few works on the modification of SIBS have been reported for improving biocompatibility.^15,16 Zhu et al. introduced hydrophilic sulfonic acid groups into the SIBS via sulfonation (S-SIBS), and found that the S-SIBS-coated drug-eluting stents (DES) was associated with high anti-restenotic performance and that its safety was superior to that of the SIBS-coated DES platform in terms of inflammation induction in coronary vessel walls.¹⁵

As well recognized, a desired biocompatible surface can be tailored by introducing poly(ethylene glycol) (PEG),^17–22 poly(vinylpyrrolidone),^23–27 zwitterionic material,^28–31 peptide,³² and natural polysaccharides including heparin,^33–35 chitosan (CS),^36,37 and hyaluronic acid (HA).^38–41 Among these substances, CS is a cationic polysaccharide whose cell adhesion-enhancing nature and antibacterial properties have received great interest.⁴² The polysaccharide HA, an important component of extracellular matrix (ECM) in many tissues, is capable of providing an environment that both hemocompatible and supportive of cell migration, differentiation and proliferation, but suppress cell adhesion probably because of its hydrated effect.^43,44 A substrate could be imparted with excellent biological performances via integrating the advantages of both CS and HA.^45–47

In this work, the SIBS samples were functionalized with both CS and HA assisted by a poly(dopamine) reactive layer (Fig. 1). The biological properties of the modified samples were evaluated by a series of experiments, such as in vitro response of L929 fibroblast and antibacterial activity, relative to the references.

Fig. 1 Schematics of the biostable SIBS elastomers modified with both CS and HA through a poly(dopamine) reactive layer.

Experimental section

Materials and reagents

Poly(styrene-b-isobutylene-b-styrene) (SIBS) with 30 wt% PS hard blocks was kindly provided by Kaneka Americas. Tris (hydroxymethyl) aminomethane (Tris) and 3,4-dihydroxyphenethylamine (dopamine) were obtained from Sigma-Aldrich. Chitosan (CS) (low molecular weight, MW ≤ 2000 Da) was provided by Dalian GlycoBio Co., Ltd. Hyaluronic acid (HA) (molecular weight (MW) = 17 kDa) was purchased from Liuzhou Chemicals. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride and N-hydroxysuccinimide (NHS) were obtained from Alfa Aesar. Phosphate buffered solution (PBS, 0.1 mol L⁻¹, pH = 7.4), bovine serum fibrinogen (BFg) and sodium dodecyl sulfate (SDS) were provided by Dingguo Bio-technology. Micro BCA™ protein assay reagent kit (AR1110) was purchased from Boster Biological Technology. Dulbecco's modified Eagle's medium (DMEM) and 0.25 wt% trypsin were obtained from Beijing Solarbio Science & Technology. Sterile filtered fetal bovine serum (FBS) was supplied by Beijing Yuanhengjinma Biotechnology. Fluorescein isothiocyanate-labeled phalloidin (FITC-Phalloidin) and 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride were provided by Sigma-Aldrich. Gram-negative Escherichia coli (E. coli), Luria–Bertani (LB) broth medium and agar were obtained from Dingguo Biotechnology Co., Ltd. Other reagents were AR grade and all the materials were used as received without further purification.

Surface modification of the SIBS elastomer

Surface modification of SIBS was conducted as shown in Fig. 1, and the details were described as follows. Dopamine (2 mg mL⁻¹) was dissolved in Tris–HCl buffer solution (10 mM, pH = 8.5), and the SIBS samples were dipped into the dopamine solution for a certain time. The as-prepared samples (abbreviated as SIBS-PDA) were rinsed with ultrapure water and dried under N₂ for use.

According to the literature that the polysaccharide containing –NH₂ (–NH–) can be firmly immobilized onto the poly(dopamine) (PDA) reactive layer,⁴⁸ SIBS-PDA were immersed into the CS solution (PBS buffer solution as solvent, pH = 7.4) with a concentration of 8 mg mL⁻¹ at 50 °C for 24 h. The obtained samples (denoted as SIBS-CS) were washed with ultrapure water in order to remove physically adsorbed polysaccharide and dried under N₂. For chemically grafting HA onto SIBS-PDA or SIBS-CS, HA solution (8 mg mL⁻¹) in PBS buffer was prepared, followed by adding EDC/NHS. Then SIBS-PDA or SIBS-CS was incubated in the HA solution at room temperature for 24 h. The resulting samples (denoted as SIBS-HA or SIBS-CS-g-HA) were washed with ultrapure water for removing physically adsorbed HA and dried under N₂.

Surface characterization

Surface elemental compositions were examined by X-ray photoelectron spectroscopy (XPS, VG Scientific ESCA MK II Thermo Avantage V 3.20 analyzer) with Al/K (hν = 1486.6 eV) anode mono-X-ray source at the detection angle of 90°. The spectra were collected over a range of 0–1200 eV and high-resolution spectra of C_1s regions were given.

Surface morphology was observed by an atomic force microscopy (AFM) with contact mode (SPA300HV with a SPI 3800 controller, Seiko Instruments Industry, Japan). The root-mean-square (RMS) roughness was calculated from AFM images.

After rinsing with deionized water and drying with an argon flow, water contact angle (WCA) of the samples was measured with a drop shape analysis instrument (DSA, KRÜSS GMBH, Germany) at room temperature. The values of WCA were an average of five measurements on different areas of each sample.

Nonspecific protein adsorption

After soaking in PBS solution at room temperature for 12 h, the samples were dipped into PBS solution containing BFg (1.0 mg mL⁻¹) at 37 °C for 2 h. Each sample was sequentially rinsed five times with fresh PBS, soaked in an aqueous solution containing 1.0 wt% SDS, and oscillated at 37 °C for 1.0 h to remove the adsorbed proteins from the samples. Based on bicinchoninic acid (BCA) protein assay kit, the absorbance values of the SDS solution containing proteins was tested at 570 nm with a microplate reader (TECAN SUNRISE, Swiss), and the amount of the adsorbed proteins was calculated. Each result is an average of at least three parallel results.

Cell adhesion and proliferation

Cells were cultured in DMEM supplemented with 10 vol% FBS, 4.5 g L⁻¹ glucose, 100 units per mL penicillin, 5958 mL L⁻¹ N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 100 μg mL⁻¹ streptomycin and maintained in a humidified 5 vol% CO₂/95 vol% air incubator at 37 °C.

The samples with diameter of ∼16 mm were sterilized with 75 vol% ethanol for 1 h, followed by 6 times washing in PBS. The samples were then placed in a 24-well culture plate. L929 were trypsinized and seeded into the wells at a density of 2 × 10⁵ cells per well and incubated at 37 °C under a 5 vol% CO₂ humidified atmosphere. After the L929 were cultured in the medium containing 10 vol% FBS for 1, and 2 days, the samples were carefully washed with PBS five times, and then were fixed with 4 wt% paraformaldehyde at 37 °C for 30 min, followed by five times washing in PBS. Then they were further stained with FITC-phalloidin or DAPI for 30 min at room temperature, followed by three times washing in PBS and vacuum freeze dehydration. The cells were observed under confocal laser scanning microscopy (CLSM, LSM 700, Carl Zeiss). To quantify cell adhesion, spreading and proliferation, images were analyzed with ImageJ software to determine average cell density and cell spreading area. Cell density was measured by counting the number of DAPI-stained nuclei on three different areas (center, top, bottom) in each image. The projected area was obtained by measuring the actin-stained cells.

Antibacterial property

Escherichia coli (E. coli, ATCC 25922) were used to evaluate the bacterial adhesion and bactericidal efficacy of the samples according to the literature.⁴⁹ E. coli were grown in the LB broth medium, at 37 °C overnight. Then the bacterial suspension was centrifuged and supernatant decanted. Bacterial cell concentration was calculated by testing the absorbance of cell dispersions at 540 nm relative to a standard calibration curve. An optical density of 1.0 at 540 nm is equivalent to ∼10⁹ cells per mL. After removing the supernatant, the bacterial cells were diluted with PBS to 10⁶ cells per mL. Each sample was then soaked in the bacterial suspension at 37 °C for 4 h. After fixing with 3 vol% glutaraldehyde and dehydrating with a series of ethanol aqueous solution (10, 30, 50, 70, 90, 100 vol%), the adhered bacterial cells were observed under scanning electron microscope (SEM, XL30 ESEM FEG, FEI Company, USA).

The bacterial viability on the samples was investigated using a spread plate method. The samples were immersed into 1 mL PBS solution containing bacterial suspension (cell concentration = 10⁶ cells per mL) in 24-well plates at 37 °C. After 4 h, the samples were gently washed three times with sterile PBS, and cleaned in 2 mL sterile PBS solution under mild ultrasonic for 10 min, followed by 10-fold dilution. 100 μL diluted suspension was spread onto the solid agar. After incubation at 37 °C for 24 h, the number of the viable cells was counted manually. The results were expressed as the relative viability of bacterial which was defined as the percentage of the viable cells on the modified samples relative to that on the virgin SIBS.

Statistical analysis

The statistical significance was assessed by analysis of variance (ANOVA) method, * (p < 0.05), ** (p < 0.01), *** (p < 0.001). Each result is an average of at least three parallel experiments.

Results and discussion

Surface composition

The PDA adherent layer is an extremely versatile platform for secondary reactions with various organic species.^50,51 Herein, the inert SIBS elastomers were pre-coated with a thin PDA layer, and then CS was chemically attached via Michael addition or Schiff base reactions, followed by introducing HA through the EDC/NHS chemistry.

Surface compositions of the samples were analyzed by XPS (Fig. S1†). A small amount of oxygen was detected on the virgin SIBS references owing to the oxygen contamination. After the PDA attachment, N_1s peaks appeared in XPS curves. No new peaks were observed after the additional immobilization of CS and HA, because they are made up of carbon, hydrogen, oxygen and nitrogen. In detail, the atomic N/C ratios of SIBS, SIBS-PDA, SIBS-CS and SIBS-CS-g-HA were 0, 0.045, 0.072 and 0.055, respectively (Table 1). These fluctuations in the atomic N/C ratio confirmed the immobilization of CS and HA as illustrated in Fig. 1.

Table 1 Analysis of XPS for the various SIBS samples

Sample	Atomic concentration (%)			N/C
	C	N	O
SIBS	95.05	0	4.95	0
SIBS-PDA	81.11	3.7	15.19	0.045
SIBS-CS	68.94	4.97	26.09	0.072
SIBS-HA	67.02	3.14	29.84	0.047
SIBS-CS-g-HA	69.2	3.83	26.97	0.055

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The high-resolution C_1s spectra and their peak fitting curves of the samples were shown in Fig. 2. It can be found that the C_1s curve of the virgin SIBS (Fig. 2(a)) reference was just composed of one peak C–C (C–H), while SIBS-PDA (Fig. 2(b)) consisting of three peaks, i.e., a C–C (C–H) peak, a C–N peak, and a C–OH peak. The high-resolution C_1s spectra of SIBS-CS-g-HA were decomposed into five peaks: a C–C (C–H) peak, a C–N peak, a C–O peak, a N–C=O peak and a O–C=O peak, respectively (Fig. 2(c)). Among these peaks, the N–C=O peak was attributed to both the CS and HA grafts, the O–C=O peak just corresponded to the HA grafts.

Fig. 2 High-resolution C_1s spectra and their peak fitting curves of (a) SIBS, (b) SIBS-PDA, and (c) SIBS-CS-g-HA samples.

Surface morphology and wettability

Selective control of the interactions at the tissue and biomaterial interface, depend on the synergistic parameters including surface roughness, morphology, positive/negative charges, surface chemistry, and surface free energy. It's well acknowledged that surface roughness plays an important role in manipulating cell behavior for implant biomaterials. Herein, the morphology and the typical three-dimensional images of the samples were investigated using AFM, and presented in Fig. 3.

Fig. 3 AFM three-dimensional images of (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA.

The root mean square (RMS) roughness value of the samples generally rose with the introduction of PDA and the polysaccharides, relative to the virgin SIBS reference. Although the RMS roughness values changed, their morphologies at the submicron level had little effect on cell behavior. The wettability of biomaterials can also mediate cell-matrix anchorage via the protein adhered on their surface. The WCAs for the virgin and modified SIBS samples were presented in Fig. 4. The WCA of the virgin SIBS reference reached ∼106.5° due to its hydrophobic nature. The dopamine self-polymerization to producing a PDA layer involved in the oxidation of catechol and rearrangement into dihydroxyindole, with plenty of hydroxyl and amine group left on the surface. Hence, the WCA for the SIBS-PDA surface decreased to ∼50.5°. As for the immobilization of CS or HA onto the SIBS-PDA surface, the more hydrophilic surfaces with the WCAs of ∼23.6° or ∼32.1° were respectively presented. After grafting SIBS-CS with HA, the WCA of SIBS-CS-g-HA was further enhanced to ∼15.4°.

Fig. 4 Water contact angles of (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA samples.

Nonspecific protein adsorption

The interaction between protein and polymer surface is a critical issue for biomedical applications. Protein adsorption is thought to be a key factor in determining the subsequent responses, such as the adhesion and activation of platelets, and ultimate blood coagulation. On the other hand, the adsorbed proteins can assist cell adhesion and spreading through integrin binding, and regulate cell signaling events.

Herein, BFg adsorption on the surfaces was assayed by a BCA method. From Fig. 5, the amount of the adsorbed BFg was SIBS-PDA > SIBS-CS > SIBS > SIBS-HA > SIBS-CS-HA. The isoelectric point of BFg is about 5.5, therefore BFg carries negative charge in the PBS solution (pH = 7.4), meanwhile the SIBS-CS surface was positively charged. Thus, the amount of protein adsorbed on the SIBS-CS surface was still higher than that of the virgin SIBS. In contrast, protein adsorption on the SIBS-HA and SIBS-CS-g-HA surfaces was suppressed under the synergy of hydration layer and electrostatic repulsion arising from the HA grafts.

Fig. 5 The amount of BFg adsorbed on (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA surfaces. Data analyzed using a one-way ANOVA, * (p < 0.05), ** (p < 0.01), *** (p < 0.001) significant differences compared with the virgin SIBS reference (the error bars: standard deviations, n = 3).

Cell adhesion and proliferation

The CLSM images of the samples after L929 cell incubation were given in Fig. 6, S2 and S3.† Their cell density, projected area per cell and total projected area were calculated (Fig. 7). As shown in Fig. 7(A), the order of the cell attaching amount was SIBS-PDA > SIBS-CS > SIBS-CS-HA > SIBS > SIBS-HA. The difference between SIBS-HA and SIBS-CS suggested that the negatively charged HA would suppress the attachment of fibroblast, while the positively charged CS could improve this behavior. Thus, the cell density on the SIBS-CS-g-HA surface was significantly higher than those of SIBS-HA (** p < 0.01) and the virgin SIBS (* p < 0.05) (Fig. 7(A)).

Fig. 6 CLSM images of L929 cell at different time points on the samples for 1 day and 2 days incubation and stained for nuclei (blue), actin (green). (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA (size of the scale bars: 50 μm).

Fig. 7 Cell density (A), total projected area (B), and projected area per cell (C) for 1 day incubation on the samples. (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA. Data analyzed using a one-way ANOVA, * (p < 0.05), ** (p < 0.01), *** (p < 0.001) significant differences compared with each other for (A). Significant difference compared with SIBS-CS-g-HA for (B) and (C) (the error bars: standard deviations, n = 3).

To gain insight into the interaction of cell with surfaces, the total cell spreading area and the mean spreading area were analyzed (Fig. 7(B) and (C)). The total area of cell adhesion increased from 12184 μm² (SIBS) to 56707 μm² (SIBS-CS-g-HA). The results suggested that the co-immobilization of CS and HA can greatly enhanced cell adhesion. We also quantitatively evaluated the projected area per cell, which can measure an individual cell's spread. According to the analysis, the projected areas of SIBS were approximately 297 μm² per cell, but on the SIBS-CS-g-HA the projected areas increased up to 787 μm² per cell. In addition to the enhanced cell spreading on SIBS-CS-g-HA, an accelerated development of cell cytoskeleton was also observed when stained actin filaments (Fig. 6). All the results from observation on the cell morphology of L929 confirmed that SIBS-CS-g-HA had a better cytocompatibility in this study.

Fig. 8 presented the proliferation of L929 on the various surfaces. It has been reported that HA did not support cell adhesion, but it significantly promoted cell proliferation. As expected, SIBS-HA presented the largest growth rate in the cell density (*** p < 0.001) (Fig. 8(d)). No significant changes in cell proliferation were observed for the virgin SIBS and SIBS-CS (Fig. 8(a and c)). Although the PDA layer facilitated cell adhesion (** p < 0.01, compared with virgin SIBS, Fig. 7(A)),⁵² it was harmful to cell proliferation. The calculated cell number was declined by half after 2 days incubation (** p < 0.01, compared with 1 day incubation Fig. 8(b)). The similar phenomena have also been found by Sileika and coworkers.⁵³ The low adhesiveness of HA to cells was avoided in the presence of CS, meanwhile the good cell proliferation behavior from HA was well maintained.

Fig. 8 Quantification of cell proliferation on various SIBS samples. (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA. Data analyzed using a one-way ANOVA, * (p < 0.05), ** (p < 0.01), *** (p < 0.001) (the error bars: standard deviations, n = 3).

Antibacterial activity

Preventing bacterial attachment and biofilm formation is a great issue for the optimal performances of the implanted material and medical device.⁵⁴ Anti-adhesion of bacteria was assessed via an attachment assay, in which the samples were immersed in the bacterial suspension at 37 °C for 4 h, followed by washing away the unattached bacteria on the surfaces. We found that the bacterial cells adhered readily on the virgin SIBS reference (Fig. 9(a)). Both HA and CS graft layers are hydrophilic, the hydration layers were formed under an aqueous environment. Thus, the bacteria adhesion on the samples modified with the polysaccharides was generally suppressed compared with the virgin SIBS and SIBS-PDA (Fig. 9). In detail, the electrostatic repulsion between the negatively charged HA grafts and bacteria also prevent bacteria from attaching on the HA-modified substrates (SIBS-HA and SIBS-CS-g-HA), while the positively charged CS grafts a little facilitated the bacteria adhesion (SIBS-CS and SIBS-CS-g-HA).

Fig. 9 SEM images of (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA samples after exposure to E. coli for 4 h (size of the scale bars: 10 μm).

The anti-adhesion assay measured the number of bacteria attached to the surface, but did not discriminate live or dead. Therefore, the killing efficiency of bacteria was assessed by the spread plate method, a quantitative in vitro antibacterial assay. As shown in Fig. 10, the relative bacteria viabilities on the samples modified with the polysaccharides were significantly lower than that of the virgin SIBS (*** p < 0.001). In detail, the amine groups in the CS repeating unit could easily change into cationic –NH₃⁺ groups, and altered the membrane permeability of bacterial.⁵⁵ Thus, the CS-modified substrates (SIBS-CS and SIBS-CS-g-HA) had the much better antibacterial properties. If we consider the above antibacterial activity assays together, it is clear that SIBS-CS-g-HA is effective at preventing bacteria attachment and killing them.

Fig. 10 Relative viability of E. coli in PBS in contact with the samples at 37 °C for 4 h. (a) SIBS, (b) SIBS-PDA, (c) SIBS-CS, (d) SIBS-HA, and (e) SIBS-CS-g-HA. The cell number was determined by spread plate method. Data analyzed using a one-way ANOVA, ** (p < 0.01), *** (p < 0.001) significant differences compared with the virgin SIBS reference (the error bars: standard deviations, n = 3).

Conclusions

The biostable SIBS elastomers, for the first time, were modified with both CS and HA through a poly(dopamine) reactive layer. The as-prepared samples (SIBS-CS-g-HA) showed the excellent cytocompatibility compared with the references. In addition, the HA grafts suppressed bacterial adhesion, they preserved the antibacterial efficacy of the CS grafts, therefore, good antibacterial activity was obtained for the SIBS-CS-g-HA sample. The CS/HA-modified SIBS elastomer have high potential for implant applications.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: XPS curves of the various SIBS samples, CLSM images of cell actin (green) and nuclei (blue). See DOI: 10.1039/c4ra04523h

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