Surface Modification of Poly(dimethylsiloxane) with Polydopamine and

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Surface Modification of Polydimethylsiloxane with Polydopamine and Hyaluronic Acid to Enhance Hemocompatibility for Potential Applications in Medical Implants or Devices Peng Xue, Qian Li, Yuan Li, Lihong Sun, Lei Zhang, Zhigang Xu, and Yuejun Kang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10260 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017

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ACS Applied Materials & Interfaces

Surface Modification of Polydimethylsiloxane with Polydopamine and Hyaluronic Acid to Enhance Hemocompatibility for Potential Applications in Medical Implants or Devices Peng Xue*,†,‡, Qian Li†,‡, Yuan Li§, Lihong Sun†,‡, Lei Zhang∥, Zhigang Xu†,‡, Yuejun Kang*,†,‡ †

Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy,

Southwest University, Chongqing 400715, China. ‡

Chongqing Engineering Research Center for Micro-

Nano Biomedical Materials and Devices, Chongqing 400715, China. §

Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China.

∥

State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing

400716, China.

Abstract Polydimethylsiloxane (PDMS) has been widely utilized in microelectromechanical systems (MEMS) and implantable devices. To improve the hemocompatibility of PDMSbased implant, a facile technique was developed by modifying PDMS with a hyaluronic acid (HA) and polydopamine (PDA) composite (HA/PDA). Under appropriate ratio of HA to PDA, platelet adhesion and activation was considerably reduced on modified PDMS substrates, indicating an enhanced hemocompatibility compared to native PDMS or those coated with HA or PDA solely.

HA/PDA coating also posed minimal

cytotoxicity on the adhesion and proliferation of endothelial cells (HUVECs). The antiinflammation effect of the modified PDMS surface was characterized based on the expression of critical cytokines in adherent macrophages. This study revealed that the hemocompatibility, cytotoxicity and anti-inflammation properties could be tailored conveniently by adjusting the ratio of HA and PDA composite on the modified PDMS surface, which has an exceptional potential as the core or packaging material for constructing implantable devices in biomedical applications. Keywords: polydimethylsiloxane, polydopamine, hyaluronic acid, hemocompatibility, anti-inflammation, surface functionalization.

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1 Introduction In modern healthcare industry, medical implants and devices have been designed and invented for various therapeutic and rehabilitative applications1. These functional objects range from physical supports such as joint replacement implants and artificial blood vessels, to human organ improvement accessories such as pacemakers and intravascular stents2. A variety of materials have been used for constructing medical implants with advantages of mechanical strength, toughness and biochemical stability3. Metallic materials, such as lead, titanium, stainless steel and cobalt alloy, are the earliest pioneers as hard tissue substitutes4-8. Meanwhile, ceramics, usually inorganics, including silicates, carbides and metallic oxide, contribute to the development of dental crown in dentistry due to their resemblance of natural teeth9, 10. Recently, polymers with high molecular weights have been extensively applied in biomedical implants and packing devices, including polyvinylidene fluoride (PVDF), polyethylene (PE), polypropylene (PP) and poly(methyl methacrylate) (PMMA) 11-14. The placement of these materials within human body varies depending on specific applications. Additionally, special requirement of each implant determines the material design and properties15. As an important class of polymers, silicones are generally inert and exist in various forms. Silicone-based implants are widely used to adjust human aesthetics in surgeries with excellent protective functions and low infection rates16,

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. Moreover,

silicone is advantageous for long-term immobilization in human body due to low surface energy and smooth surface topography18. These features prevent cells and molecules from being adsorbed on polymeric surfaces, which is a highly desirable protective feature for medical implants. As a popular silicone derivative, polydimethylsiloxane (PDMS) is an elastomer with attractive properties for developing microelectromechanical systems (MEMS) and implantable devices19, 20. For instance, PDMS has been widely used as accessories or essential components of pacemakers, blood pumps, shunts, esophagus replacement, and packaging materials for integrated electronic devices and sensors20, 21. Particularly, PDMS-based drug reservoir was fabricated to constantly release drug into a human eye for treatment of ocular posterior segment diseases22, 23. PDMS was also applied as a neuronal culture substrate, exhibiting a good potential for fabricating flexible microelectrode array implants24. However, there are still some critical concerns that

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hinder the applications of PDMS-based implants for long-term utilization in human body. Particularly, adverse immunological reactions are predominant issues that result in the overall failure of PDMS-based implants in many applications25. Surface modifications by conjugating biomolecules have been extensively applied on biomedical devices for tissue engineering to prevent plasmatic coagulation and suppress immunological responses26. To improve the hemocompatibility of implants, various biomolecules including heparin, hyaluronic acid, polyethylene glycol (PEG), CD34, zwitterions (e.g., sulfobetaine) and RGD peptides have been coated on the implant surfaces based on physical adsorption, electrostatic interactions or covalent bonding27-32. The effect of surface modifications on hemocompatibility varies depending on specific implant materials. Although the blood compatibility and endothelization of PDMS-based implants are better than many other materials, there is still plenty of room to further enhance the hemocompatibility particularly in terms of anticoagulation, antiinflammation and anti-hyperplasia33, 34. PEG-grafted and carboxybetaine-modified PDMS surfaces have enhanced hemocompatibility compared to native PDMS27, 35. However, such molecular conjugations require multiple steps and harsh reaction conditions during surface functionalization. It is desirable to develop more convenient coating techniques for PDMS modifications. Polydopamine (PDA) has been proposed as a biocompatible coating material, which can be strongly adsorbed onto a majority of substrates through covalent conjugation or physical interactions36, 37. Previously, PDA was used to pre-treat PDMS surface to support long-term cell culture for stem cell differentiations or development of drug screening platforms owing to the rich amino groups of PDA molecules38, 39. Moreover, PDA also acts as a surface mediator to provide active binding sites for conjugating other biomolecules26, 40. However, the hemocompatibility of PDA is even poorer than native PDMS substrate41. On the other hand, hyaluronic acid (HA) is a major component of extracellular matrix (ECM) with key functions of anticoagulation and anti-inflammation42.

Surface coating with HA may be an efficient method to

improve the anticoagulant properties of PDMS substrates43,

44

. In fact, Wu and co-

workers have developed a novel multi-coating technique based on a HA/PDA composite to modify the stainless steel surfaces of cardiovascular implanted devices45. This promising multifunctional surface treatment has proved to effectively enhance the

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hemocompatibility of metal implants, induce desirable cell-material interactions, while suppress inflammatory reactions and cause milder tissue responses in vivo. It has been reported that PDA can be efficiently adsorbed on PDMS surfaces38, and there is a strong electrostatic interaction and hydrogen bonding between PDA and HA molecules46. Therefore, it may be a rational design to include HA with PDA coating to improve the hemocompatibility of PDMS-based medical implants. Inspired by Wu and co-workers’ original work using HA/PDA multi-coating on metal materials45, we applied a similar method but on a polymeric material to achieve optimal anticoagulant and anti-inflammatory functionalization of PDMS-based implants or medical devices (Figure 1). In this study, PDMS surfaces was processed with three different HA/PDA conjugates by varying the amount ratio of these two components, and characterized by Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Compared to previous PDMS modification approaches that usually require complex reactions, HA/PDA coating needs only two steps free from harsh reaction conditions. The hemocompatibility of the modified PDMS substrates was studied based on platelet adhesion and activation assays. Further investigations on macrophage adhesion, activation and release of inflammatory cytokines were also performed for assessing the anti-inflammation property. These important in vitro assays, as used previously for characterizing HA/PDA-coated metal materials45, were also followed to elucidate the enhanced surface hemocompatibility of PDMS-based devices for potential biomedical implant applications.

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Figure 1. Schematic of PDMS surface modification procedure using PDA and HA, and the hemocompatibility evaluation using multiple types of cells.

2 Materials and methods 2.1 Materials Polydimethylsiloxane SYLGARD 184 silicone elastomer kit was purchased from Dow Corning Inc., USA. Formalin, Triton X-100, bovine serum albumin (BSA) and dopamine hydrochloride (Mw=189.64) were obtained from Sigma Aldrich, USA. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, TrypLE™ Express, PrestoBlue cell viability assay kit, micro-BCA protein assay kit, CyQUANT cell proliferation assay kit, fibronectin, DAPI, Alexa Fluor 633 phalloidin, Dead cell Apoptosis Kit with Annexin V Alexa FluorTM 488 & Propidium Iodide and 1×PBS (pH=7.4) were provided by Life Technologies, USA. Ninhydrin (≥98%, MW =178.14) and hyaluronic acid (≥95%) were purchased from Aladdin, China. The mouse HA ELISA Kit was obtained from Shanghai Crystal Biological Engineering, China. Human Alpha-granular membrane protein (GMP-140) ELISA Kit, A020-02LDH Kit, Mouse Interleukin 6 (IL-6) ELISA Kit, Mouse Interleukin 12 (IL-12) ELISA Kit and Mouse Tumors necrosis factor α (TNF-α) ELISA Kit were purchased from Beyotime, China. Fresh human whole blood was obtained by the volunteer from Yongchuan

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Hospital affiliated to Chongqing Medical University. Deionized (DI) water with a resistivity of 18.2 MΩ·cm was collected from Millipore Synthesis A10 (Molsheim, France).

2.2 PDMS surface modification with HA and PDA Native PDMS substrates were fabricated based on traditional prototyping method. Briefly, PDMS prepolymer prepared by mixing elastomer and curing agent at a mass ratio of 10:1 was dropwise added into individual reservoirs of 24-well tissue culture plates (TCPs). Subsequently, PDMS-loaded TCPs were transferred into a vacuum oven and degassed for 1 h, followed by hard baking at 70 oC for 2 h. Then, the crosslinked PDMS slices were ready for surface functionalization. To modify PDMS surface with PDA, dopamine solution was first prepared by dissolving dopamine in 10 mM Tris buffer (pH = 8.5). Then, 1 mL of dopamine solution (2 mg mL-1) was introduced into each well at 25oC for 24 h. After that, the substrate was washed with DI water three times to remove the unbound molecules, and the PDA-coated PDMS substrate was ready for use after drying. To further label the PDMS substrate with HA, the PDA-coated PDMS was immersed into HA solutions with a range of concentrations (1, 2 and 5 mg mL-1) at 37oC for 12 h, and labeled as HA-PDA-1, HA-PDA-2, HA-PDA-3, respectively. The final PDMS surface with HA/PDA functionalization was obtained by thoroughly cleaning with DI water. Native PDMS and PDMS incubated with HA (2 mg mL-1) were used as control groups.

2.3 Characterizations of modified PDMS PDMS surface characterizations in terms of physical and chemical properties are critical to understand the cell behavior during hemocompatibility testing. Water contact angle on PDMS was measured using a Theta Optical Tensiometer (Attension, Finland) to evaluate surface wettability. Briefly, 1 µL of DI water was dropped onto the samples to form static sessile drops, where the contact angles were analyzed using a tangent method. Three random points on each sample were measured. Surface morphology and roughness were determined by atomic force microscopy (AFM, Bruker, Germany). Topographical images of a scanning area of 2 µm × 2 µm were measured in a tapping mode at a frequency of 0.8 Hz. Three sampling points on each PDMS substrate were analyzed for surface roughness.

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Additionally, modified PDMS surface chemical composition was analyzed using ninhydrin-based colorimetric assay and ELISA assay. Free amine groups were quantified by immersing PDA-coated PDMS substrates with 2% ninhydrin dissolved in 60% ethanol at 100 °C for 15 min, followed by measuring UV-vis-NIR absorbance of the reagent at a wavelength of 570 nm. The correlation between the amount of free amine groups and the concentration of DA solution was determined using ninhydrin assay36. The quantitation of immobilized HA on various PDMS substrates was determined using mouse HA ELISA assay following a provided protocol47.

2.4 Blood compatibility of modified PDMS Platelets isolated from human whole blood were used to assess the blood compatibility of HA/PDA-coated PDMS surface in vitro. The native, PDA-coated and HA-coated PDMS were used as control. The experimental groups included HA/PDA-coated PDMS obtained using three different HA coating concentrations. All blood samples were collected from healthy donors with their informed consent and analyzed in Yongchuan Hospital of Chongqing Medical University. The recruitment of donors and utilization of blood samples were performed strictly in compliance with the approval and guidelines of the Institutional Review Board (IRB) of Chongqing Medical University. Platelets were first isolated from whole blood for adhesion tests. Briefly, fresh whole blood was centrifuged at 1500 g for 6 min. The supernatant plasma containing platelets was collected and transferred into another centrifuge tube to reach equilibrium, followed by another centrifugation at 1000 g for 6 min. Three quarters of the supernatant plasma containing small amount of platelets were discarded, and the remaining supernatant was collected as platelet-rich plasma (PRP). Afterwards, 1 mL of PRP (3×108 cells mL-1) was uniformly added onto various PDMS substrates, and incubated at 37 oC for 1 h. To investigate the adhesion behavior of adsorbed platelets, surface morphology was characterized with scanning electronic microscopy (SEM) and surface distribution was measured with confocal laser scanning microscopy (CLSM). For SEM analysis, the substrates were washed with 1×PBS (pH = 7.4) three times and the adhered platelets were fixed with 2.5% glutaraldehyde solution at 4oC for 2 h. Surface dehydration was performed by washing with ethanol solution under gradient concentrations (50%, 75%,

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90% and 100%) progressively. Dealcoholization was conducted by washing with isoamylacetate dissolved in ethanol under gradient concentrations (50%, 75%, 90% and 100%) serially. All the samples were dried in ambient environment and sputtered with gold for SEM characterization (JSM-6510LV, JEOL, Japan). For 3D imaging of the adhered platelets, the substrates were washed with 1×PBS (pH = 7.4) five times. Then, the samples were fixed with formaldehyde solution for 30 min, followed by rinsing with PBS three times. After drying in ambient environment, 3D micro-morphology of platelets were measured using CLSM (VK-X150, Keyence, Japan). To calculate the amount of adhered and activated platelets on various PDMS substrates, LDH and GMP 140 assays were performed following the kit manuals, respectively48.

2.5 HUVEC adhesion and proliferation on modified PDMS HUVECs were purchased from the cell repository of Chinese Academy of Sciences, and cultured at 37oC with 5% CO2 under the humidified atmosphere. Cells in the passages between 3 and 5 were used for testing. Various PDMS surfaces were prepared individually in a 24-well plate as described in Section 2.2. Before cell seeding, all the samples were sterilized by UV light irradiation for 1 min. Afterwards, HUVECs were seeded onto each sample at a density of 2×104 cells in each well, and grown at 37oC for 4 h, 24 h and 72 h. Cell adhesion efficiency and proliferation were evaluated using CyQUANT proliferation assay. Specifically, cells were washed three times with PBS after incubation for 4 h, 24 h or 72 h. Then, cells were retrieved with TrypLE™ Express and frozen at −80oC for 1 h. Afterwards, cells were rapidly thawed and lysed by adding 200 µL of lysis buffer containing 1×CyQUANT GR dye. After staining for 2 min, cell lysates were added into a 96-well plate and the fluorescence intensity was measured using a microplate reader (SPARK 10M, TECAN) under an excitation (Ex) wavelength of 485 nm and an emission (Em) wavelength of 535 nm. The calibration curve of the fluorescence intensity as the function of cell number was measured using a standard solution. Hence, cell numbers on various PDMS substrates were calculated based on the standard curve after fluorescence measurement. The viability of HUVECs was evaluated using PrestoBlue assays. PDMS substrates were washed with PBS three times after incubation for 4 h, 24 h

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or 72 h. Then, 10% PrestoBlue reagent (in DMEM) was added. After 1 h of incubation, reaction reagent was transferred into a 96-well plate and UV-vis-NIR absorbance was measured under the wavelengths of 570 nm and 600 nm with a microplate reader (SPARK 10M, TECAN). To observe the cell morphology during adhesion and proliferation, the cells were stained with commercial fluorescent dyes, including Rhodamine-phalloidin reagent and 4',6-diamidino-2-phenylindole (DAPI). Briefly, cells were fixed with formalin for 30 min, and then permeabilized with Triton X-100 (0.1% in 1 × PBS, v/v) for 5 min and blocked with BSA (1% in 1 × PBS, w/v) for 30 min. Before microscopic imaging, cells were stained with Rhodamine-phalloidin (20 nM) for 1 h and DAPI (1 µg mL-1) for 5 min. Finally, the fluorescence images were acquired using CLSM with two individual laser channels (Ex = 405 nm and Em = 410-480 nm for DAPI, Ex = 488 nm and Em = 490-630 nm for Rhodamine-phalloidin).

2.6 HUVEC apoptosis on modified PDMS To analyze HUVEC apoptosis on the DA/HA-coated PDMS substrate, various PDMS substrates were prepared and placed on a 12-well plate. Then, cells were seeded at a density of 1×105 in each well. After 12 h and 24 h of incubation, cells were collected and concentrated by using TrypLE™ Express. The cells were washed with PBS and loaded into 100 µL of 1×annexin-binding buffer. Then, 5 µL Alexa Fluor 488 annexin V (stock solution) and 1 µL of PI (100 µg mL-1) was introduced to each 100 µL of cell suspension. Then, cells were incubated with the reagents for 15 min in the dark, followed by adding 400 µL of 1×annexin-binding buffer. The stained cells were analyzed using flow cytometry (Ex = 488 nm, Em = 530-575 nm).

2.7 Anti-inflammation property of modified PDMS Inflammation induced by implantable devices is a critical problem and may cause thrombosis, hyperplasia or other unpredictable complications49. During inflammation, blood monocytes migrate into the tissue and transform into macrophages. The activation of macrophages can be evaluated and confirmed by the released amount of cytokines and protein markers related to inflammation, including tumor necrosis factor-alpha (TNF-α),

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interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12)50. The amount of adhered macrophages and released cytokines are direct indicators to evaluate the antiinflammation properties of HA/DA-coated PDMS surface. In this study, TNF-α, IL-6 and IL-12 were selected as the typical markers for the assessment of inflammation process. The macrophages were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). To measure the amount of released cytokines, macrophages were seeded into a 24-well plate coated with PDMS at a density of 1×104 in each well and incubated for 24 h. Then, the supernatant of culture medium was harvested, and the amount of TNF-α, IL-6 and IL-12 released were measured using the corresponding ELISA kits following the provided protocols. To characterize the morphology of the adhered macrophages on various PDMS surfaces, cells were seeded into a 24-well plate coated with PDMS at a density of 1×104 per well and grown for 24 h. The attached macrophages was stained with Rhodaminephalloidin reagent and DAPI. The adherence efficiency of macrophages was measured using CyQUANT cell proliferation assay, as described in Section 2.5. Finally, fluorescence images of the cells on each sample were acquired using CLSM.

2.8 Statistic analyses Statistical analyses were performed using one-way analysis of variance (ANOVA). A probability p-value smaller than 0.05 was considered significant. The data were analyzed using Origin (OriginLab, MA, USA) and presented as the mean ± standard deviation (SD).

3 Results and discussion Dopamine is a natural chemical in brain as a neuro-transmitter, which can be spontaneously polymerized into PDA under alkaline conditions without oxidants51. PDA can potentially self-assemble on PDMS surface under pH of 8~9. Meanwhile, hyaluronic acid (HA) with excellent hemocompatibility can be adsorbed onto the PDA layer based on a strong electrostatic interaction or hydrogen bonding interaction. To evaluate the HA adsorption on the hemocompatibility of PDMS surface, HA solutions with a range of

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concentrations (1 mg mL−1, 2 mg mL−1 and 5 mg mL−1) were used to construct the HA/PDA-modified PDMS surface (denoted as HA-PDA-1, HA-PDA-2 and HA-PDA-3), respectively. 3.1 Surface characterizations Surface properties such as wettability and topography have fundamental effects on cellmaterial interactions37,

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. The water contact angles of various PDMS substrates are

shown in Figure 2a. The uncoated PDMS surface exhibited a contact angle of 111.8o, which was typical for a hydrophobic surface. After modification with PDA, the contact angle dramatically decreased to 84.3o, indicating the conversion into a hydrophilic surface. These results were consistent with the findings in the previous report52, demonstrating that PDA coating could reduce the substrate hydrophobicity. On the other hand, the surface wettability was not significantly altered and remained hydrophobic (contact angle = 104.8o) after PDMS surface was coated with HA. Furthermore, the contact angle of PDA-coated PDMS after HA immobilization slightly increased compared to those before HA treatment, implying that the effect of HA coating on the wettability of PDMS was limited. The contact angle of HA/PDA-modified PDMS surface was only 10~15% less than that of native PDMS, indicating a slight hydrophilization effect of HA/PDA composite coating.

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Figure 2. Surface characterizations of various PDMS substrates: (a) water contact angle measurement based on sessile drops on native (a1) and treated PDMS surfaces coated with PDA (a2), HA (a3) and HA/PDA (a4-a6); the corresponding contact angles measured on various substrates (a7). (b) AFM characterizations of various PDMS substrates: surface topography of native (b1) and treated PDMS surfaces coated with PDA (b2), HA (b3) and HA/PDA (b4-b6); the corresponding RMS roughness on various substrates (b7). Surface roughness also influences cell behavior either individually or in terms of mass population53. The topography and roughness of various PDMS surfaces were analyzed using AFM (Figure 2b). The root-mean-square (RMS) roughness of native PDMS surface was measured as 2.37 nm and no apparent irregularity was observed. However, the RMS roughness increased to 7.52 nm after PDA deposition, which was probably due to the aggregation of PDA molecules during polymerization process54 and

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PDA nanoparticles formed on the PDMS surface by self-assembly55. Meanwhile, the PDA-coated PDMS became much smoother after HA immobilization with a roughness of approximately 2~6 nm. More specifically, the roughness of HA/PDA-coated PDMS decreased with the increase of HA concentration in the coating process (HA-PDA-1: 5.94 nm; HA-PDA-2: 2.91 nm, HA-PDA-3: 2.15 nm). Because HA molecule is highly soluble and hygroscopic, higher amount of HA may form a hydrogel layer on the PDMS substrate56 and thus create a smoother surface. Additionally, PDMS solely coated with HA exhibited an average roughness of 1.8 nm, which was slightly less than that of native PDMS (2.37 nm). These results indicated that immobilized HA had a notable effect on the PDMS surface topography and roughness.

3.2 Surface coating with PDA and HA The amount of PDA immobilized on PDMS surfaces was evaluated following a ninhydrin assay by comparing the free amine concentrations before and after coating37. Ninhydrin, which has a yellow color originally, reacts with the amino groups in PDA molecules. The resultant yields a purple color product through the reduction process of ninhydrin, and the color intensity produced is proportional to the amount of amino groups. Therefore, the UV-NIR absorbance of the purple product generated after the reaction is measured at a wavelength of 570 nm to quantitate the amount of amino groups using ninhydrin reagents. As shown in Figure 3a, no PDA was detected on the native PDMS substrate or those solely coated with HA, indicating an excellent specificity of ninhydrin assay for PDA quantitation. In contrast, PDA-modified PDMS exhibited a considerably higher PDA concentration of 0.166 mg cm-2, showing successful immobilization of PDA onto PDMS surface. During the PDA coating process, 2 mg mL-1 was used as the optimal concentration of DA because the increment of immobilized PDA was not significant when further increasing DA concentration to above 2 mg mL-1. The amount of immobilized HA was measured by traditional ELISA assays46 (Figure 3b). The PDMS substrates coated with PDA only were used as a negative control and those coated with HA only as a positive control. It was observed that surface-bound HA significantly increased with its concentration for all HA/PDA-modified PDMS. Specifically, the amount of immobilized HA on HA-PDA-3 was approximately 3 folds

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higher than that on HA-PDA-1 (*p-value < 0.05). Although the HA-coated and HAPDA-2 PDMS were prepared under the same HA concentration (2 mg mL−1), the latter one pre-treated with a layer of PDA showed a notable improvement in HA immobilization. The enhanced HA binding on top of a PDA layer was probably attributed to the Michael reaction and covalent conjugation between PDA and HA molecules46. This efficient method for HA immobilization is very promising and highly desirable to improve the hemocompatibility of PDMS surface.

Figure 3. (a) Quantification of immobilized PDA on various PDMS surfaces; (b) Quantification of immobilized HA on various PDMS surfaces. Data are shown as the mean ± SD (n = 4). *p-value < 0.05 between two groups under comparison.

3.3 Platelet adhesion and activation Platelet adhesion is a straightforward test to evaluate the blood compatibility of HA/PDA-coated PDMS. The adhered platelets on various PDMS substrates are shown in the SEM images (Figure 4a). The PDA-coated surface exhibited a vein-like rough texture due to the aggregation of DA molecules after polymerization, which was consistent to the highest roughness found in AFM characterizations. Moreover, the surfaces became smoother under higher coating concentrations of HA. We observed that there was a massive amount of platelets aggregated on native and PDA-coated PDMS, indicating poor hemocompatibility that may induce platelet coagulations. In fact, surface modification with PDA favored platelet adhesion on PDMS. However, the amount of

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adherent platelets dramatically decreased on HA-coated PDMS, showing better hemocompatibility. For HA/PDA-coated PDMS, the platelet adhesion further reduced on the surfaces treated with higher HA concentrations, which could be due to the adsorbed fibrinogen under excessive HA that hampered platelet adherence and coagulation57. The confocal laser scanning microscopy (CLSM) showed that the platelets on HA-PDA-2/3 were randomly distributed and there was no significant coagulation induced by platelet activation (Figure 4b). Meanwhile, the HA immobilized on HA-PDA-1 was not sufficient to completely block the active sites of PDA, which caused notable platelet adhesion. Considering the surface coating effect on HA immobilization (Figure 3b), this adhesion test suggested that PDA might act as an effective mediator to tailor the functionality of HA for inhibition of platelet attachments.

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Figure 4. Platelet adhesions on native PDMS and those treated with PDA, HA or HA/PDA composites: (a) SEM characterizations (red arrows indicate typical platelet aggregates; scale bars: 20 µm); (b) 3-dimensional CLSM characterizations.

Generally, the quantity of adherent platelets is closely correlated to the relative amount of lactate dehydrogenase (LDH) expressed in a platelet population, which can be released by cell lysing and quantitated based on a colorimetric assay. We characterized the adhered platelets on various PDMS surfaces using LDH assays (Figure 5a). Compared to native PDMS, PDA-coated group showed a higher level of LDH due to the better bioaffinity of PDA molecules. On the other hand, the LDH expression on HA- and

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HA/PDA-coated PDMS was much less than the PDA-coated group, because HA has been proved to inhibit cell adhesion42, 44. In particular, there was a significantly lower level of LDH on HA-PDA-2 compared to other groups. Moreover, platelet activation is a critical step prior to thrombus formation during hemostasis. The activation of adhered platelets was characterized based on the expression of GMP-140, which is an alpha-granule membrane protein on the plasma membrane of a platelet after activation. Therefore, the amount of GMP-140 can indirectly show the platelet activation levels. Figure 5b indicated that the expressions of GMP-140 on HA-PDA-2/3 were significantly lower than those on other surfaces, which implied that platelet activation was suppressed by HA/PDA composite coating with higher HA concentrations. HA is known as a critical natural component of extracellular matrix (ECM) and does not favor activation-induced platelet coagulation42. These results demonstrated the poor adaptability of platelets on HA/PDA-coated PDMS, while such surface inertness may contribute to the improved hemocompatibility of PDMS-based devices.

Figure 5. (a) LDH assays and (b) GMP-140 assays to characterize platelet adhesion and activation, respectively. All data are shown as the mean ± SD (n = 4). *p-value < 0.05 between experimental and control groups.

3.4 Cellular behaviors on modified PDMS To evaluate the biocompatibility of the treated PDMS, the cytotoxicity of the composite coating was assessed using HUVECs. The cellular metabolic activity and cell quantity

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were determined using PrestoBlue viability assay and CyQUANT proliferation assay, respectively (Figure 6). The data were recorded and analyzed after 4 h, 24 h and 72 h of incubation on the substrates of native, PDA-coated, HA-coated and HA/PDA-coated PDMS. CyQUANT proliferation assay is a direct method to quantify the attached cells based on the total DNA (Figure 6a). It was observed that cell quantity exhibited a typical time-dependent increase, implying that all the substrates supported cell growth within 72 h of incubation. Particularly, the cell quantity on the PDA-coated PDMS was the highest among all groups of substrates, which was consistent with the previous finding that PDA coating could effectively improve the attachment and proliferation of cells39,

58

.

Meanwhile, the cell quantity on HA/PDA-coated PDMS was generally lower than that on PDA-coated substrates, and was negatively correlated to the HA concentration. These results suggested that excessive HA could inhibit HUVEC adhesion and proliferation59. However, unsaturated coating under lower HA concentrations exposed more active sites of PDA molecules, which facilitated HUVEC proliferation through inducing the intracellular secretion of type IV collagen41. Moreover, the cell proliferation on HAPDA-1 was higher than that on native PDMS or those solely coated with HA, indicating a more biocompatible surface multi-coated with HA/PDA. To evaluate cell viability, we used standard assays based on PrestoBlue reduction, because it was closely related to the metabolic activity of living cells. The viability of HUVECs on various PDMS substrates (Figure 6b) exhibited a profile similar to the proliferation assays, in which the PDAcoated PDMS supported the highest viability while those on HA/PDA-coated substrates decreased under higher HA concentrations.

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Figure 6. The proliferation assays (a) and viability assays (b) of HUVECs on native PDMS and those coated with PDA, HA and HA/PDA composites for 4 h, 24 h and 72 h. #

p-value < 0.05 between two groups under comparison; *p-value < 0.05 as compared to

the native PDMS as a negative control; ∆p-value < 0.05 as compared to the PDA-coated PDMS as a positive control; @p-value < 0.05 as compared to the HA-coated PDMS as a positive control.

The adherent HUVECs on various PDMS substrates were also examined using optical microscopy and CLSM after 4 h, 24 h and 72 h (Figure 7). The cells on native PDMS exhibited the lowest density due to the poor surface biocompatibility, while those on PDA-coated substrates showed optimal cell spreading and reached confluence after 72 h. The cell density generally decreased on the HA/PDA-coated substrates with higher HA concentrations, which was consistent with the proliferation results in Figure 6a. CLSM imaging indicated abnormal cell morphologies and shrinkage on native PDMS, HA- and HA/PDA-coated substrates (Figure 7b). Meanwhile, the cells on PDMS coated with HA, HA-PDA-1 and HA-PDA-2 showed similar or slightly higher viability as compared to those on native PDMS (Figure 6b). However, the cell viability on these substrates was still suboptimal as compared to PDA-coated PDMS. In addition, the cells on PDMS coated with HA-PDA-3 showed even further reduction in viability. Although native or HA/PDA-coated PDMS could support viable cells in short-term incubation, they could not fully assist regular actin polymerization and myosin contraction, resulting in abnormal morphologies, cell shrinkage and thus, limited proliferation and viability. The

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results in platelet adhesion and activation, however, revealed that the single coating only with PDA had poor hemocompatibility (Figures 4 and 5). Therefore, there is a balance between bioaffinity and hemocompatibility, which may be achieved by adjusting the ratio between HA and PDA components in the composite coating.

Figure 7. Bright field and fluorescence images of HUVECs adhered on native, PDAcoated, HA-coated and HA/PDA-coated PDMS after incubation of 4 h, 24 h and 72 h. Scale bars: 100 µm for bright field images; 50 µm for fluorescence images.

We further examined the cellular fate of HUVECs cultivated on various PDMS substrates using flow cytometry-based apoptosis assay (Figure 8). It was observed that 20


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the percentage of apoptotic cells increased with the incubation time. Specifically, 22.65% of HUVECs incubated on native PDMS entered the end stage of apoptosis after 24 h, while the apoptotic rate was below 10% on PDA-coated substrate. Meanwhile, the cell apoptosis rate ranged from 12.34% to 17.87% on HA-coated and HA/PDA-coated substrates, suggesting improved biocompatibility compared to native PDMS.

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Figure 8. Apoptosis assays of HUVECs incubated on native PDMS and those coated with PDA, HA and HA/PDA composite for 12 h (a) and 24 h (b). The upper right quadrant represents the end stage of apoptotic cells.

(c) Comparison of HUVEC

apoptotic rate on various PDMS substrates.

3.5 Anti-inflammation property of modified PDMS Macrophages are usually the precursors that migrate to the interface between native tissues and implants. The potential inflammation induced by implant materials can be monitored based on the relevant cytokines released from the activated macrophages60, which target on inflammatory cells and accumulate at the wound areas to induce inflammatory reaction61. In this study, several important cytokines including TNF-α, IL-6 and IL-12 that were released from macrophages cultured on various PDMS substrates were measured using ELISA. First, the morphologies and quantities of adhered macrophages were characterized using CLSM (Figure 9a-f) and CyQUANT cell proliferation assay (Figure 9g), respectively. Obviously, the macrophages exhibited the highest density and spread normally on PDA-coated PDMS. However, extended pseudopods and cytoplasmic fringes of the sparse macrophages were observed on HAcoated PDMS. In addition, the native and HA-coated PDMS demonstrated much weaker affinity with macrophages, suggesting a notable inhibition effect of HA on macrophage adhesion. Similar to above results for HUVECs and platelets, macrophage adhesion was also compromised under a higher level of HA immobilization on the HA/PDA-coated PDMS. The amount of released cytokines was closely related to the quantity of adhered macrophages and thereby the surface anti-inflammation property62. Inflammation is a typical cascade process and TNF-α, IL-6 and IL-12 are the direct indicators to monitor the inflammation progress. As shown in Figure 9h, the released TNF-α decreased considerably with the increase of HA in the HA/PDA-coated substrates. IL-12 exhibited a trend similar to TNF-α but with less significant difference (Figure 9j). Interestingly, the expression of IL-6 (Figure 9i) was not sensitive to the substrate modifications except under very high HA concentrations (HA-PDA-3). Because TNF-α and IL-12 are Th1 cytokines, while IL-6 is a typical pro-inflammatory cytokine63, their different responses to surface modifications could be related to their particular biological functions. These

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results suggested that PDMS surface modification with HA/PDA composite had an evident effect on reducing the expression of Th1 cytokines and thus suppressing inflammatory responses.

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Figure 9. Fluorescence images of adherent macrophages on native (a), PDA-coated (b), HA-coated (c) and HA/PDA-coated (d-f) PDMS after 24 h of incubation (Scale bar: 50

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µm); The quantities of adherent macrophages (g) and expression of three typical cytokines (h: TNF-α, i: IL-6 and j: IL-12) after 24 h of incubation on various PDMS substrates. The data are shown as the mean ± SD (n = 4). *p-value < 0.05 between two groups under comparison. #p-value < 0.05 as compared to the PDA-coated PDMS as negative control.

∆

p-value < 0.05 as compared to the HA-coated PDMS as positive

control.

4 Conclusions We investigated the surface modification of PDMS using a composite of hyaluronic acid (HA) and polydopamine (PDA) to enhance the hemocompatibility while suppress inflammation responses, which is a crucial requirement for PDMS-based implantable devices. This convenient surface treatment is based on the strong interactions between HA and PDA molecules, taking advantage of both bioaffinity of PDA and the anticoagulation and anti-inflammation properties of HA. More importantly, a balance between bioaffinity and hemocompatibility could be achieved by adjusting the ratio between HA and PDA components in the coating composite. This in vitro study demonstrated a notable improvement on the modified surface in terms of platelet inactivation, inhibition of inflammation reaction, and reduction of cytotoxicity as compared to native PDMS. This convenient surface treatment is a promising method that may effectively improve the functionality of PDMS as the core or packaging materials for design and fabrication of medical implantable devices.

Author information Corresponding authors *E-mail: [email protected]. Tel: +86-23-68253792. Fax: +86-23-68254969. *E-mail: [email protected]. Tel: +86-23-68254056. Fax: +86-23-68254969. Author contributions Peng Xue and Qian Li contributed equally to this work. Notes The authors declare no competing financial interest.

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Acknowledgements P. X. is grateful to the start-up grant from Southwest University (SWU116032), Fundamental Research Funds for Central Universities (XDJK2017C001) and National Natural Science Foundation of China (51703186). Y.K. acknowledges the Fundamental Research Funds for Central Universities (SWU115059 and XDJK2016A010) and the National Natural Science Foundation of China (31671037).

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55. Yang, Y.; Lu, Y. F.; Lu, M. C.; Huang, J. M.; Haddad, R.; Xomeritakis, G.; Liu, N. G.; Malanoski, A. P.; Sturmayr, D.; Fan, H. Y.; Sasaki, D. Y.; Assink, R. A.; Shelnutt, J. A.; van Swol, F.; Lopez, G. P.; Burns, A. R.; Brinker, C. J. Functional Nanocomposites Prepared by Self-assembly and Polymerization of Diacetylene Surfactants and Silicic Acid. J. Am. Chem. Soc. 2003, 125, 1269-1277. 56. Schante, C. E.; Zuber, G.; Herlin, C.; Vandamme, T. F. Chemical Modifications of Hyaluronic Acid for the Synthesis of Derivatives for a Broad Range of Biomedical Applications. Carbohydr. Polym. 2011, 85, 469-489. 57. Lee, F.; Kurisawa, M. Formation and Stability of Interpenetrating Polymer Network Hydrogels Consisting of Fibrin and Hyaluronic Acid for Tissue Engineering. Acta Biomater. 2013, 9, 5143-5152. 58. Ryu, J.; Ku, S. H.; Lee, H.; Park, C. B. Mussel-Inspired Polydopamine Coating as a Universal Route to Hydroxyapatite Crystallization. Adv. Funct. Mater. 2010, 20, 21322139. 59. Li, J. G.; Zhang, K.; Wu, J. J.; Zhang, L. J.; Yang, P.; Tu, Q. F.; Huang, N. Tailoring of the Titanium Surface by Preparing Cardiovascular Endothelial Extracellular Matrix Layer on the Hyaluronic Acid Micro-pattern for Improving Biocompatibility. Colloid. Surf., B 2015, 128, 201-210. 60. Fujiwara, N.; Kobayashi, K. Macrophages in Inflammation. Curr. Drug Targets: Inflammation Allergy 2005, 4, 281-286. 61. Moore, K. J.; Tabas, I. Macrophages in the Pathogenesis of Atherosclerosis. Cell 2011, 145, 341-355. 62. Rooney, P.; Srivastava, A.; Watson, L.; Quinlan, L. R.; Pandit, A. Hyaluronic Acid Decreases IL-6 and IL-8 Secretion and Permeability in an Inflammatory Model of Interstitial Cystitis. Acta Biomater. 2015, 19, 66-75. 63. Arican, O.; Aral, M.; Sasmaz, S.; Ciragil, P. Serum Levels of TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in Patients with Active Psoriasis and Correlation with Disease Severity. Mediators Inflammation 2005, 2005, 273-279.

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Table of Content Graphic

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Table of Content Graphic 375x249mm (96 x 96 DPI)


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Figure 1. Schematic of PDMS surface modification procedure using PDA and HA, and the hemocompatibility evaluation using multiple types of cells. 200x146mm (120 x 120 DPI)



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Figure 2. Surface characterizations of various PDMS substrates: (a) water contact angle measurement based on sessile drops on native (a1) and treated PDMS surfaces coated with PDA (a2), HA (a3) and HA/PDA (a4a6); the corresponding contact angles measured on various substrates (a7). (b) AFM characterizations of various PDMS substrates: surface topography of native (b1) and treated PDMS surfaces coated with PDA (b2), HA (b3) and HA/PDA (b4-b6); the corresponding RMS roughness on various substrates (b7). 264x206mm (120 x 120 DPI)


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Figure 3. (a) Quantification of immobilized PDA on various PDMS surfaces; (b) Quantification of immobilized HA on various PDMS surfaces. Data are shown as the mean ± SD (n = 4). *p-value < 0.05 between two groups under comparison. 239x103mm (120 x 120 DPI)



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Figure 4. Platelet adhesions on native PDMS and those treated with PDA, HA or HA/PDA composites: (a) SEM characterizations (red arrows indicate typical platelet aggregates; scale bars: 20 µm); (b) 3-dimensional CLSM characterizations. 281x279mm (96 x 96 DPI)


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Figure 5. (a) LDH assays and (b) GMP-140 assays to characterize platelet adhesion and activation, respectively. All data are shown as the mean ± SD (n = 4). *p-value < 0.05 between experimental and control groups. 365x157mm (120 x 120 DPI)



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Figure 6. The proliferation assays (a) and viability assays (b) of HUVECs on native PDMS and those coated with PDA, HA and HA/PDA composites for 4 h, 24 h and 72 h. #p-value < 0.05 between two groups under comparison; *p-value < 0.05 as compared to the native PDMS as a negative control; ∆p-value < 0.05 as compared to the PDA-coated PDMS as a positive control; @p-value < 0.05 as compared to the HA-coated PDMS as a positive control. 367x154mm (120 x 120 DPI)


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Figure 7. Bright field and fluorescence images of HUVECs adhered on native, PDA-coated, HA-coated and HA/PDA-coated PDMS after incubation of 4 h, 24 h and 72 h. Scale bars: 100 µm for bright field images; 50 µm for fluorescence images. 213x213mm (120 x 120 DPI)



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Figure 8. Apoptosis assays of HUVECs incubated on native PDMS and those coated with PDA, HA and HA/PDA composite for 12 h (a) and 24 h (b). The upper right quadrant represents the end stage of apoptotic cells. (c) Comparison of HUVEC apoptotic rate on various PDMS substrates. 111x221mm (120 x 120 DPI)


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Figure 9. Fluorescence images of adherent macrophages on native (a), PDA-coated (b), HA-coated (c) and HA/PDA-coated (d-f) PDMS after 24 h of incubation (Scale bar: 50 µm); The quantities of adherent macrophages (g) and expression of three typical cytokines (h: TNF-α, i: IL-6 and j: IL-12) after 24 h of incubation on various PDMS substrates. The data are shown as the mean ± SD (n = 4). *p-value < 0.05 between two groups under comparison. #p-value < 0.05 as compared to the PDA-coated PDMS as negative control. ∆ p-value < 0.05 as compared to the HA-coated PDMS as positive control. 142x217mm (120 x 120 DPI)


Surface Modification of Poly(dimethylsiloxane) with Polydopamine and

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