Controlling Molecular Weight of Hyaluronic Acid Conjugated on Amine

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Controlling Molecular Weight of Hyaluronic Acid Conjugated on Amine-rich Surface: Towards Better Multifunctional Biomaterials for Cardiovascular Implants Jingan Li, Feng Wu, Kun Zhang, Zikun He, Dan Zou, Xiao Luo, Yonghong Fan, Ping Yang, Ansha Zhao, and Nan Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07444 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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ACS Applied Materials & Interfaces

Controlling

Molecular

Conjugated

on

Weight

Amine-rich

of

Hyaluronic

Surface: Towards

Acid Better

Multifunctional Biomaterials for Cardiovascular Implants Jingan Li#, *, 1, Feng Wu#, 1, Kun Zhang *, 1, 2, 3 , Zikun He 1, Dan Zou 1, Xiao Luo 1, Yonghong Fan 1, Ping Yang*, 1, Ansha Zhao 1, Nan Huang 1 1.

Key Lab. for Advanced Technologies of Materials, Ministry of Education, School

of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China 2.

School of Life Science, Zhengzhou University, 100 Science Road, Zhengzhou

450001, PR China 3.

Center of stem cell and regenerative medicine, First Affiliated Hospital of

Zhengzhou University, 40 University Road, Zhengzhou 450052, PR China

*Corresponding author: [email protected] (Jingan Li); [email protected] (Kun Zhang); [email protected] (Ping Yang) FAX: +86-28-87600625 TEL: +86-13551284797 #

Joint first authors

1

ACS Paragon Plus Environment


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Abstract The molecular weights (MW) of hyaluronic acid (HA) in extracellular matrix (ECM) secreted from both vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMC) play crucial roles in the cardiovascular physiology, as HA with appropriate MW influences important pathways of cardiovascular homeostasis, inhibits VSMC synthetic phenotype change and proliferation, inhibits platelet activation and aggregation, promotes endothelial mono-layer repair and functionalization, and prevents inflammation and atherosclerosis. In this study, HA with gradients of MW (4×103 Da, 1×105 Da and 5×105 Da) were prepared by covalent conjugation to a copolymerized film of dopamine and hexamethylendiamine (PDA/HD) as multifunctional coatings (PDA/HD-HA) with potential to improve the biocompatibility of cardiovascular biomaterials. The coatings immobilized high MW-HA (PDA/HD-HA2: 1×105 Da; PDA/HD-HA-3: 5×105 Da) exhibited a remarkable suppression of platelet activation/aggregation and thrombosis under 15 dyn/cm2 blood flow, simultaneously suppressed the adhesion and proliferation of VSMC, and adhesion, activation and inflammatory cytokine release of macrophages. Wherein, PDA/HDHA-2 significantly enhanced VEC adhesion, proliferation, migration and functional factors release, as well as the captured number of endothelial progenitor cells (EPC) in dynamic condition. The in vivo results indicated that the multi-functional surface (PDA/HD-HA-2) created a favorable microenvironment of endothelial mono-layer formation and functionalization for promoting re-endothelialization and reducing restenosis of cardiovascular biomaterials. Keywords:

Multi-functional

coating;

Hyaluronic

acid;

Molecular

Weight;

Biocompatibility; Cardiovascular biomaterials

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1. Introduction Cardiovascular disease (CVD) is a class of diseases that usually result from malfunction in coronary arteries and keeps the leading cause of morbidity and mortality globally for many years.1 Interventional therapy of CVDs with cardiovascular implants, such as vascular stents, emerged as the typical clinical methods, and the surface biocompatibility associated multi-functions including anticoagulation, anti-hyperplasia, anti-inflammation and pro-endothelialization has been proved to play essential roles after the implantation.2-4 The application of drug-eluting stents (DES) has been reported to effectively inhibit the pathological migration and proliferation of the smooth muscle cells (SMC) and the adhesion of macrophages, treating hyperplasia and inflammation temporarily.5 Nevertheless, the drugs (usually paclitaxel and/or rapamycin) loaded on DES delayed vascular healing and reendothelialization, which may lead to high risk of late thrombosis.6-7 Like we know, endothelial cells (EC) maintain the blood vessel patency via forming a endothelium monolayer and releasing functional factors including nitric oxide (NO) and prostacyclin (PGI2), etc.8-9 Therefore, the strategy of endowing the cardiovascular stent excellent pro-endothelialization property on basis of good anti-coagulation, anti-hyperplasia and anti-inflammation should be addressed urgently. Surface modification via biomolecule conjugation has been generally accepted as an effective method to improve the biocompatibility, and it has been widely applied on biomedical devices and tissue engineering.10-11 However, developing a biocompatible surface by biomolecule conjugation which possesses cardiovascular associated multi-function simultaneously is still a great challenge, but this may make great implication for the interventional therapy of CVDs. In the previous study, we tried to develop a novel multi-functional HA/PDA (hyaluronic acid and polydopamine)

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coating by electrostatic adsorption and hydrogen bonding, but after implantation of pig femoral artery, excessive high molecular weight hyaluronic acid (HMW-HA) stimulated fibroblasts to elaborate hyaluronidase, which cleaves the HA macromolecule into smaller polymers, and this triggered thrombosis, hyperplasia and inflammation.12 Hyaluronic acid as the main component of extracellular matrix (ECM) is a natural biopolymer whose molecular structure is highly conserved,13 while this biomolecule owns different functions including anti-coagulation, anti-hyperplasia, anti-inflammation and pro-endothelialization which depend on its molecular weights (MW).14 HMW-HA not only inhibits platelet, SMC and macrophage adhesion, further endows surface anti-coagulation, anti-hyperplasia, anti-inflammation function, but also gives the surface a non-immunogenic property which is crucial for the implants.15-16 Yet HA with extreme HMW also inhibit endothelial progenitor cells (EPC) adhesion and EC migration which is obviously not conducive to the rapid endothelialization.17 On the contrary, low molecular weight hyaluronic acid (LMWHA) has been reported as the main participant during the thrombosis, and it also contributes to the inflammation.18 Therefore, it is paramount for obtaining better biocompatibility associated multi-functions to control the MW of the conjugated HA in the process of surface modification.

Considering better stability of the modification platform, we deposited a copolymerized coating of dopamine and hexamethylendiamine (PDA/HD) rich in amine groups onto the implants as Yang et al. reported, instead of the previous PDA coating.19 In their description, the PDA/HD coating displayed good adhesion strength to the substrate and resistance to the deformation behavior of compression and expansion of a stent. The primary amine groups of the coating could be effectively immobilized to the carboxylic groups of HA. The rich amine groups of the PDA/HD

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coating may create an endothelial friendly microenvironment which could compensate the negative influence on EC from the HMW-HA.20 In this contribution, we control the MW of the HA (4×103 Da, 1×105 Da and 5×105 Da according to the reported research) conjugated onto the amine-rich surface (PDA/HD) towards better multi-functions for cardiovascular biomaterials.12, 21-22 The multi-functional coatings were prepared on 316L stainless steel (316L SS), and they were characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurement and atomic force microscopy (AFM). The conjugated HA was quantified by a Toluidine blue-O (TBO) method and a quartz crystal microbalance equipped with dissipation monitoring (QCM-D, Q-sense AB, Sweden). The evaluation of hemocompatibility, pro-endothelialization property, anti-hyperplasia, anti-inflammation and in vivo tissue response (including wire implantation in SD rats’ abdominal artery and stent implantation in New Zealand white rabbit’s iliac artery) were also performed via specific experiments. We also explored the relationship between the MW of HA and bio-functions, further studied the mechanism how the PDA/HD-HA coatings improved the surface biocompatibility.

2. Experimental section 2.1 Fabrication of the PDA/HD-HA coatings The PDA/HD coating was deposited on mirror polished 316L stainless steel (316LSS) substrates (Φ10mm, Baoji, China) as Yang et al. described .6, 19 The copolymerization of HD and dopamine was mainly based on oxidative polymerization with the formation of covalent bonding and physical adsorption.

19

2 mg/ml of

hyaluronic acid (HA, Sangon Biological Engineering Co. Ltd., China) with gradient molecular weight (MW) of 4×103 Da, 1×105 Da and 5×105 Da (The pH of each HA solution remained 4.5±0.1) was in advance activated in water-soluble carbodiimide

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solution for 15 min. 6 Then, the 316L SS coated with PDA/HD were immersed into the above HA solution for incubation. After reaction for 12 h, the specimens were washed with phosphate buffered saline (PBS) (3 times, 5 min) and dH2O (3 times, 5 min) before surface analysis.19 Samples were labeled as PDA/HD-HA-1, PDA/HDHA-2 and PDA/HD-HA-3, respectively. The fabrication process of the PDA/HD-HA coatings was displayed in Figure 1.

Figure 1. The scheme of preparing PDA/HD-HA coatings on the 316L SS surface

2.2 Characterization of PDA/HD-HA coatings The surface chemical compositions of the specimens were measured by X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Electron, USA).

23

The density

of amine groups on the PDA/HD coating was determined using Acid Orange II (AO II) colorimetric method. 24 The carboxyl group quantification of HA bound to the surface of coatings was performed by a Toluidine blue-O (TBO) method.24-25 Quantification of HA immobilization was carried out using a quartz crystal microbalance equipped with dissipation monitoring (QCM-D, Q-sense AB, Sweden).26 The surface morphology and roughness of coatings were analyzed by atomic force microscopy 6


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(AFM, Key sight 7500, USA).6 To evaluate the hydrophilicity of the PDA/HD-HA coatings, the water contact angles (WCA) of the 316LSS, PDA/HD, PDA/HD-HA-1, PDA/HD-HA-2 and PDA/HD-HA-3 samples were detected by a contact angle apparatus (DSA 100, Krüss, GmbH, Germany).27 2.3 Blood compatibility of PDA/HD-HA coatings The blood compatibility of the PDA/HD-HA coatings was investigated by the platelet adhesion and the whole blood test respectively, and the 316LSS and PDA/HD samples were used as control. 12, 28 2.4 HUVEC attachment, proliferation, migration and functional factor release Human umbilical vein endothelial cells (HUVEC) obtained from the newborn umbilical cord (Huaxi Hospital, Chengdu, China) were applied in our study. HUVEC between 3rd and 5th passages were seeded onto the samples with the concentration of 5×104 cells/ml, and then cultured at 37 oC for 4 h, 1 day and 3 days, respectively. After the sequentially washed step, the samples were fixed with 4% paraformaldehyde (Sigma, USA) for 2 h at room temperature and stained by rhodanmine (Sigma, USA) for 15 min, finally examined and recorded by a fluorescence microscope (DMRX, Leica, Germany).29 Platelet endothelial cell adhesion molecule-1 (CD31) and intercellular cell adhesion molecule-1 (ICAM-1) on the HUVEC membrane were also stained and observed by confocal laser scanning microscopy (CLSM, Nikon Eclipse Ti, Japan). A CCK-8 assay was performed to investigate the HUVEC attachment and proliferation on the samples.12, 30 Nitric oxide (NO) release from the HUVEC was detected using a chemiluminescence NO analyzer (NOA) (Seivers 280i, Boulder, CO): In brief, the used medium of HUVEC cultured on each sample were collected and applied for NO quantitative analysis. NO released by HUVEC was purged from the collected medium and transported to the NO analyzer by a stream of N2 (g).31 The

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calculation of the amount of NO released by HUVEC was based on the calibration curves of the NOA, which was in detail showed elsewhere.30 After implantation, the endothelialization on the device surface mainly come from the migration of the neighbor healthy EC. Thus, the EC migration experiment was performed as Yang et al. to further evaluate the endothelialization of the PDA/HD-HA coatings.6 2.5 HUEPC capture and proliferation Endothelial progenitor cells (EPC) capture is another pathway for the surface endothelialization of cardiovascular implanted devices,32 so dynamic EPC capture was performed in this study for more endothelialization investigation. Human umbilical endothelial progenitor cells (HUEPC) were differentiated by inducing human mesenchymal stem cells (HUMSC), and the HUMSC were isolated from Wharton’s jelly tissue of newborn umbilical cord as a traditional method.33 The HUMSC between 3rd and 5th passages were cultured using M199 medium (Sigma) containing 20% FBS and 20 ng/ml vascular endothelial growth factor (VEGF, Sigma) to differentiate into HUEPC. The induced HUMSC after 3rd passage may differentiate into HUEPC. The HUEPC were fed with fresh medium F12 (Sigma) containing 20% FBS every 48 h. HUEPC between 3rd and 5th passages were used for experiments to ensure the genetic stability of the cultures. The PDA/HD-HA-1, PDA/HD-HA-2, PDA/HD-HA-3, PDA/HD and 316LSS samples were placed in the flow chamber device, and the DMEM medium flow containing HUEPC (concentration: 5×104 cells/ml) with 15 dyn/cm2 speed was applied in the devices in a humidified incubator with 95% air and 5% CO2 for 4 h and 10 h, respectively. Then, the samples were picked out, washed with PBS (pH=7.4, 3 times, 5 min), and fixed with 4% paraformaldehyde and stained by rhodamine, also examined and recorded by

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the fluorescence microscope. The captured HUEPC on each sample were counted statistically from three parallel samples (fifteen images each sample).34 To investigate the proliferation of the captured HUEPC, the samples were set into a 24-well culture plate after capture for 10 h, and incubation in a humidified incubator with 95% air and 5% CO2 for 1 day and 3 days respectively. The morphology of the HUEPC was observed under the fluorescence microscope after the fixed step and typical rhodamine staining. The HUEPC number was also investigated by the CCK-8 assay. 2.6 Anti-HUASMC-proliferation property of PDA/HD-HA coatings Human umbilical arterial smooth muscle cells (HUASMC) were also derived from the newborn umbilical cord by the method described in the previous work,35 and cultured in a standard condition elaborated above. HUASMC between 2nd and 7th passages were seeded on the PDA/HD-HA-1, PDA/HD-HA-2, PDA/HD-HA-3, PDA/HD and 316LSS samples with the concentration of 5×104 cells/ml, and cultured at 37 oC for 1 day and 3 days, respectively. The morphology and behavior of the HUASMC was observed under the fluorescence microscope after the fixed step and stained with rhodamine. The HUASMC number was also examined. 36 2.7 Competitive adhesion of HUVEC and HUASMC on PDA/HD-HA coatings The co-culture of HUVEC and HUASMC was performed to investigate competitive adhesion and growth behaviors of the two cell types.6 Cell trackers with different colors were used to distinguish HUVEC (Green chloromethylfluorescein diacetate, CMFDA) and HUASMC (Red chloromethyl trimethyl rhodamine, CMTMR) in the co-culture assay. Cells were incubated with the cell tracker dyes for labeling and then seeded on the samples by mixing the HUVEC and HASMC suspensions at a volume ratio of 1:1 using DMEM-F12 medium supplemented with 10% FBS (The densities of both cell types: 5×104 cells/cm2 ). The competitive cell adhesion was

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examined after 6 h incubation in a humidified atmosphere with 5% CO2 and 95% air. The cells were observed and photographed using fluorescence microscope. The average density of adherent cells was determined from more than 15 images. 2.8 Anti-inflammation property of PDA/HD-HA coatings The anti-inflammation property of the PDA/HD-HA coatings was investigated via the macrophages culture test and the detection of their inflammatory cytokines release, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). The peritoneal macrophages obtained from the SD rats (Huaxi Hospital, Chengdu, China) were cultured in the standard culture condition. The cells were added onto each samples with the concentration of 5×104 cells/ml, and incubated at 37 oC for 24 h. To study the extent of cell morphology and spreading, the fluorescence staining of the attached macrophages was performed using rhodamine, and the numbers of the cells on each sample were statistically counted from 15 random pictures.37 The supernatant harvested after macrophages cultured on samples for 24 h was applied as the specimen, and the TNF-α and IL-6 release were measured using related kits according to the manual.38-39 2.9 In vivo tissue response test of PDA/HD-HA coatings All procedures were in compliance with the China Council on Animal Care and Southwest Jiaotong University animal use protocol, following all the ethical guidelines for experimental animals. Bare 316L SS wires (Φ0.1mm×10mm), PDA/HD coated 316L SS wires and PDA/HD-HA coated 316L SS wires were implanted in the lumen of SD rats’ abdominal aorta for 30 days. The wire remains in contact with flowing blood within the aorta to simulate the presence of a stent strut with some regions of the wire in direct contact with the arterial wall and some regions of the wire not in contact. After 30 days, the aortas containing the implanted wires

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were harvested for histological analysis.40 Cross sections were ethanol fixed and then stained with antibodies specific for EC (CD31, Sigma), SMC (α-SMA and OPN, Sigma) and macrophages (TNF-α, Sigma).41-43 The nucleus of all the cells were stained by 4,6-diamino-2-phenyl indole (DAPI, Sigma). The stained images were observed by confocal laser scanning microscopy (CLSM, Nikon Eclipse Ti, Japan). To further explore the multi-functions of the PDA/HD-HA coatings, stent implantation of New Zealand white rabbit’s iliac artery was performed as Zhang et al. reported.44 2.10 Statistical analysis The data more than two groups were statistically evaluated using ANOVA by homogeneity test of variances firstly, and post hoc test was prepared subsequently in LSD method for comparison. Two groups of data were statistically evaluated using Student’s paired t test. They were expressed as mean ± standard deviation (SD). The probability value p < 0.05 was considered as a significant difference. The data analysis was performed using the software SPSS 11.5 (Chicago, IL).

3. Results and discussion 3.1 Determination of amine concentration by the AO test To confirm that the PDA/HD coating was successfully fabricated onto the 316LSS substrate, quantitative characterization for amine concentrations on the 316LSS and PDA/HD surfaces were performed using AO test, and the results were presented in Figure 2. Compared with the 316LSS samples, the PDA/HD samples showed a significantly higher (p 11 nmol/cm2, suggesting the successful preparation of the PDA/HD coating on the 316LSS. The 316LSS showed a false positive result of < 5nmol/cm2, and this made no negative effect on the demonstration, because the photos obtained by Iphone 6.0 also proved

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that the preparation of PDA/HD coating made the color of the 316LSS surface change from silver white to the typical golden yellow.

Figure 2 Surface amine concentration of 316LSS and PDA/HD samples (*pPDA/HD-HA-2>PDA/HD-HA-3. This experiment also revealed that PDA/HD surface preferred HA-1 because of its low MW (LMW) which contributed to the exposure of the carboxyl group, and the carboxyl group could be conjugated with the amino group of the PDA/HD surface. Most of the carboxyl group on the PDA/HD-HA-2 and PDA/HD-HA-3 coatings was blocked by the spatial conformation of the HA-2 and HA-3 because of their high MW (HMW). In addition, carboxyl group density may be strongly depending on various factors, such as the viscosity of HA solutions, the feed ratio, the incubation time, etc. Wherein, the viscosity of HA solutions is closely related to the concentration and MW of HA solutions, and the concentrations of all the HA solutions are 2mg/ml, therefore only HA MW influenced the carboxyl group density. In this work, the incubation time of PDA/HD-HA-1, PDA/HD-HA-2 and PDA/HD-HA-3 are consistent at 12 h, thus the feed ratio is also influenced by the HA MW.

Figure 4 Surface carboxyl concentration of PDA/HD, PDA/HD-HA-1, PDA/HD-HA-2 and PDA/HD-HA-3 samples (*p HA-3. The QCM-D result further indicated that the LMW HA (HA-1) may be immobilized onto the PDA/HD surface more easily and rapidly compared with the HMW HA (HA-2 and HA-3).

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Figure 5 Dynamic adsorption of HA-1, HA-2 and HA-3 on the PDA/HD-modified Au surface via QCM-D real-time monitoring.

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3.4 AFM characterization Figure S1A shows the morphology and roughness of the PDA/HD-HA-1, PDA/HD-HA-2, PDA/HD-HA-3, PDA/HD and 316L SS samples detected by the AFM characterization. The 316L SS substrate presented a rough surface with the roughness of 4.3 ± 0.4 nm. The PDA/HD deposition made the 316L SS surface smoother (3.0 ± 0.5 nm) as shown in Figure S1A, which was different from the single dopamine deposited surfaces.45 The PDA/HD-HA-1 coating showed a smoother surface with the roughness of 2.3 ± 0.5 nm, attributing to the lubricant produced by HA and the absorbed water from the air. However, the PDA/HD-HA-2 and PDA/HDHA-3, with the roughness of 3.1 ± 0.1 nm and 4.3 ± 0.9 nm, showed rougher surfaces compared with PDA/HD-HA-1, wherein PDA/HD-HA-3 possessed the largest roughness, which was just negatively correlated with the amount of the immobilized HA. 3.5 Water contact angle Water contact angle was measured to examine the wettability of the PDA/HDHA-1, PDA/HD-HA-2, PDA/HD-HA-3, PDA/HD and 316L SS surfaces (Figure S1B). Compared with 316L SS, water contact angles dramatically increased after coated with PDA/HD, while decreased again after the HA immobilized, indicating that the PDA/HD-HA coatings were more hydrophilic than PDA/HD surface, and made no wettability change compared with the pristine 316L SS substrate. 3.6 Blood compatibility The in vitro platelet adhesion test was used to investigate the blood compatibility of the PDA/HD-HA coatings. The quantity of platelets on different samples detected by the LDH method was presented in Figure 6A. The data showed that the 316L SS substrate and the PDA/HD coating facilitated a much higher level of platelet adhesion

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compared with samples of PDA/HD-HA coatings (p PDA/HD > PDA/HDHA-1 > PDA/HD-HA-2 > PDA/HD-HA-3. The PDA/HD-HA coatings possessed fewer platelets because of their anti-coagulant group carboxyl,46 thus fewer platelets should be attributed to more carboxyl group, while PDA/HD-HA-2 and PDA/HDHA-3 coatings possessed almost the same carboxyl group amounts, but the PDA/HDHA-3 coating possessed fewer platelets compared with the PDA/HD-HA-2 coating. The reason may be that the longer spatial chain structure of the HA-3 contributed to inhibiting platelet adhesion.47 Activated ratio of the adherent platelet on different samples examined by the GMP140 assay was displayed in Figure 6B. The quantity of activated platelets decreased in the order: PDA/HD > PDA/HD-HA-1 > 316L SS > PDA/HD-HA-2 and PDA/HD-HA-3. It was unexpected that the PDA/HD-HA-1 caused a much higher level of platelet activation compared with the 316L SS surface because the carboxyl group on the surface inhibited platelets adhesion. We speculated the reason may be that the shorter spatial chain structure of the HA-1 could not completely cover the PDA/HD substrate beneath, and the exposed PDA/HD caused the platelet activation.48 SEM images were applied for evaluating the adhesion and morphology of platelets on each sample surfaces. The representative typical images of platelets adhesion behavior on these samples are depicted in Figure 6C. There were more platelets attached and aggregated markedly on 316L SS substrate with marked shape change (spreading) and pseudopod formation. Platelets aggregation and spreading further exacerbated on PDA/HD and PDA/HD-HA-1 coatings although their adhesion were significantly reduced, even parts of the platelets almost spread entirely and broke, suggesting seriously activated phenotype. Platelets on the PDA/HD-HA-2 and

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PDA/HD-HA-3 coatings maintained their round shapes with no formation of pseudopods, indicating non-activated phenotype.

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Figure 6 (A) Amount and (B) activated ratio of platelets on 316L SS, PDA/HD, PDA/HD-HA-1, PDA/HD-HA-2 and PDA/HD-HA-3. The amount was obtained by the LDH assay, and the activated ratio was obtained by the GMP140 assay (*p

Controlling Molecular Weight of Hyaluronic Acid Conjugated on Amine

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