Laminin-Loaded

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Surface modification with ECM-inspired SDF-1#/ laminin-loaded nanocoating for vascular wound healing Tao Liu, Xin Wang, Xiaohan Tang, Tao Gong, Wei Ye, Changjiang Pan, Hongyan Ding, Xun Luo, Xia Li, and Qing Mei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08516 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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ACS Applied Materials & Interfaces

Surface modification with ECM-inspired SDF-1α α/laminin-loaded nanocoating for vascular wound healing Tao Liu a, b, Xin Wang b, c, #, Xiaohan Tang a, Tao Gong a, Wei Ye a, Changjiang Pan a, *, Hongyan Ding a, Xun Luo d, Xia Li e,**, Qing Mei Wang b, *** a Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai'an, China b Stroke Biological Recovery Laboratory, Spaulding Rehabilitation Hospital, Harvard Medical School, Charlestown, MA c Department of Rehabilitation, Clinical Medical College, Yangzhou University, Northern Jiangsu Province Hospital, Yangzhou, China d Kerry Rehabilitation Medicine Research Institute, Shenzhen, China e Department of Geriatrics, The Affiliated Huai'an Hospital of Xuzhou Medical College, Huai'an, China # The author contributed equally to the work and should be considered as co-first author * Corresponding author Tel: +86 151-8966-1181

E-mail address: [email protected]

** Corresponding author Tel: +86 130-1656-3180


*** Corresponding author Tel: +1 617-952-6184


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Abstract Surface biomimetic modification with extra-cellular matrix (ECM)-derived biomolecules is an emerging potential method of accelerating the healing of vascular stent lesions. However, insufficient capacity of the constructed biofunctional layer in maintaining its long-term efficiency and preventing thrombus and neointimal hyperplasia continue to be major limitations in clinical application. Based on the structure and function of ECM, in this study, we constructed a novel stromal cell-derived factor-1α (SDF-1α)/laminin-loaded nanocoating on the 316L stainless steel (SS) surface to provide improved function in modulation of vascular remodeling. The modified surface was found to control delivery of biomolecules and exhibit promising potential to provide stage-adjusted treatment after injury. An in vitro biocompatibility study suggested that the constructed layer may effectively prevent thrombosis formation by inhibiting platelet adhesion and activation, while accelerating endothelium regeneration by inducing endothelial cell (EC) migration and endothelial progenitor cell (EPC) aggregation. An in vivo animal test further demonstrated that the nanocoating may prevent thrombus and neointimal hyperplasia after implantation for 3 months. Therefore, the ECM-inspired nanocoating described in this study is a promising novel approach for vascular stent surface modification. Keywords: bioinspired; nanoparticle; SDF-1α; endothelialization; restenosis.


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1. Introduction Percutaneous coronary intervention (PCI) by using an expandable metal stent to open the narrowed or blocked vessel is the primary treatment for patients with coronary artery diseases (CADs). To date, the most commonly used stent platform continues to be drug-eluting stents (DESs), which are widely reported may trigger delayed healing of injury and raise the risk of late thrombus and restenosis.1 Therefore, decreasing the risk of side effects after surgery by improving the surface of the stent has been a major theme in vascular stent research. In particular, surface biofunctional modification with specific biomolecules or chemical compositions to regulate the biological response in the tissue-material-blood interface and to prevent thrombus and restenosis has attracted much attention in recent years. The vascular endothelium layer as a natural barrier between blood and vascular tissue can express or secret various anti-coagulation and anti-proliferation factors to prevent intravascular thrombus and intimal hyperplasia.2 As a result, it’s widely suggested that accelerating endothelialization after stent implantation is an ideal way to decrease the risk of adverse effects after PCI.3 Researchers found that the ECM components played important roles in endothelium regeneration and angiogenesis. The ECM surrounded by endothelium is organized by a basement membrane and interstitial matrix.4 The former is mainly composed of laminin (Ln), collagen IV, entactin, heparan sulfate proteoglycans, and some attached cytokines such as vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α),5 while the latter includes fibrillar collagens, fibronectin (Fn), and vitronectin (Vn).



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During endothelium injury, the exposed ECM may contribute to accelerating endothelialization by releasing cytokines to activate surrounded ECs and by providing abundant binding sites for enhanced cell adhesion, migration, and proliferation. Furthermore, the vascular basement membrane, especially laminin, has also been found to maintain mature vessels in a stable quiescent state,6 as well as induce the morphological differentiation of ECs into capillary-like structures.7 Based on the importance of the ECM in the healing of vascular injury, numerous studies have tried to incorporate one or more ECM components onto material surfaces to mimic the function in accelerating endothelialization.8,9 Tu et al.10 reported a novel method of incorporating the complete ECM secreted by ECs or smooth muscle cells (SMCs) onto the titanium surface via decellation technology, and they found the EC-derived ECM may have contributed to promoting the proliferation and functional expression of ECs. However, in many studies, the ECM component-modified surfaces were also found to have their own major shortcomings. It has been reported that ECM component-modified surfaces triggered platelet adhesion, and SMC over-proliferation was associated with coagulation and intimal hyperplasia after endothelium injury.11-13 In addition, ECM was found to promote inflammation via inducing activation and enhancing penetration of immune cells.14 This may further increase the risk of in-stent thrombus and restenosis. Furthermore, the major components of ECM are biodegradable in vivo, especially at the site of injury,15 resulting in a short half-life of the ECM component-modified surface after implantation without a protective barrier. Therefore, the biomaterial surface simply modified with single or multiple ECM


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components may not be able to meet the biocompatibility requirements in a clinical setting. In recent decades, the nanoparticle system has garnered great attention in vascular stent surface modification and has been demonstrated effective in promoting drug loading, optimizing delivery behavior, and preventing adverse side effects.16-18 Previously, we synthesized a novel heparin/poly-l-lysine nanoparticle for vascular stent surface modification.19 The nanoparticle-modified surface may provide proper biofunction and long-term efficiency for preventing thrombosis and restenosis. Based on the advantages and shortcomings of the ECM, an innovative bioinspired heparin/poly-l-lysine nanoparticle loaded with Ln and SDF-1α was constructed in this study for surface modification. According to an in vitro biocompatibility evaluation and in vivo animal testing, the nanoparticle-modified surface may provide adequate biofunction and long-term efficiency for decreasing the risk of thrombus and restenosis, as well as accelerating endothelialization by enhancing EC migration and EPC aggregation. As a result, this may be a promising approach to transforming the conventional polymer-based DESs system into a nanoparticle-based stent platform to meet the clinical requirements of vascular stent modification. 2. Materials and methods 2.1 Materials and Reagents 316L stainless steel (316L SS) was processed into round shape (Φ 10 mm, ~1.2 mm in thickness) and mirror-polished. Dopamine (DA), poly-l-lysine (PLL, 150-300 kDa), laminin (Ln-1, from Engelbreth Holm Swarm murine), and SDF-1α were



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purchased from Sigma-Aldrich. Low molecular weight heparin sodium (Hep, ≥180 USP units/mg) was bought from Shanghai Aladdin BioChem Technology Co., LTD. Toluidine blue O (TBO), Acid orange II (AO II), and rabbit anti RGD antibody were purchased from Sigma-Aldrich. Mouse monoclonal anti human p-selectin antibody, rabbit anti human fibrinogen antibody, and mouse monoclonal anti human fibrinogen (FGN) γ chain antibody were purchased from Abcam. Rhodamine 123, crystal violet, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. Also, 0.01 M phosphate-buffered saline (PBS, pH 7.4) was used for PLL, Hep Ln, and SDF-1α solution preparation. 2.2 Nanocoating construction A schematic drawing of nanoparticle construction and immobilization is shown in Figure 1. First, poly-dopamine was coated on the 316L SS surface by using the method that described in our previous study.20 Then, 10 mg/ml of heparin was mixed with equal volume of 50 mg/ml Ln solution and incubated at 37 ℃ for 3 hours. Afterward, an equal volume of the Hep/Ln solution was added dropwise to 0.5 mg/ml of PLL under high-frequency ultrasonic conditions to formulate a Ln-loaded heparin/poly-l-lysine nanoparticle suspension. The suspension was then centrifuged at 15,000 rpm for 10 minutes to collect the nanoparticle. Subsequently, the precipitated nanoparticles were re-suspended in PBS. Then, dopamine-coated 316L SS was placed in a 24-well plate, and 0.5 ml nanoparticle suspension was added to the sample surface. After that, the plate was placed in a air bath shaking table and incubated at 37 ℃ for 12 hours with gentle shaking (60~65 rpm) (termed as SS-DA-NPL). Finally,


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the samples were thoroughly rinsed three times with PBS, and then immersed into 0.5 ml 200 ng/ml of SDF-1α solution and incubated at 37 ℃ for 3 hours to construct the bio-inspired nanocoating (termed as SS-DA-NPLS). The sample modified with Hep/PLL nanoparticle only was set as the control group, SS-DA-NP. 2.3 FTIR and XPS assay Fourier transform infrared spectroscopy (FTIR) and X-ray photo-electron spectroscopy (XPS) were used to detect the alteration of the surface chemical structure and elemental composition during nanoparticle immobilization. The FTIR assay was performed on a TENSOR 27 infrared spectroscopy (Bruker, Germany) using the model of attenuated total reflection. Infrared adsorption between 4000 and 650 cm 1 was recorded at room temperature and under atmospheric conditions. The −

XPS analysis was carried out by using VGESCALAB MK II spectrometer with a monochromatic Mg Ka X-ray source (1253.6 eV). The pressure of testing chamber was set as 8 × 10 8 Pa. The scale of binding energy was referenced by adjusting the −

C1s peak at 284.6 eV. 2.4 AFM assay The morphology of the nanoparticle-modified surface was detected on an INNOVA atomic force microscope (Bruker, Germany) by using tapping mode. The experiment was conducted at room temperature, and the image was processed by NanoScope Analysis software. 2.5 Surface hydrophilicity The change in surface hydrophilicity during nanoparticle immobilization was



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characterized by measuring the water contact angle. The test was performed on a DSA25 contact angle goniometer (Krüss GmbH, Germany) at room temperature. A droplet of ultrapure (UP) water was added onto sample surfaces, and the contact angle was calculated immediately by using DSA 1.8 software. Six parallel samples were prepared with at least 3 different points of each sample surface measured. 2.6 Quantitative characterization of amine group, heparin, and RGD exposing density Surface amine density was detected by the AO II method. The samples were initially immersed in 1 ml of AO II solution (0.5 mM, dissolved in pH 3 HCl) and incubated for 6 hours (37 ℃) with gentle shaking. Afterward, the samples were thoroughly rinsed with HCl solution (pH 3). Then, the samples were placed on a filter paper and the surface was dried with a gentle blow. Next, the samples were immersed in 1 ml of NaOH water solution (pH 12) and shaken at 37 ℃ for 30 minutes. Finally, the NaOH solution mixed with eluted AO II (150 µl) was transferred into a 96-well plate, and the absorbance value was detected at 485 nm. The AO II molar concentration was parallel to that of the amine group. TBO

assay

was

prepared

to

detect

heparin

exposure

density

of

nanoparticle-modified surfaces. First, the samples were placed in a 10 ml round-bottom centrifuge tube and 5 ml of TBO solution (0.04 wt.%, dissolved in 0.01 M HCl/0.2 wt.% NaCl solution) was added. The tube was subsequently placed in an air bath shaker and incubated for 4 hours (37 ℃) with gentle shaking (~60 rpm). Next, the samples were thoroughly rinsed with UP water and immersed in 5 ml of an 80% ethanol/0.1M NaOH (v/v=4/1) solution. After being shaken for 10 minutes, the


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solution mixed with eluted TBO was transferred to a 96-well plate, and the absorbance value was detected at 530 nm. The RGD exposure density on the nanoparticle-modified surface was detected by an immunochemistry method. The sample was first immersed in 1 wt.% of bovine serum album (BSA, dissolved in PBS) and incubated at 37 ℃ for 30 minutes. After that, the sample was thoroughly rinsed with PBS. Next, 50 µl of rabbit anti RGD antibody (primary antibody, 1:200 diluted in BSA solution) was added to the sample surface and incubated for 1 hour (37 ℃). After PBS rinsing, 50 µl of HRP-conjugated goat anti rabbit IgG antibody (secondary antibody, 1:100 diluted in PBS solution) was added to each sample surface and incubated for 1 h (37 ℃). Afterward, 150 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) was added to the surface and incubated under darkness for 10 minutes. Finally, 50 µl of H2SO4 (1 M) was added to the sample surface to terminate the peroxidase catalyzed reaction, and the absorbance value was detected at 450 nm. 2.7 Heparin and SDF-1α release assay The release profiles of heparin and SDF-1α from the nanoparticle-modified surface under fluid condition were characterized by TBO assay and the ELISA method, respectively. Peristaltic pump (Masterflex 7518-10, Cole Parmer, USA)and flow chamber system (Figure S3A) were used in this study and the shear stress was set as 15 dyn/cm2 to mimic artery blood flow. For heparin release assay, the NP, NPL, and NPLS modified samples (n=8) were placed in different flow chambers, respectively. The SS-DA was set as a blank control.



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Then, 100 ml of DMEM/F12 culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was pumped into to each tube and incubated at 37 ℃ for 1, 3, 5, 7, 10, 14, and 21 days. At each time point, the medium was collected and new fresh medium was added. Then, 5 ml collected medium was transferred to a 50 ml centrifuge tube and mixed with equal volume of the TBO solution. After 6 h incubation (37 ℃), the mixture was centrifuged at 3500 rpm for 10 minutes and the supernatant was removed. Then, 5 ml of 80% ethanol/0.1M NaOH (v/v=4/1) was added to each tube. The absorbance value was detected using the same method described in section 2.6. For SDF-1α release assay, the NPLS modified sample (n=8) was placed in the flow chamber. The SS-DA-NPL was set as a blank control. Then the dynamic release of SDF-1α was conducted using the same method described in heparin release assay. The medium was collected at each time point, and the SDF-1α content was detected by using the ELISA kit. The release ratio was calculated using the following equation: Release ratio (%) =

Where

Ca × 100% C t − Cr

Ct means

total

SDF-1α concentration,

Cr

indicates

residual

SDF-1α concentration after surface modification, and Ca refers to accumulated SDF-1α concentration released from day 1. 2.8 In vitro evaluation of blood compatibility 2.8.1 Platelet adhesion and activation assay Fresh blood draw from healthy volunteer was anticoagulated by using 3.8 wt.% sodium citrate (v/v=9/1). The blood was first centrifuged at 1500 rpm for 15 minutes


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to acquire platelet-rich plasma (PRP). Next, 50 µl of PRP was added to sample surface. Then the sample was placed in a air bath incubator and incubated at 37 ℃ for 2 hour. Afterward, the sample was gently rinsed with normal saline and used for fluorescence staining and p-selectin expression assay. For fluorescence staining, the sample was first fixed in 2.5% glutaraldehyde for at least 6 hours. Then, 50 µl of rhodamine 123 solution was added to each sample surface and incubated for 30 minutes in room temperature . After that, the sample was thoroughly rinsed with normal saline. The distribution of adhered platelets were observed by an inverted fluorescent microscopy (Carl Zeiss A2). The p-selectin expression level was detected by using the same method described in 2.6 (RGD exposure density assay). The only difference is that the primary and secondary antibodies used are mouse monoclonal anti human p-selectin antibody and HRP-conjugated goat anti mouse IgG antibody, respectively. 2.8.2 FGN adsorption and activation profile For fibrinogen (FGN) adsorption and activation evaluation, the PRP was initially centrifuged at 3000 rpm for 15 minutes to acquire platelet-poor plasma (PPP). Next, 50 µl of PPP was added to each sample surface. Then the sample was placed in a air bath incubator and incubated at 37 ℃ for 1 hour. The subsequent procedures were similar to that of RGD density assay that described in section 2.6. The only difference is that rabbit anti human FGN antibody was used for FGN adsorption assay, and mouse monoclonal anti human FGN γ chain antibody was used for activation assay. The activation ratio of FGN was calculated using following equation:



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Activation ratio =

OD FGN activation × 100% OD FGN adsorption

2.8.3 Clotting time assay Activated partial thromboplastin time (APTT) and thrombin time (TT) were measured to evaluate the anticoagulation property of nanocoating. In this experiment, nanoparticles were immobilized on 316L SS foil with the same procedure described in section 2.2. Then the foil which was cut into small pieces (0.3 × 1.0 cm2) was placed in a test tube and 300 µl of PPP was added. After incubation at 37 ℃ for 3 min, the APTT and TT values were detected by using an automatic blood coagulation analyzer (ACL-200). 2.9 In vitro evaluation of cellular compatibility 2.9.1 EC proliferation and migration HUVECs were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 20 µg/ml endothelial cell growth supplement. Before cell seeding, the SS-DA was initially sterilized by autoclaving, and the subsequent nanocoating construction was also carried out under sterile conditions. For cell proliferation assay, 1 ml of ECs suspension (5×104 cells/ml) were added to each sample and incubated at 37 ℃ under 5% CO2 for 1 day and 3 days. At each time point, the supernatant was removed, and 500 µl of fresh culture medium/CCK-8 mixture (v/v=9/1) was added and incubated for another 4 hours. After that, The absorbance of the supernatant was measured at 450 nm. The samples were rinsed with normal saline and fixed in 2.5% glutaraldehyde for at least 6 hours. Subsequently, 50 µl of rhodamine 123 solution was dropped upon each sample surface and incubated in


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darkness for 30 minutes. Then, 30 µl of DAPI solution was added to and the samples were incubated in darkness for 10 minutes. Finally, the samples were rinsed three times with normal saline and observed by inverted fluorescent microscopy (Carl Zeiss A2). For EC migration evaluation, the nanocoating was constructed on a 316L SS foil surface. The detection procedure was referenced from Liu et al.21 2.9.2 EC biofunction evaluation The biofunction of adhered ECs was evaluated by measuring the synthesis activity of nitric oxide (NO), prostacyclin (PGI2), and VEGF. In brief, 1 ml of high density ECs suspension (5×105 cells/ml) was added to different samples and incubated at 37 ℃ for 6 hours to establish the formation of a confluent monolayer. After that, all the samples were transferred to a new 24-well plate, and 500 µl of non phenol red culture medium was added. After incubation for 12 hours, the supernatant was collected for detection. The NO release profile was detected using the Griess test. First, 100 µl of Griess reagent was mixed with equal volume of above supernatant, and the mixture was incubated at room temperature for 15 minutes. After that, the absorbance value of the mixture was measured at 540 nm. PGI2 and VEGF release profiles were detected by an ELISA kit according to the manual. The NO, PGI2, and VEGF release capacities were finally normalized to cell number. 2.9.3 EPC proliferation and mobilization assay The protocol of EPC proliferation assay and fluorescence staining was similar to that of ECs described in section 2.9.1. The only difference is that the culture medium



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used is α-MEM containing 10% FBS. EPC mobilization assay was carried out using a millipore transwell. In this experiment, nanocoating was constructed on 316L SS substrate with a diameter of 15 mm. During test, fresh culture medium was initially added to a 6-well plate (3 ml/well). The transwell was subsequently inserted into the pore of the plate, and 2 ml of EPC suspension (5×105 cells/ml) was added. After incubation at 37 ℃ for 2 hours, the samples were placed into the 6-well plate and incubated at 37 ℃ for 24 hours. Then, the bottom of the transwell was gently rinsed with normal saline and subsequently fixed in 90% ethanol solution at room temperature for 30 minutes. After that, the EPCs adhered to the upper surface of the bottom were removed using a swab, and the cells across the bottom and those that had migrated to the under surface were stained using 0.1% crystal violet. The mobilized cells were observed using inverted microscopy (Carl Zeiss A2). 2.10 Animal test An in vivo animal test was carried out by implanting 316L SS (blank control), NPL- and NPLS-modified disks into dog femoral arteries for 3 months. The disks were elliptical in shape (3 mm × 5 mm) and a tiny hole (0.5~0.8 mm) was predrilled at each end to facilitate sewing. Before nanocoating construction, the disks were mirror-polished on both sides. Six healthy adult dogs (~25 Kg) were used in this test and divided into two groups. In the first group (n=3), each dog receiving two blank controls in one femoral artery and two NPL-modified disks in another fermoral artery. In another group (n=3),


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each dog receiving two blank controls in one femoral artery and two NPLS-modified disks in another fermoral artery. During implantation, the disks were sewed to the inner face of the femoral artery by using a non-biodegradable suture. After surgery, the dogs received antibiotic treatment by injecting penicillin (8 million units per day) for 3 days. At 3 months, the disks were carefully sliced from the artery and rinsed with heparin solution immediately. After that, the entire morphology of new formed tissue was observed by a tridimensional microscope (Motic Images Plus 2.0 ML). Subsequently, the disks were immersed in 4% paraformaldehyde and incubated at room temperature for at least 12 hours. Afterward, the neointimal was carefully stripped from the material surface and sliced using a histotome. The tissue slices were then stained using hematoxylin eosin (HE) and Masson's trichrome staining protocol. The stained tissue was observed using an upright microscope. Mean thickness of neointimal was calculated using an image analysis system (Image Pro Plus). 2.11 Statistic analysis All the biological experiments were performed at least three times. The statistical data was analyzed using SPSS 11.5 software. Statistical evaluation of the data was performed using one-way ANOVA. The probability value P < 0.05 was considered significant. 3. Results 3.1 FTIR and XPS Changes in the surface chemical groups after certain modifications are shown in Figure 2. According to FTIR spectra (Figure 2A), the peaks at 1600 cm-1 and 1502



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cm-1 indicating benzene ring vibration were present on the poly-dopamine deposited 316L SS surface (SS-DM). The NP- and NPL-modified surface showed new peaks at 1666 cm-1 and 1555 cm-1, which approximate to the amide I and II that mainly derived from PLL.The characteristic peaks of heparin absorption could also be observed at 1230 cm-1 (C-O-S and S=O vibration) and at 1056 cm-1 (C-O-C vibration). The incorporation of SDF-1α had no influence on FTIR peak position, as its chemical composition and structure were similar to that of PLL and laminin. The result of XPS wide-scan spectra is shown in Figure 2B. Compared with SS-DA, new peaks at ~234.6 eV (S2s) and ~168.8 eV (S2p) are appeared on SS-DA-NP, SS-DA-NPL, and SS-DA-NPLS, which demonstrate the existence of heparin. Furthermore, as the protein is nitrogen-rich, the surface nitrogen content (shown in Table 1) gradually increased with the incorporation of laminin and SDF-1α. However, the sulfur content increased on the NPL-modified surface but decreased after assembling SDF-1α. This result indicated that the incorporation of laminin may contribute to raising the nanoparticle binding density, while the assembly of SDF-1α may cause a physical shielding effect from the exposure of heparin. 3.2 Surface morphology and hydrophilicity Figure 3A shows the AFM images of SS-DA and nanoparticle-modified surfaces. Polydopamine-coated 316L SS surface is relatively smooth, with only a few tiny particles derived from

dopamine self-polymerization. For SS-DA-NP and

SS-DA-NPL, it is easy to see that the nanoparticles are successfully immobilized and evenly distributed on the DA-coated surface, and the particle sizes are consistent with


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the results presented in Figure S1. The assembly of SDF-1α has no significant impact on surface morphology. The alteration of surface hydrophilicity after certain modifications was detected by water contact angle assay. According to Figure 3B, the water contact angle of SS-DA-NP (32.8 ± 3.7°) was greatly decreased when compared with SS-DA (68.9 ± 1.4°), as both Hep and PLL contains large amount of hydrophilic chemical groups, such as carboxyl, amine, hydroxyl, and sulfonic acid groups (Figure 3B). On the NPL-modified surface, the water contact angle is increased when compared with SS-DA-NP, suggesting that the assembly of laminin may decrease the hydrophilicity of nanocoating. The assembly of SDF-1α, however, did not cause significant changes in the water contact angle when compared with SS-DA-NPL; the reason may partly due to the relatively low SDF-1α concentration and binding density. 3.3 Heparin, amine, and RGD exposure density AO II and TBO assay were used for characterization of exposed amine groups and heparin, and the results are shown in Figure 3C. As a protein, laminin is rich in amine groups; therefore, the incorporation of laminin and assembly of SDF-1α were found to increase the amine exposure density. As the amino group played a vital role in the grafting of NPs to the DA-coated surface, the increase in amine exposure density may enhance the binding efficiency of NP to the DA coating. As a result, the heparin exposure density was significantly increased (*p

Laminin-Loaded

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