Catechol Film for in Situ Generation of

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ACS Biomaterials Science & Engineering

Copper-Incorporated Collagen/Catechol Film for in situ Generation of Nitric Oxide Rifang Luo, †,‡, ƾ Yujie Liu, †,‡, ƾ , Hang Yao, † Lang Jiang, † Jin Wang, *,†,‡ Yajun Weng, †,‡, Ansha Zhao, † Nan Huang†,‡ †

School of Material Science and Engineering，Southwest Jiaotong University，Chengdu，610031，

China ‡

Key Lab of Advanced Technology of Materials of Education Ministry, Southwest of Jiaotong

University, Chengdu, 610031, China ƾ

These authors contributed equally.

*Corresponding author：Tel：+86 28 87634148. Fax：+86 28 87600625.

E-mail address：[email protected] (Jin Wang)

ABSTRACT Local and continuous release of nitric oxide (NO) has been suggested to be a potential and desirable demand for blood contacting implants. However, the life time of NO release from polymer films is limited by the reservoir of loaded NO donor. In situ generation of NO via catalytic decomposing the endogenous S-nitrosothiols (RSNOs) at the blood/material interface is a novel and challenging approach. Herein, a copper-incorporated film was constructed with the copolymerization of catechols (catechol or epigallocatechin gallate (EGCG)) and collagen. FT-IR results suggested the successful deposition of catechol/collagen copolymer film. The XPS results demonstrated the existence of copper on the surfaces. AFM results demonstrated that copper particles were formed in the thin polymeric film. Copper-incorporated samples presented a capability of generating physiological levels of NO. Difference of the generated amount of NO was associated with the Cu (I) concentration during the testing period, demonstrated by micro-BCA assay. NO-generating films not only

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showed significant properties on inhibiting platelet activation and adhesion, but also dramatically decreased smooth muscle cell adhesion. Such copper-incorporated film might suggest potential in the design of vascular devices. Key words: nitxic oxide (NO); NO generating catalysis; collagen; catechol; copper; polymerization

INTRODUCTION Since the discovery of nitric oxide (NO) in 1980s, this signal molecule has been shown to play various significant roles in biological process, including cardiovascular homeostasis,

wound

neurotransmission

healing,

immune

response,

bone

metabolism,

and

1-4

. Recent investigations have been working on the development

of efficient NO-related materials for clinical demands 5-7. In relation to cardiovascular disease (CVD) therapies, although implants like vascular stents have demonstrated its benefit effects, thrombus formation and intimal hyperplasia are still concerns

6-8

. One

of the main topics of stent design is ‘rapid endothelialization’, mainly ascribed to the essentially inherent properties of generating NO via eNOS in the endothelial cell (EC) layer

9, 10

. Biomimetic designs for doing endothelium function are interesting and

challenging. Owing to the benefit effects of NO, blood/material interface which could release NO seems to be helpful in inhibiting platelet adhesion and smooth muscle cell (SMC) proliferation, which are both significantly to be concerned in vascular interface design 11-15. Indeed, a number of NO-releasing polymer coatings using polymer-embedded NO donor molecules, such as diazeniumdiolated dibutyl hexanediamine (DBHD/N2O2), have been studied over the last decade

16-19

. Such materials had shown greatly

improved blood compatibility in various animal models

20, 21

. However, despite the

demonstrated effect so far, due to the limited reservoir of the NO donors in the polymer coatings, the NO-releasing approach is only suitable for biomedical devices that require short-term contact, such as hemodialysis and coronary artery bypass surgery, but not for long-term implantations. NO-generating approach might be a potential candidate for long-term implants design. Polymers containing an


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immobilized catalyst such as glutathione peroxidase (GPx)-like catalytic mimics (i.e. selenocystamine, cystamine) and copper (Cu) nanoparticles or Cu (II) ion/ligand complex have been reported by liberating NO from circulating S-nitrosothiols (RSNO) 22-26

at the plasma/polymer interface

. During circulation, NO production may be

possible to take advantage of endogenous species (RSNOs) that are constantly produced in the body 27, 28. It is known that catalytic species of copper to decompose RSNOs is either Cu(I) or Cu(II), which could also be obtained via corrosion of copper nanoparticles

29, 30

.

Along with a low dose of copper embedding, efficient NO-generating was also achieved with no toxic effect

28, 30

. Based on these observations, in this work, we put

forward a mussel inspired approach to construct a polymeric thin film for embedding copper catalysts. As demonstrated by Lee and Messersmith, the co-existence of catechols and amines was crucial when performing dopamine self-polymerization process 32, 33. Here, we used separate molecules which respectively played the roles as catechol and amine. Briefly, catechol and epigallocatechin gallate (EGCG) were chosen as the catechol donors and collagen which was also the primary component of extracellular matrix of endothelial cell was set as the amine donor. As reported, the oxidation of catechol to quinone and crosslinking among amines and quinones made sense in dopamine polymerization-like process. This catechol and amine crosslinking polymer could coat various substrates like metals and polymers. Along with the redox and crosslinking reaction, copper was embedded into the thin film. Within the redox and chelation reactions between catechols and copper ions, it might cause diverse copper type, including Cu(I), Cu(II) and Cu(0). All these three types of copper would make sense in decomposing RSNO

29, 30

. Thus, further copper induced catalytic

generation of NO might become reliable (Scheme 1). Besides, facing the potential use in vascular devices, the in vitro evaluations of blood/material interactions were performed, including hemocompatibility test and vascular cell (EC and SMC) interactions. Due to the adhesive and crosslinking properties of catechol compounds, this work provided a facile strategy of preparing copper-incorporated films. This work also aimed at enriching the research of copper-containing surface for in situ catalytic



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generation of NO from endogenous RSNOs.

Scheme 1. The illustration of (a) preparation of copper-incorporated polyphenol/collagen film and (b) the hypothesis of in-situ generation of nitric oxide from RSNOs.

MATERIALS and METHODS Material CuCl2·2H2O (Kelong Chemical Reagent Co., Ltd., China) acted as the precursor of copper catalyst species. S-nitroso-N-acetyl-DL-pencillamine (SNAP), L-glutathione (GSH), catechol and Griess reagent were all purchased from Sigma Aldrich Chemical Co. EGCG was bought from Taiyang Lvbao Co. (Jiangsu, China). Acid soluble type I collagen from bovine skin was provided from Sichuan University 31. Deionized water was used in the NO releasing experiment. Micro-BCA was obtained from Pierce Biotechnology Inc. (Rockford, U. S. A.). Various reagents used for the evaluation of hemocompatibility

and

cytocompatibility

were

provided

from

professional

manufacturers which were mentioned in the experimental part. Other reagents were local products of analytical grade. Preparation of copper-incorporated thin film


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Copper-incorporated thin films were fabricated on 316L stainless steel (SS) or 24 well cell culture plate at room temperature in the mixture solution of collagen (0.5 mg/ml), catechol (0.5 mg/ml) or EGCG (2 mg/ml), CuCl2·2H2O (0.5 mg/ml) in acetic acid buffer solution overnight (by using 1 M HCl or 1 M NaOH to adjust the pH value to 5.5). After deposition, samples were ultrasonically cleaned with deionized (D.I.) water and then labeled. Films deposited on 316L SS were labeled as SS-C-CA (collagen/catechol copolymerization) and SS-C-E (collagen/EGCG copolymerization), respectively. Characterization of the films The chemical structures of the films were investigated by attenuated total reflectance

Fourier

transform-infrared

(ATR-FTIR,

NICOLET

5700)

spectrophotometer at the range of 4000–500 cm−1. The surface chemical compositions were characterized by X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos Ltd, UK). The instrument was equipped with a monochromatic Al Kα (hν=1486.6 eV) The C1s peak (binding energy 284.7 eV) was used as a reference for charge correction. The contact angle was measured by static drops using DSA100 (Krüss, Hamburg, Germany) with the method depicted by the manufacturer at 25 oC and 60 % relative humidity. Atomic Force Microscope (AFM, Digital Instruments, Santa Barbara, California) was used to investigated the morphology of the film. In vitro Cu (I) content analysis The mechanism of NO generation is dependent upon the blood-borne low molecular weight S-nitrosothiols (RSNO), to come in contact with the reduced ionized Cu (I) species and generate NO from the RSNOs. Thus during the test period, the real Cu (I) content might affect the catalytic effect. Copper analysis via the colorimetric BCA assay has excellent sensitivity and selectivity, and the results have been reported to correlate very well with a conventional atomic absorption 34. In the traditional micro-BCA test, Cu (II) will be reduced to Cu (I) and form BCA/Cu+ complex which could be read at 562 nm. In this modified micro-BCA test, the BCA solution did not contain any Cu (II) ions and the real reactive copper ions were



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coming from the copper-incorporated films (via releasing or corrosion). Samples were immersed into the modified micro-BCA solution (300 uL) and incubated at 37 oC for 30 min, after that 160 uL of BCA/Cu+ complex solution was taken out and transferred to a 96 well plate and exhibited a strong linear absorbance at 562 nm. This test could directly show the relative content of Cu (I) which will affect the catalytic effect of generating NO form RSNO. Catalytic release of nitric oxide in vitro The NO release catalyzed by sample was examined using Saville-Griess reagent reported before

11, 35, 36

. Briefly, a 10 mL solution containing 65 µM SNAP, 30 µM

GSH was added into a three-neck bottle, and the releasing NO during the RSNO decomposition was continuously purged from the test solution with argon. Saville-Griess reagent was prepared by adding HgCl2 into Griess solution to obtain a final concentration of 2 µM. Then RSNOs were detected before and after the catalytic decomposition for 30 min by reacting with equal-volume Saville-Griess reagent, and the absorption was read at 540 nm. The amount of releasing NO was obtained by calculating the decrease of RSNO after catalytic decomposition. Platelet adhesion test and cGMP analysis The amounts of the samples used for statistical count were no less than five, and each test was carried out more than three times. The fresh human whole blood (citrated blood) for the experiments was legally obtained from Blood Center of Chengdu, China. The analysis was performed within 24 h after the blood donation. Platelet rich plasma (PRP) was prepared by centrifuging (1500 rpm, 15 min) fresh human whole blood. Due to its chemical instability, little endogenous RSNO can be preserved in the final PRP 11. Therefore, RSNO was added for compensation. For each sample 1 ml of PRP was added, supplemented solutions of SNAP (a commonly used RSNO in vitro) and GSH were added subsequently, and finally 65 µM SNAP and 30 µM GSH were obtained. 500 µL of fresh PRP was distributed on the samples (10 mm diameter) and incubated for 0.5 h at 37 ◦C in humidified air. After washing with PBS buffer solution, they were fixed using 0.5 % glutaraldehyde solution for 12 h, then


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washed again with 0.9 % PBS for three times, and subsequently immersed in 40, 50, 75, 90, 100 % ethanol solutions for 15 min. After critical point drying, samples were gold sputtered, and then examined by scanning electron microscopy (SEM, Quanta 200, FEI, Holland). In this test, samples without addition of SNAP were set as the controls. cGMP of platelets after contacting different samples was analyzed using human cGMP ELISA kit (Hufeng biotechnical Co, Shanghai, China). PRP with NO donor (SNAP) was freshly prepared similar to the platelet adhesion experiment. After platelet incubation for 30 min, 100 µL 10 % triton was added and followed by sonication, then centrifuged with 2500 rpm to separate cell fragments and the supernatant was used for analysis. Vascular ECs culture and MTT assay Human umbilical vein endothelial cells (HUVECs) were isolated from new-born umbilical cord according to Jaffe et al’ work 37, using enzymatic digestion. Following isolation, HUVECs were cultured in M199 media (Gibco, U.S.A.) supplemented with 15% FBS (Sigma, U.S.). Cells were cultured in humidified air containing 5 % CO2 at 37 °C. Cells in passage two were used. For cell adhesion study, the samples were placed inside the 24 well polystyrene culture plates. Also, all the samples were double prepared, one for compensation of SNAP donor series (65 µM SNAP, 30µM GSH) and one for the series without donor as the controls. HUVEC were seeded at the density of 5×104 cells /cm2 with endothelial basal medium. For the positive control group, HUVEC were seeded at the same density on SS surfaces under the same media condition. At predetermined time point (0, 2, 6, 12, 24 h), the culture medium was 80 % taken out and freshly prepared donor containing culture medium was replenished into the plates. This operation was trying to mimic the approach for the continuous NO interaction with endothelial cells during cell culture period. After each time point, samples were taken out and washed in PBS, fixed with 5 % glutaraldehyde solution for 4 h, After that ,cells were Rhodamine-phalloidin stained (50 µL per sample, lucifuge for 20 min). After that samples were visualized by fluorescent microscopy



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(Zeiss, Germany). In brief, images were obtained and then processed using IPWin60C (software), and the average cell spreading area and cell number was then converted to evaluate the cell adhesion .For each sample, 10 images (100 X) were taken and the number of the cells at each image was obtained by manually counting and the results were averaged. The averaged adhered cell number was then converted to cell adhesion density. To investigate the endothelial cell metabolic activity (viability) on different samples, (3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out. ECs were seeded onto each sample at 5 × 104 cells per well. After 1 day and 3 days culture, M199 media was removed and 400 µL of MTT was added into each well and incubated for another 4 h to form water insoluble formazan in cells, then MTT was removed and 400 µL DMSO was added into each well, shaken for 15 minutes to dissolve water insoluble formazan. Then 120 µL of the above formazan solution was taken out from each sample and added into one well of a 96-well plate. The optical density was measured at 570 nm wavelength with the micro-plate reader. SMCs adhesion test Human umbilical artery smooth muscle cells (HUASMCs) were isolated from new-born umbilical cord by the explant method as described previously 38. Following isolation, SMCs were cultured in DMEM-F12 media (Gibco, U.S.A.) supplemented with 10 % FBS (Sigma, U.S.). Cells were cultured in humidified air containing 5 % CO2 at 37 °C and cells in passage two-five were used. Also the samples were double prepared as mentioned before. To investigate the cell proliferation behavior, SMCs were seeded at a density of 5 × 104 cells/per sample and incubated at 37 °C in DMEM-F12 media supplemented with 10% FBS for 2 h. Then samples were taken out and washed in PBS. Then all the samples were fixed, fluorescence stained, and visualized by fluorescent microscopy as the same procedure mentioned in ECs evaluation. RESULTS and DISCUSSION Film characterization


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Inspired by mussel adhesive proteins, Lee and Messersmith did a fantastic job via a straightforward

method

for

the

synthesis of

polydopamine

by

oxidative

polymerization of catecholic dopamine 32, 33. There were also some reports indicating the oxidant-induced-polymerization of dopamine to expand the application of polydopamine film

39-41

. It is believed that even at an acidic environment, dopamine

will perform its role as a reductant via the catechol oxidation to quinone with the input of oxidant. In this work, copper ion will perform its role as an oxidant, together with the co-existence of amine (collagen) and catechol compounds, such dopamine-polymerization-like process might be achieved. Typically, collagen (0.5 mg/ml), EGCG (2 mg/ml) and CuCl2·2H2O (0.5 mg/ml) were co-solved in an acetic acid buffer solution (pH = 5.5). Under aerobic conditions, the phenol hydroxyl groups of EGCG would suffer oxidation with the colour gradually turning to light and then dark yellow, suggesting that the catechol moieties were oxidized to quinones. Normally, the process of catechol oxidation to quinone is quite low at acidic condition, thus we believed that copper ions here played the role as oxidants. Moreover, the crosslinking among amines and quinones could also be performed under weak acid conditions 33. After reaction for about 6 h, obvious floc formation could be seen in the solution suggesting the polymerization was done like what happened during dopamine polymerization. Owing to the catechol groups, such polymerized coating was believed to coat various substrates. Noteworthily, the copper-free collagen/catechol or collagen/EGCG film could not be prepared. The co-polymerization process between amines and catechols should be achieved under a mild basic pH solution, however the solubility of collagen (amine donor) is quite low in basic pH solution which is the main obstacle to form collagen/catechol film. The chemical structure of the modified surfaces was analyzed by ATR-FTIR spectroscopy. As shown in Fig. 1 (a), both copper-incorporated collagen/catechol thin films had a broad peak around 3400 cm−1 which were ascribed to aromatic -NHx and –OH stretching vibrations. The films also presented peaks at 1680 cm-1 (amine I peak) and 1520 cm−1 (the overlap of C=C resonance vibrations in aromatic ring). It meant that amines and phenol hydroxyls groups were partially retained after polymerization



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process. As shown in Fig. 1 (b), compared with stainless steel, the presence of nitrogen and copper suggested the successful deposition of copper-incorporated collagen/catechol or collagen/EGCG films. The atomic ratio of the elements was listed in Table 1.

Fig. 1. (a) ATR-FTIR results (red for SS-C-CA sample and blue for SS-C-E sample), (b) the XPS results, (c) the surface water contact angle results of different samples, (d) high resolution of Cu 2p peak of SS-C-E sample, (e) high resolution of N1s (SS-C-E). Table 1 Elemental composition and ratios of each coating via XPS analysis. Sample

Cu (%)

C (%)

N (%)


O (%)

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SS-C-CA

6.90

75.09

7.03

10.50

SS-C-E

4.58

77.45

4.15

13.83

The water contact angle (WCA) of each sample was shown in Fig. 1 (c). Noteworthily, the original 316L SS presented a value of 75° while the WCA values of the treated samples reduced and reached to almost 45.0°. The sharp decrease of the WCA might be ascribed to the introduction of the amine and hydroxyl groups which were hydrophilic. In principle, Cu(I)/(II) ions will be chelated by surrounding phenol hydroxyl groups, and on the other hands copper ions would play the role as a oxidant and lead to the oxidation of catechol to quinone, associated with the Cu(0) formation. Taking SS-C-E as an example, according to Fig.1 (d), the peak of Cu 2p3/2 and 2p1/2 were wide and could be fitted to two parts, one for Cu (I) and (II) and the other part for Cu (0). This result suggested that, copper ions which played the role as an oxidant were reduced to Cu (0) state during polymerization process. Besides, Fig. 1 (e) presented the N1s signal of the SS-C-E sample. The fitted curves showed the obvious aromatic N signal which was ascribed to the Schiff base or Michael addition between amines (collagen) and catechols/quinones (polyphenols). These observations could also suggest that the 316L SS was successfully coated with collagen/catechol thin films. Due to the presence of catechol groups, such coating was believed to be deposited onto various substrates because of the intrinsic adhesive nature of catecholamine. For a typical AFM and TEM evaluation, coatings were prepared onto silicon wafers and TEM copper grid. After the deposition of copper-incorporated collagen/catechol thin films, copper particles might form and embedded within the film (Fig. 2 a and b). Recently published papers have shown the possibility of catechols and copper ions to form copper nanoparticles with size from tens to hundreds nm

42, 43

. Briefly, catechol

groups were involved both in Cu (II) reduction and nanoparticle surface binding. In our job, the embedded copper particles in the outer surface presented a nano-size (20 – 250 nm) which showed a similar agreement with those catechol-induced copper nanoparticle formation results. Although the detailed ratio of copper state was still



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unclear, yet in this study, no matter the state of copper, Cu(I) would be the direct ion to decompose RSNO to generate NO. Cu(II) and Cu(0) might also make sense to change its state to Cu(I) because of the convenience via redox or corrosion respectively 29.

Fig. 2. (a) AFM results of Si, Si-C-CA and Si-C-E samples and (b) TEM results of C-E samples prepared on uncoated TEM copper grid.

Catalytic generating of nitric oxide in vitro As mentioned before, in the presence of reductant GSH, copper can catalysis decomposition of S-nitrosothiols. In this work, in vitro catalytic degradation of SNAP was evaluated using Saville-Griess reagent (Fig. 3 (a)). It could be seen that the Cu-incorporated samples has considerable NO generating catalytic activity. It was reported healthy endothelial cells continuously synthesize and secrete NO with a rate of approximately 0.5-4×10-10 mol/cm2/min

44

. The copper-incorporated samples

catalytically generate NO with a rate about 0.77×10-10 mol/cm2/min with catechol and 0.28×10-10 mol/cm2/min with EGCG. The rate of all Cu-incorporated samples is within the physiological level. As known, Cu (I) was the principal for direct catalyzing of decomposing RSNOs to generate NO 29, 30. The relative content of Cu (I) might directly reflect the effect decomposing of NO from SNAP in this study. As seen in Fig. 3 (b), modified micro-BCA assay suggested that during test incubation period (30 min), the real Cu (I) concentration on SS-C-CA sample solutions was the highest (almost 18 µM) which also showed good agreement with the NO releasing rate


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results.

Fig. 3 (a) Catalytic effect of generated NO releasing rate and (b) released Cu (I) concentration during the testing period using modified Micro-BCA method (within 30 min).

Platelet adhesion, activation and cGMP analysis The effect of NO release catalyzed by Cu-incorporated samples on platelet adhesion was investigated. As seen in Fig. 4 and 5 (a), compared to the bared SS, an obvious enhanced inhibition of platelet activation and aggregation can be observed on Cu-incorporated samples. The shape of platelet adhered on biomaterials can be classified into five kinds: round, dendritic, spread, dendritic spread and fully spread 45. Without addition of SNAP, platelets adhered on copper incorporated samples were nearly fully spread. Yet, after the addition of donor, with the help of copper ions, NO was released via the decomposing of SNAP and showed significant inhibition effect on platelet activation. This phenomenon could not be found on 316L SS surfaces because of no copper release. These results suggested the successful catalytic effect of decomposing NO from SNAP on copper-incorporated sample surfaces. The result of cGMP analysis revealed that intracellular cGMP levels after incubation of Cu-incorporated sample with SNAP for 30 min are significantly increased (Fig. 5 (b). There are many studies suggesting that NO can inhibit the activation of platelets

46-48

. Most likely, in our work, the possible effect of NO

generating films on inhibiting platelet adhesion was mediated via the elevation of cGMP. In this study, although the film was constructed with collagen components, which was quite positive in platelets aggregation and activation, with the incorporated copper, a dramatic effect in inhibiting platelet activation was observed. This collagen



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based surface could also mimic the injured tissue surface like stent implantation (with collagen exposure in the vessel wall). Herein, copper-incorporated film was potent in decomposing RSNO to release NO and thus present enhanced hemocompatibility via up-regulating cGMP level of platelets.

Fig. 4. (a) Morphology of adhered platelets on different samples with and without addition of donor and (b) the coverage ratio of platelets on different samples.

Fig. 5. (a) Staining of platelets and (b) the cGMP level of platelets on different samples with the addition of SNAP (bar 25 µm).

ECs adhesion, proliferation and viability One of the inherent properties of endothelial cell (EC) is to release NO to maintain


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vascular micro-environment balance. Besides, some published results suggested that released NO in physiological level would promote EC proliferation

49, 50

. Rapid

endothelialization had also been frequently demonstrated in the survival of implants. Hereon, the effect of the catalytic generation of NO on ECs adhesion, spreading and proliferation was evaluated. As is well accepted, good cell adhesion property does a great favor in cell survival. The morphology of adhered ECs after 2 h culture was distinctly listed in Fig 6. Compared to SS, the number of adhered ECs on Cu-incorporated samples surfaces was higher, especially when in the presence of donor. After being cultured for 1 day and 3 days (Fig 8), the number of attached ECs on the samples generally increased during cell culture, and ECs on Cu-incorporated surfaces presented higher cell viability compared with samples in the presence of donor. Moreover, the results of cell counting and MTT (Fig 7) suggested good adhesion and proliferation which could not be found on SS surfaces.

Fig. 6. Endothelial cells adhered onto different samples after culture for 2 h (bar 50µm).

Fig. 7. Cell viability results of ECs cultured on different samples for 1 day and 3 days.



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Fig. 8. Cell staining of ECs cultured on different samples with and without addition of NO donor when cultured for 1 day and 3 days.

Note that, even at SS samples which did not have catalytic effect of decomposing SNAP, also showed a little bit enhanced cell viability and with the addition of NO donor. The injected SNAP could also suffer self-decomposition. Although the amount of self-decomposed SNAP is not as much higher as copper-generated decomposition, a low concentration of NO would also make sense for a certain enhanced EC viability 51

.

SMC adhesion The effect of the catalytic generation of NO on SMCs adhesion and spreading was evaluated. Significant differences were found in Fig. 9. SMCs cultured on copper-incorporated samples were spread greater than those samples cultured with addition of SNAP. This phenomenon could not be found on the cells cultured on316L SS. The inhibition of SMCs attachment by catalytic generation of NO was also


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demonstrated.

Fig. 9. Cell staining of SMCs cultured on different samples with and without addition of NO donor within 2 h culture.

The present results have demonstrated the effect of copper-incorporated films on in-situ generating of NO from decomposing SNAP. In vitro study indicated that, with the NO releasing from the nearby surface, platelet activation was dramatically decreased. Moreover, NO releasing is at a safe physiological level which could enhance the proliferation of endothelial cells and on the other hand, inhibit adhesion of smooth muscle cells. However, the in vivo tests of such film are urgent, because the copper particles might cause reactive oxidative species (ROS) production in vivo and cause complicated cell/material interactions. Moreover, detailed work on long-term performance and cytotoxicity, especially the cytotoxicity associated with copper content or NO generation level is an urgent job to facing the potential application. Especially for copper induced in vitro cytotoxicity, not only the released copper ions concentration in culture medium, but also the state of copper would make contributions. As well known, higher concentration of copper ions and the existence of copper particles (induce ROS production) are responsible for cytotoxicity

52-54

. In

this study, a complexity could not be ignored is the coexistence of NO and copper particles. Moreover, NO might be a double-edged sword for protecting cells or performed like ROS. The next step of this study is to investigate above concerns. Nevertheless, current results presented potential application of such film for the



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modification of vascular stent. CONCLUSION In this work, copper incorporated polymeric collagen/catechol thin film was successfully prepared. Compared with controls, such copper embedded samples presented significant enhanced nitric oxide (NO) generating effect. The catalytic effect was associated with the available Cu (I) content during tested immersion period. Obvious inhibition of platelet activation and aggregation were seen on the copper incorporated samples. Besides, in vitro cell culture tests showed that biological level of NO releasing could also promote the adhesion and proliferation of ECs, and on the other hand inhibit the adhesion of SMCs. This approach also suggested the potential for the surface modification of vascular devices.

ACKNOWLEDGEMENTS This work is supported by National Natural Science Foundation of China (Project 51173149 and 81330031), Ministry of Scientific and Technical Project of China (Key Basic Research Project No. 2011CB606204). REFERENCES [1] Kuo, P. C.; Schroeder, R.A. The emerging multifaceted roles of nitric oxide. Ann.

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Catechol Film for in Situ Generation of

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