Copper-based SURMOFs for nitric oxide generation - ACS Publications

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Biological and Medical Applications of Materials and Interfaces

Copper-based SURMOFs for nitric oxide generation: hemocompatibility, vascular cell growth, and tissue response Qian Zhao, Yonghong Fan, Yu Zhang, Junfeng Liu, Weijie Li, and Yajun Weng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22731 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 10, 2019

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ACS Applied Materials & Interfaces

Copper-Based SURMOFs for Nitric Oxide Generation: Hemocompatibility, Vascular Cell Growth, and Tissue Response Authors: Qian Zhao1, 2, Yonghong Fan1, 2, Yu Zhang1, 2, Junfeng Liu1, 2, Weijie Li1, 2 and Yajun Weng1, 2 *

Authors address: 1

Key Laboratory of Advanced Technologies of Materials, Ministry of Education,

Southwest Jiaotong University, Chengdu 610031, China 2

School of Materials Science and Engineering, Southwest Jiaotong University,

Chengdu 610031, China

Keywords: Metal-organic frameworks, nitric oxide, surface modification, cardiovascular stents

Abstract: A coating that can generate nitric oxide (NO) for surface modification of cardiovascular stents with adaptable NO release is an efficient approach to prevent thrombosis and neointimal hyperplasia. Herein, we prepared a copper-based surface-attached

metal−organic

framework

(Cu-SURMOFs)

of

copper

(II)

benzene-1,3,5-tricarboxylate (CuBTC) using a layer-by-layer assembly method (LBL) for NO generation on the surface of alkali-activated titanium. It was easy to control surface chemistry and NO release by changing the number of LBL deposition cycles. The obtained CuBTC coating was characterized by XRD, SEM, FTIR, and XPS analysis and was able to decompose endogenous S-nitrosoglutathoine (GSNO) to catalytically produce NO. The resulting NO flux increased with increased deposition cycles. The coating prepared with ten cycles of deposition showed ideal NO release

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and promoted proliferation of endothelial cells (EC), suppressed growth of smooth muscle cells (SMC) and macrophages (MA), and inhibited platelet adhesion and activation. Further evaluation of thrombogenicity in an arteriovenous shunt model showed that the CuBTC coating had great ability to prevent thrombosis, and in vivo implantation of CuBTC-coated titanium wire demonstrated a significant inhibition of intimal hyperplasia. The results showed that use of copper-based SURMOFs could be a promising strategy for the surface modification of cardiovascular stents.

1 Introduction Nitric oxide (NO) is secreted by vascular endothelial cells (EC) and is a key signaling molecule in the blood circulation system. The biological activity of NO includes anti-platelet aggregation and activation, regulation of blood pressure, maintenance of vasodilatation, and anti-proliferation of smooth muscle cells (SMC)1. Hence many researchers have developed materials capable of NO generation for devices that contact the blood2. These materials can generate NO continuously by decomposing an endogenous NO donor (i.e. GSNO) catalytically. Cardiovascular stents constructed mainly of metal are widely used to treat atherosclerosis. These stents require good anticoagulant property and the ability to inhibit neointimal hyperplasia3. Therefore, NO-generating materials are considered a good candidate for surface modification of cardiovascular stents to inhibit platelet adhesion and activation, promote endothelialization, and suppress excessive proliferation of SMC4. In our previous work5, 6, diselenide molecules were immobilized onto stents for NO generation and the modified stents showed greatly improved anti-thrombosis property. However, the application of these stents was limited due to the degradation of the coating and selenium loss without supplementation. Metal organic frameworks (MOFs) represent a new class of coordination materials assembled from metal connecting points and organic bridging ligands7. The modifiable structure and property of MOFs can permit a wide range of applications8, 9.


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To date, the use of MOFs in biomedicine includes diagnosis, bio-imaging, drug delivery, and gasotransmitter release10. NO loaded in MOFs and reported to inhibit platelet aggregation11, relax coronary arteries12, and inhibit microbial proliferation13 in vitro. More recently, copper-based MOFs such as CuBTC

14

and CuBTTri15, were

reported to catalytically generate NO from an endogenous NO donor. Grown in situ on a polymer surface or blended into the polymer, the structure of the copper-based MOFs was maintained and catalytic efficiency remained high even after long-term immersion

16-18.

However, few studies have examined the biocompatibility of

copper-based MOFs, particularly for blood contact. Although many approaches have been explored to prepare MOFs on a material surface (SURMOFs), including direct seeded growth, dip-coating, gel synthesis, electrochemical deposition, and hydrothermal methods19, 20, most approaches require tedious chemical reactions and hazardous reagents, thus they are obviously not suitable for the preparation of biocompatible materials. The layer-by-layer (LBL) method was applied to prepare zinc- and copper-based SURMOFs on –COOH, -OH, or pyridine-terminated surfaces in ethanol solution21,

22.

The LBL method offers advantages of mild reaction

conditions23 , a simplified operational procedure, and no requirement for harmful residual reagents. Neufeld et.al18 deposited CuBTC crystals directly onto the surface of carboxyl-functionalized cotton using a LBL method for the generation of NO from endogenous sources, and the prepared material showed great potential in biomedical applications. However, little is known about preparing SURMOFs on metal surfaces and the ability of SUMORFs on metal surfaces to generate NO in vitro or in vivo. Herein, we used LBL method to prepare SURMOFs of CuBTC on titanium for NO generation. First, the titanium was alkali activated to form –OH terminated surface and then CuBTC crystals were deposited using LBL method. The resulting CuBTC deposited surface was characterized by X-ray diffraction (XRD), FTIR, SEM, and XPS. The NO release rate was evaluated by a chemiluminiscence analyzer (NOA 280i). A platelet activation test and thrombogenicity analysis ex vivo were performed to evaluate the hemocompatibilty of these materials. The proliferation of EC, SMC,



and macrophages (MA) was assayed in vitro, and the tissue response was investigated by implanting the CuBTC-modified titanium wire into the abdominal aortas of SD rats.

2 Experimental section 2.1 Materials. Pure titanium foil (99.5 ％ , 0.025×110 mm) was purchased from Zhong Nuo Advanced Material (Beijing) Technology CO. Ltd. Copper (П) acetate (Cu(OAc)2), Benzene-1,3,5-tricarboxylic acid (BTC, 98%), S-nitrosoglutathoine (GSNO), ethylenediamine-tetraacetic acid disodium salt (EDTA-2Na), L-glutathione (GSH), and bovine serum albumin (BSA) were all purchased from Sigma Aldrich Chemical Co. Ethanol and Phosphate Buffered Saline (PBS, 0.01 M, pH = 7.4) were used in experiments.

2.2 Hydroxylation of Titanium foil. The titanium foil (Ti) was immersed into 5mM NaOH solution and incubated at 80℃ for 12h to obtain anchoring functional groups (-OH) for LBL deposition of CuBTC. The resulting hydroxylated Ti surface (Ti-OH) was thoroughly cleaned by ultrasonic treatment in deionized water for 15 min.

2.3 LBL deposition of CuBTC. BTC and copper (II) acetate were dissolved into ethanol to obtain a concentration of 10 mM, respectively. The hydroxylated titanium foil was immersed into the copper (II) acetate solution for 15 min and rinsed thoroughly with ethanol for 5 min to remove the free copper ions. The foil was then immersed into the BTC ligand solution for 30min, and rinsed again with ethanol for 5 min. The samples were prepared by repeating the procedure for a total of 10, 20 and 30 complete cycles, and the resulting materials are referred to as M10, M20, and M30 in the following discussion. The samples were then immersed into ethanol for 5 days to remove the physically


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adsorbed copper ions. The ethanol was replaced every 12 hours, and the samples were air dried and cut into squares (1.0 cm×1.0 cm) for use.

2.4 Material Characterization. X-ray diffraction (XRD) measurement was carried out to detect the crystal structure of CuBTC. The samples were analyzed by using an X’Pert diffractometer (X’pert PRO, Philips, The Netherlands) with Cu Kα irradiation (λ = 1.5406 Å) at 30 kv/30 mA, as the 2θ was varied from 5° to 20°. The chemical structures of the sample surfaces were determined by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, NICOLET 5700) with diffuse reflectance mode at 4000–400 cm-1. X-ray photoelectron spectroscopy (XPS) analysis was carried out to detect the surface chemical compositions using a PHI-5400 X-ray photoelectron spectrometer (PerkinElmer, USA) with a monochromatic Al Kα (hν = 1486.6 eV) was employed as the excitation source. Analysis was performed at 10–20 kV working voltage and 45 mA emission current under a pressure of 2 × 10-9 Torr in the chamber. The binding energy scale was standardized by setting the C1s peak at 284.6 eV. Scanning electron microscopy (SEM, Quanta FEG 250, FEI, Holland) was used to observe the surface morphology and the elemental distribution of copper was analyzed by an energy dispersive X-ray detector accessory device (EDX).

2.5 Evaluation of stability of the SURMOFs and NO release. The mechanical stability of the prepared SURMOFs of CuBTC was evaluated by bending the samples repeatedly 10 or 20 times and then observed the samples by SEM. The loss of copper ion was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Briefly, the samples were immersed in 1 mL of PBS (pH=7.4) for 1, 3, 5, 7, 14, and 28 days at 37℃ and then shaken in an oscillator. The cumulative copper release with time was then measured. NO release from GSNO was recorded by a Sievers 280i chemiluminescence NO analyzer (NOA 280i). Nitrogen was used as the carrier gas, the reaction temperature was set at 37 ° C, and the oxygen supply was 6.0 ±0.2 psig. The instrument was



calibrated each time before testing using the calibrating gas (NO/nitrogen, 44.5 ppm). Then 5 ml of PBS was added to the reaction vessel and the samples (0.5 cm×1.0 cm) were hung above the reaction solution. Subsequently, GSNO (an endogenous RSNO species) and GSH (an endogenous reducing agent) were added. After baseline collection, the sample was pushed into the solution and the NO flux was measured and recorded by computer.

2.6 Cell growth on samples. The EC cell line was purchased from Servicebio Technology Co. Wuhan, China. The SMC were obtained by outgrowth from the fragments of human umbilical artery. EC and SMC were cultured in DMEM/F12 supplemented with 15% FBS. Mouse RAW264.7 macrophage lineage cells were purchased from the American Type Culture Collection (ATCC, USA) and cultured in DMEM/High Glucose with 10% FBS. The samples were placed in the wells of a 24-well cell culture plate and 1 mL cell suspension was added to each well. The density of seeded cells were as follows: EC for 1 × 104 cells/cm2, SMC for 5 × 104 cells/cm2, and MA for 1 × 105 cells/cm2. GSH and GSNO were added into the cell culture medium to obtain final concentrations of 20μM, and the culture medium was changed every 12 h for the experimental group. The control group did not receive GSNO and GSH. After additions, the samples were placed in a cell incubator at 37°C in damp air with 5% CO2. After the prescribed time, the samples were taken out, washed with PBS (pH=7.4), and then fixed with glutaraldehyde (2.5%). The cells were then stained with rhodamine and observed by fluorescence microscope. The density or coverage ratio of cells was calculated using Image J software from more than 10 photos obtained by the fluorescence microscope.

2.7 In vitro evaluation of platelet adhesion and activation. Fresh whole blood was obtained from the Blood Center of Chengdu, China. Platelet rich plasma (PRP) was obtained by centrifuging the blood samples at 1500 rpm for 15


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min. The samples were placed in the wells of a 24-well cell culture plate, and half the samples did not receive the NO donor and the other received the NO donor. Next, 500 μL of PRP was added to each sample. For the group receiving NO donor, GSH and GSNO were added to final concentrations of 80μM and 40 μM, respectively. Additionally, 10 μL of collagen (10 mg/ml) were added to activate the platelets in the NO donor group. After incubation for 45 min at 37 ℃ in the dark, the samples were then washed three times with PBS (pH=7.4), and then fixed by 2.5% glutaraldehyde overnight. The samples were stained with rhodamine and observed under a fluorescence microscope (Olympus, IX 51). After gradient dehydration, the prepared samples were then examined by SEM. The cGMP concentration of the platelets was detected after 45 min of incubation by using a human cGMP ELISA kit (Hufeng Biotechnical Co., Shanghai, China), using a test procedure that was similar to that used for platelet adhesion.

2.8 Ex-vivo blood circulation to test the thrombogenicity of samples. All procedures were performed in accordance with the Animal Protection Agreement of the China Animal Protection Association and Southwest Jiaotong University, and followed all appropriate ethical guidelines. The samples Ti and M10 (0.8 cm×1.0 cm) were rolled into a tube and placed in a heparinized extracorporeal circulation catheter. Adult New Zealand white rabbits (3.0–3.5 kg) were anaesthetized by injection of 30 mg/mL of pentobarbital sodium, and then the left carotid artery and right external jugular vein were isolated. A blood circuit was constructed by connecting blood vessels with the extracorporeal circulation catheter immediately after the injection of GSNO (1 mL/kg, 2mM) . The samples were removed and photographed after 30 min. The occlusion ratio was calculated by measuring the cross section of the tube. After dehydration, de-alcoholization, and critical point drying, the samples were observed by SEM. The flow rate (%) was measured by recording the time for 5mL of effluent simulated body fluid. At the end of the arteriovenous shunt experiment, the circuit was separated from the blood vessel as a two-ended catheter. Next, 5 ml of simulated body fluid was injected into one side of the catheter, and collected at the other end.



An empty catheter was used as the control, and its flow rate was set as 100%, and the reciprocal of the consumed time of the sample divided by that of the control was the relative flow rate of the sample.

2.9 In vivo animal implantation. Eight male SD rats (~300 g) were used in this experiment. The samples were prepared on a titanium wire and implanted into the abdominal aortas of the SD rats. Briefly, the rats were first anaesthetized by pentobarbital sodium, and then the abdominal aortas were isolated. The unmodified and MOF-coated titanium wires were implanted into the vessels. After one month, the samples were removed along with the surrounding blood vessel and fixed in 4% paraformaldehyde. Hematoxylin and eosin (HE) staining was performed by serially cutting the blood vessel after careful removal of the samples.

2.10 Statistical analysis. The experiments were performed at least three times and the results were expressed as the mean ± standard deviation (SD). Statistical significance was evaluated using a one-way analysis of variance (ANOVA) and the statistical differences between two groups were considered significant for p < 0.05.

Figure 1 Schematic diagram of LBL deposition of CuBTC coating on alkali-activated


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titanium surface and Optical micrographs showing the sample color change.

3 Results and discussion 3.1 Preparation of CuBTC on alkali activated titanium surface. CuBTC was deposited on –OH terminated surfaces using the LBL method as described by Wöll et al.24 Here, the titanium was alkali activated by NaOH solution to form the –OH terminated surface. New strong absorption bands appeared on Ti-OH in the range of 2850 ~ 3650 cm-1 (-OH, stretching absorption) and 700~1000 cm-1 (Ti-O, stretching absorption) that were not present in the FTIR spectra for Ti alone, indicating the presence of –OH groups on the surface (Figure 2A). Upon CuBTC deposition, strong absorption bands were observed at ~1640 cm−1 (COO-, asymmetric stretching absorption), ~1370 cm−1 (COO- symmetric stretching absorption), ~729 cm−1 (C−H, out-of-plane bending absorption), and 1430~1650 cm−1 (-C=C-, benzene ring vibration) corresponding to BTC coordination with Cu2+.18 The XRD patterns clearly showed a new peak at 2θ = 11.7º on the surface with the deposited CuBTC compared with titanium and alkali-activated titanium. This peak was identified as (222) face of CuBTC based on the simulated XRD pattern. The oriented crystal growth of CuBTC on the –OH surface was also reported by Shekhah et.al25. In addition, the intensity of the peak increased with consecutive cycles of deposition, indicating continual growth of the CuBTC crystals (Figure 2B). There were obvious peaks of Cu2p and C1s for the CuBTC deposited surfaces in the XPS full spectra, and an -O-C- peak at 532.28 eV in the O1s high resolution fitting spectra (Figure 2C, D). The atomic concentration of Cu increased, but the amounts of Ti and O were reduced with increased CuBTC deposition cycles (Table 1). There was an obvious color change of the surface, as observed by visual inspection (Figure 1). The SEM image revealed that the growth of CuBTC crystals with consecutive deposition cycles blocked the pits on the Ti-OH (Figure 3). The crystals were ultrasonically stripped from the surface and then examined by TEM. Homogeneous and robust CuBTC crystals were observed (Figure S1). The copper



distribution was determined by EDX and revealed a uniform distribution of CuBTC crystals on the surface (Figure 3). Together, the results showed that CuBTC is a regular complex of Cu2+ and BTC and there was successful deposition of CuBTC onto the alkali-activated titanium surface.

Figure 2 Surface characterization of Ti, alkali-activated Ti, and CuBTC deposited surfaces. (A) FTIR spectra of samples. (B) XRD analysis of samples, with 2θ varied from 5° to 20°; the patterns were contrasted with the simulated CuBTC pattern. (C) XPS full spectra for the samples. (D) Curve fitting of O1s high resolution spectra of sample Ti-OH and sample M10.


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Figure 3. SEM images of samples and EDX analysis of Cu on SURMOFs of CuBTC.

Table 1 Elemental compositions of Ti, alkali activated Ti, and CuBTC deposited surfaces determined by XPS (Atm. %).

Sample

C

O

Ti

Na

Cu

Ti

0

69.5

30.5

0

0

Ti-OH

35.7

40.7

16.2

7.2

0

M10

71.1

19.2

2.1

1.4

5.5

M20

77.3

14.5

1.0

0.5

6.7

M30

76.6

14.5

0.5

0.5

7.5

3.2 NO generation and stability. Mechanical stability of the CuBTC coating was analyzed by folding the samples 10 or 20 times. The SEM image showed that some of the CuBTC crystals peeled off from the surface after folding, and the stability increased with increased deposition cycles (Figure S2). In addition, very slow leaching of copper from the CuBTC coating was observed over time (Figure S3). The results suggested good stability of the CuBTC coating, suggesting this material would persist on cardiovascular biomaterials for long-term service. Appropriate and stable NO generation is an important determinant of the good biocompatibility of NO generation materials. The NO generation was examined using a Sievers 280i Nitric Oxide Analyzer, and GSNO was used as the NO donor in this



experiment. NO release was monitored by recording the NO flux with time. To confirm that the observed increase in NO generation was due to the presence of CuBTC on the surfaces, Ti and Ti-OH were used as controls, and there was no obvious increase of NO release after immersion of Ti and Ti-OH into the NO donor (Figure S4). An initial burst of NO release was observed when the freshly prepared CuBTC coating was immersed into the donor. The GSNO was consumed rapidly, then the reaction solution was replaced and additional NO donor was added. This phenomenon occurred several times before stable release was achieved (Figure 4, S5 and S6). This may be attributed to Cu2+ leaching from the coating, increasing the burst release of NO. When the free Cu2+ in the coating was washed away, and the chelated Cu2+ in the CuBTC coating caused the stable NO release. The NO flux increased with the number of deposition cycles, and the results revealed that the NO release catalyzed by M10 reached (5.0±1.5) × 10-10 mol·cm-2·min-1, M20 reached (9.1±2.2) × 10-10 mol·cm-2·min-1, and M30 reached (13.1±2.5) × 10-10 mol·cm-2·min-1 (30μM GSNO and 30μM GSH). To confirm that the detected release of NO was due to the deposited CuBTC coating and not free Cu2+, 500μM EDTA was added into the reaction solution to silence its catalytic activity. As a strong chelating agent, EDTA can chelate free copper ions immediately and mask the catalytic activity of Cu2+, or potentially even competitively bond with the Cu2+ from CuBTC. With addition of EDTA, the NO flux catalyzed by CuBTC exhibited a dramatic decline with no initial NO burst (Figure 5A). To investigate the durability of the CuBTC coating, samples were immersed in PBS at 37℃ for 30 days, and then the catalytic activity of the coating was tested after being carefully washed. No significant decrease of NO release was observed under the same condition (30μM GSNO and 30μM GSH) (Figure 5B). The result showed efficient NO release rates of (4.0±0.8) × 10-10 mol·cm-2·min-1, (8.5±1.5) × 10-10 mol·cm-2·min-1, and (12.5±2.0) × 10-10 mol·cm-2·min-1, respectively, for M10, M20, and M30. Protein adsorption is the first event after implantation of biomaterials, and active sites of the CuBTC catalyst may be blocked by bound protein. To test this, we next measured the NO release of a CuBTC-mounted surface after protein adsorption. After being


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incubated with cell culture medium (15% FBS) for 2h, the NO release rate of sample M10 decreased to (2.8±1.2) × 10-10 mol·cm-2·min-1, that of sample M20 decreased to (8.5±1.5) × 10-10 mol·cm-2·min-1, and that of M30 decreased to (10.1±2.0) × 10-10 mol·cm-2·min-1 (20μM GSNO and 20μM GSH) (Figure 5C). Even after immersion in PBS for 30 days or incubated with 1mg/mL BSA for 2h, the NO flux of the CuBTC-coated samples was only slightly reduced (30μM GSNO and 30μM GSH). The NO flux was reduced by 50% when the concentration of NO donor was reduced to 15μM (Table 2, S7).

Figure 4 Real-time NO generation catalyzed by sample M10. The GSNO and GSH were each added to a final concentration of 30μM.



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Figure 5 NO generation of CuBTC coated samples. A. 500μM EDTA was added into the reagent (30μM GSNO, 30μM GSH). B. The samples were immersed in PBS (pH=7.4) for 30 days before testing (30μM GSNO, 30μM GSH). C. Samples were incubated with cell culture medium (F12, with 15% FBS) for 2h, 20μM GSNO and 20μM GSH were added as NO donors.

Table 2 Statistical results of NO generation catalyzed by CuBTC-coated samples (× 10-10 mol·cm-2·min-1), data are presented as mean ± SD (n ≥ 4).

Group

Sample treatment

NO donor

M10

M20

M30

A.

/

30 μM

5.0±1.5

9.1±2.2

13.1±2.5

B.

+500μM EDTA while testing

30 μM

0.8±0.2

1.8±0.3

2.2±0.6

C.

Immersed in PBS (pH=7.4) for 30 days

30 μM

4.0±0.8

8.5±1.5

12.5±2.0

D.

Immersed in cell culture medium (15% FBS) for 2h

20 μM

2.8±1.2

8.5±1.5

10.1±2.0

E.

PBS (pH=7.4, 30days) & BSA(1mg/mL, 2h)

30 μM

3.6±0.4

5.0±1.2

8.2±2.1

F.

PBS (pH=7.4, 30days) & BSA(1mg/mL, 2h)

15 μM

2.1±1.0

2.8±0.5

3.0±0.8

3.3 Cell growth on CuBTC coated samples. Good cytocompatibility is required for successful implantation of biomaterials. We further evaluated the biological effects of the CuBTC coatings on the proliferation of vascular cells (SMC and EC), as well as inflammatory cells (MA). Endothelial cells (EC) were seeded on prepared samples, cultured for 1 day and 3 days, and then observed after rhodamine staining. We performed the NO release experiment under the different conditions listed above. For cell culture, 20 μM of NO donors was added. The results revealed that better growth of EC on M10 than that on Ti and Ti-OH, especially when NO donor solution was added into the culture medium (Figure 6). However, the proliferation of EC on M20 and M30 was significantly inhibited and EC


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were almost dead on the 3rd day (Figure S8). Cu2+ and NO are considered safe and promote EC proliferation when present at moderate amounts26, but excess Cu2+ and NO may cause apoptosis and oxidative damage27, 28. The CuBTC coatings are rich in Cu2+ and the element concentration of Cu2+ was increased with increased deposition cycles, and the NO generation rate was also increased. Samples M20 and M30 exhibited NO release of 8.5±2.0×10-10 and 10.1±2.0×10-10 mol·cm-2·min-1, respectively, in cell culture, higher than the amount of NO typically produced by EC (healthy EC secrete NO in a range of 0.5-4.0× 10-10 mol·cm-2·min-1). Sample M10 had a NO release of 2.8±1.2×10-10 mol·cm-2·min-1, indicating this material should be more adaptable for EC growth and proliferation. Therefore, samples M20 and M30 were not used in the following experiments.

Figure 6 The proliferation of EC with and without the addition of NO donor (20μM GSNO and 20μM GSH; the donor was added every 12h). (A) Rhodamine staining of EC on samples after culturing for 1 day and 3 days. (B) Cell density of EC on samples. Data are presented as mean ± SD (n ≥ 4) and analyzed using one–way ANOVA, *p

Copper-based SURMOFs for nitric oxide generation - ACS Publications

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