Construction of Antithrombotic Tissue-Engineered Blood Vessel via

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Construction of Antithrombotic Tissue-Engineered Blood Vessel via Reduced Graphene Oxide Based Dual-Enzyme Biomimetic Cascade Da Huo, Ge Liu, Yanzhao Li, Yuxin Wang, Ge Guan, Mingcan Yang, Keyu Wei, Jingyuan Yang, Lingqin Zeng, Gang Li, Wen Zeng, and Chuhong Zhu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04836 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Construction of Antithrombotic Tissue-Engineered Blood Vessel via Reduced Graphene Oxide Based Dual-Enzyme Biomimetic Cascade AUTHOR NAMES Da Huo, Ge Liu, Yanzhao Li, Yuxin Wang, Ge Guan, Mingcan Yang, Keyu Wei, Jingyuan Yang, Lingqin Zeng, Gang Li, Wen Zeng*, Chuhong Zhu* AUTHOR ADDRESS Department of Anatomy, State Key Laboratory of Trauma, Burns and Combined Injury, National & Regional Engineering Laboratory of Tissue Engineering, State and Local Joint Engineering Laboratory for Vascular Implants, Key Lab for Biomechanics and Tissue Engineering of Chongqing, Third Military Medical University, Chongqing 400038, China *.Corresponding authors: Chuhong Zhu and Wen Zeng, Department of Anatomy, Third Military Medical University, Gao Tan Yan Street, Shaping Ba District, Chongqing, 400038, China. E-mail: [email protected], [email protected]

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ABSTRACT

Thrombosis is one of the biggest obstacles in the clinical application of small-diameter tissue-engineered blood vessels (TEBVs). The implantation of an unmodified TEBV will lead to platelet aggregation and further activation of the coagulation cascade, in which the high concentration of adenosine diphosphate (ADP) that is released by platelets plays an important role. Inspired by the phenomenon that endothelial cells continuously generate endogenous antiplatelet substances via enzymatic reactions, we designed a reduced graphene oxide (RGO) based dual-enzyme biomimetic cascade to successively convert ADP into adenosine monophosphate (AMP) and AMP into adenosine. We used RGO as a support and bound apyrase and 5'-nucleotidase (5'-NT) on the surface of RGO through covalent bonds; then, we modified the surface of the collagen-coated decellularized vascular matrix with the RGO-enzyme complexes, in which RGO functions as a platform with a large open surface area and minimal diffusion barriers for substrates/products to integrate two catalytic systems for cascading reactions. The experimental results demonstrate that the two enzymes can synergistically catalyze procoagulant ADP into anticoagulant AMP and adenosine successively under physiological conditions, thus reducing the concentration of ADP. AMP and adenosine can weaken or even reverse the platelet aggregation induced by ADP, thereby inhibiting thrombosis. Adenosine can also accelerate the endothelialization of TEBVs by regulating cellular energy metabolism and optimizing the microenvironment, thus ensuring the antithrombotic function and patency of TEBVs even after the RGO-enzyme complexes loses its activity.


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KEYWORDS tissue-engineered blood vessels, reduced graphene oxide, ADP, adenosine, apyrase, 5’- nucleotidase

As many as 15 million people die each year from cardiovascular and cerebrovascular diseases, which rank as the leading causes of death worldwide, so many patients need to undergo blood vessel transplantation surgery. The availability of autologous blood vessels is occasionally limited because their quality is poor when patients suffer from primary disease such as diabetes mellitus. Therefore, there is an urgent need for biocompatible artificial blood vessels.

To minimize invasive operation in patients by avoiding autologous cell harvesting, TEBVs without endothelial cells are a major area of future research. However, small-diameter TEBVs have a low patency rate due to acute thrombosis, resulting in graft failure. As an exogenous foreign body, the implantation of an unmodified TEBV will lead to platelet adhesion mediated by vWF in the blood and platelet receptor GP Ib/IX/V at first. Two kinds of collagen receptors, GP VI and integrin α2β1, are also involved in the adhesion of platelets (Scheme 1). This process is reversible and can cause platelet activation and a release reaction, in which the high concentrations of adenosine diphosphate (ADP) that are released can lead to platelet aggregation or enhance the effect of other inducers.1 After being activated by ADP, the fibrinogen receptor on GP IIb/IIIa was exposed. Adjacent platelets will be linked together, mediated by fibrinogen (Scheme 1). The aggregation of platelets will further activate the coagulation reaction, and the formation of the fibrin network will


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ultimately lead to thrombosis. Thus, inhibiting platelet aggregation induced by vascular transplantation is an effective way to prevent acute thrombosis.

Surface modification and endothelialization of the vascular biomaterials are two effective approaches for improving the hemocompatibility and long-term patency of artificial vascular grafts. Studies have shown that under the effect of ADPase in endothelial cells, platelets become insensitive to agonists, and the aggregation function is inhibited. There is some anti-thrombus research based on catalyzing ADP using apyrase (CD39). Hohmann fused CD39 to activated GP IIb/IIIa-specific single-chain antibody and made targ-CD39.2 They demonstrated that targ-CD39 was able to target activated platelets and prevent ADP-induced platelet activation or vessel occlusion without bleeding side effect. In a study by Degen, CD39 was fused to glycoprotein VI-Fc, which is the essential platelet collagen receptor in atherothrombosis. GP VI-CD39 could stimulate local ADP degradation and inhibit ADP-induced platelet aggregation. CD39 also shows inflammatory response modulation effect.3 Ziegler used the above-mentioned targ-CD39 for the treatment of myocardial infarction, they demonstrated that targ-CD39 could reduce ischemia-reperfusion injury and restore cardiac function.4 However, endothelialization is a relatively slow process and is not able function in the early period of implantation. Inspired by the antithrombotic function of endothelial cells, we designed a biomimetic synergistic catalytic integration of two enzymes to reduce the concentration of ADP in TEBVs and inhibit platelet aggregation. Apyrase and 5'-NT were able to catalyze the conversion of ADP into AMP and AMP into adenosine, respectively.5 This cascading reaction can be realized under physiological conditions, and the "bad"


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substance that promotes platelet aggregation can be transformed into a "good" substance that can inhibit aggregation.6 To enhance the stability of the two enzymes and to provide a platform for the cascading reaction, we used RGO as a support and covalently bound the two enzymes on the surface of RGO. RGO is a type of two-dimensional nanomaterial that possesses excellent physical and chemical properties, such as a high specific surface area, a high adsorption capacity and better biocompatibility and biological stability than graphene oxide (GO) or other carbon nanomaterials.7,8 Research has shown that the protein loading amount of graphene is as high as 2.25-4.81 mg mg-1, which is far higher than that of other nanomaterials.9 Most importantly, the two-dimensional structure of RGO provides a suitable geometry as a catalyst support with a large open surface area that is readily accessible to substrates/products with minimal diffusion barriers, thus facilitating the cascading reaction from ADP to adenosine. A number of studies have also shown that graphene-based materials can enhance the stability of biocatalytic systems.10,11 In this study, graphene-based nanomaterials were used as enzyme carriers to construct antithrombotic TEBVs.12 After the construction of the RGO-enzyme complexes, they are coated to the surface of the collagen-coated vascular matrix. They act as a barrier to separate collagen and blood, but more importantly, they can convert the released ADP into antiplatelet AMP and adenosine. Adenosine can act on the platelet A2 receptor, activate adenylyl cyclase, increase the content of cyclic AMP (cAMP) and inhibit platelet aggregation.13 Previous studies have shown that AMP and adenosine can attenuate or even reverse the platelet aggregation caused by ADP (Figure S1, Figure S2).14 Another study found that moderate platelet adhesion and activation


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play important roles in the recruitment, adhesion and differentiation of endothelial progenitor cells (EPCs) and accelerate the endothelialization process,15,16 but that platelet aggregation will lead to thrombosis.17 We inhibited further platelet aggregation after the release reaction stage. At the same time, adenosine regulates the microenvironment of TEBVs. After TEBV transplantation, released extracellular nucleotides (ATP and ADP) will trigger inflammation and lead to an inflammatory microenvironment. Apyrase and 5'-NT hydrolyze these extracellular nucleotides into adenosine, thus altering an ATP/ADP-driven pro-inflammatory milieu to an adenosine-induced anti-inflammatory environment and accelerating the endothelialization of TEBVs.18,19 Both in vitro and in vivo experiments have shown that the RGO-enzyme-coated TEBVs had good antiplatelet and antithrombotic functions. We believe that these easily prepared vessels can provide options for the preparation of clinically available TEBVs.

RESULTS AND DISCUSSION Construction of RGO-Enzyme Complexes. Fully dispersed RGO is a prerequisite for its further combination with enzymes. Only if RGO is fully dispersed to a few or even a single layer can the specific surface area and absorbability be enhanced. At present, the most effective method is ultrasonic dispersion. The particles are dispersed via the cavitation effect and the vibration of the ultrasonic wave itself.

The RGO dispersion was observed with a transmission electron microscope (TEM). As shown in the figure, the edge of the RGO flake was almost transparent, which means that


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RGO has been fully dispersed to even a single layer; the size of each RGO flake ranged from 0.7 µm to 2 µm (Figure 1b).

The enzymes were linked to RGO via the amphipathic molecule DSPE-PEG2000-COOH (Figure 1a).20 The strong hydrophobic force between the DSPE chain and hydrophobic RGO tightly immobilized DSPE-PEG2000-COOH on the surface of the RGO, and the hydrophilic PEG chain provided the RGO with good water dispersibility and biocompatibility.21 Enzymes were covalently linked to DSPE-PEG2000-COOH through amide bonds. Studies have shown that if an enzyme is directly linked to the amino group or carboxy group on the surface of the materials, the original structure or function of the enzyme may be impacted.22 Therefore, the DSPE-PEG2000-COOH molecule acts as a long hydrophilic spacer between the enzyme and the material surface to ensure that the activity of enzyme will not be affected.

Raman spectroscopic results indicates that the enzyme was successfully linked to RGO (Figure 1c).23 Specific Raman bands appear in the RGO-enzyme complexes measurements that do not exist for RGO alone, such as the amide band that includes C=O stretching and N-H deformation vibration. RGO was verified by the D and G bands. Zeta potentials of RGO, RGO-apyrase and RGO-apyrase-5'-NT (RGO-enzyme complexes) were also measured to demonstrate the conjugation of RGO and two enzymes (Table S2).

Because of the two-dimensional structure and size of RGO, it has a vast specific surface area and excellent protein loading ability. It has been found that the protein loading amount of RGO (2.25–4.81 mg mg−1) is far higher than that of carbon nanotubes (< 0.45 mg· mg−1).9 The amount of enzymes on each RGO flake was quantified. There were about 669


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5’-NT molecules and 805 apyrase molecules on each flake (Table S3, Figure S4). Additionally, the stability and biocompatibility of RGO are much better than those of GO because the surface oxygenic functional groups are likely to interact with and activate platelets, which will lead to platelet aggregation.24

Every RGO flake contains 5’-NT and apyrase, so it can act as a reaction unit and efficiently transform ADP into AMP and adenosine. Here, the role of RGO is similar to that of vascular endothelial cells, which can transform pro-coagulant factors into anticoagulant factors in stress microenvironments to prevent clotting and thrombus formation.

In Vitro Test of the Anti-Shearing Ability of RGO-Enzyme complexes. There are high requirements for the stability of TEBVs because they must face fluid shear stresses with high intensities in vivo. Immobilized enzymes have to resist the scouring of the blood flow and avoid being washed away. A plate flow chamber experiment was used to detect the anti-shearing ability of the RGO-enzyme complexes, and collagen-coated slides were used to simulate the lumen of the blood vessel (Figure 2a, 2b). After exposure to liquid shear stress for 48 h, few unmodified enzymes remained in the control group, but considerably more enzymes remained in the RGO-enzyme complexes group (Figure 2c). The average optical density (AOD) in the RGO-enzyme complexes group was significantly higher than that in the control group after 48 h (Figure 2d). Additionally, enzymes in the RGO group are more dispersively distributed, which could reduce enzyme agglomeration and thus enhance its activity.11


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In Vitro Determination of Enzyme Activity. The above results show that after binding to RGO, the stability of the enzyme was enhanced, but its activity still needed to be determined. Thus, we determined the rate of the enzymatic reactions by measuring the inorganic phosphate (Pi) content of the catalytic reaction products.25,26 The specific concentrations of ADP, AMP and adenosine in plasma were determined by high-performance liquid chromatography (HPLC).

The Pi concentration was monitored for 14 min of enzymatic reaction (Figure 3). The absorbance at 630 nm was linearly related to the phosphate concentration (Figure 3a). In the enzyme reaction system, the reaction was in its lag and linear phases, as the reaction rate first increased and subsequently remained constant. The Pi content in the apyrase system at 14 min was 6.2658 µmol, so the average reaction rate of apyrase reached 0.458 µmol/min (Figure 3b). Similarly, the average reaction rate of 5’-NT was 68.071 nmol/min (Figure 3c).

The results of the HPLC are shown in Figure 3. The average concentration of ADP in the RGO-enzyme complexes group decreased from 31.930 µmol/L to 20.168 µmol/L, the AMP content increased from 13.568 µmol/L to 25.184 µmol/L, and the adenosine content increased from 3.393 µmol/L to 13.155 µmol/L (Figure 3d). In contrast in the control group, the ADP concentration increased continually from 28.548 µmol/L to 39.396 µmol/L, and the AMP content decreased from 8.089 µmol/L to 4.606 µmol/L (Figure 3e). The concentrations of ADP in RGO-enzyme complexes group were significantly lower than those in the control group after 20 min. In contrast, the AMP and adenosine concentrations were significantly higher in the RGO-enzyme complexes group (Figure 3f).


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The above results show that these two immobilized enzymes showed good activity and could transform ADP into antiplatelet AMP and adenosine. Adenosine not only regulates the transplanted microenvironment but can also act on platelet A2 receptors, which can activate adenylate cyclase, increase the cAMP content, and inhibit platelet aggregation.27 Therefore, we tested the inhibitory effect of RGO-enzyme complexes on platelet activation.

In Vitro Detection of Platelet Activation and Aggregation. P-selectin (CD62P) is a type of platelet α-granule membrane glycoprotein that is expressed on the platelet surface only after the release reaction, which means that CD62P McAb binds only to degranulated platelets or activated platelets rather than resting ones.28 Flow cytometry data revealed that the mean fluorescence intensity (MFI) of the collagen-coated control group reached 1.37 times that of the RGO-enzyme complexes treated group (Figure 4a, 4b), which means that the enzyme significantly inhibited platelet activation.

ADP is the most important substance that causes platelet aggregation, especially endogenous ADP released from platelets.29 ADP will also inhibit the activity of 5'-NT, whereas AMP can activate it.30 Therefore, after ADP was converted into AMP, 5'-NT activity was enhanced, thus promoting the conversion of AMP into adenosine. After the concentration of ADP decreased, the aggregation of platelets was also suppressed.

Flow cytometry experiments showed that platelet activation was inhibited, and we further demonstrated the effect of RGO-enzyme complexes on platelet aggregation in vitro. Slides with RGO-enzyme complexes or collagen coated slides were immersed into fresh platelet rich plasma (PRP) for 2 h and then observed through a scanning electron microscope


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(SEM) (Figure 4c). There were only a few platelets dispersedly distributed in the RGO-enzyme complexes group, but there were platelet aggregations in the control group. Most of the platelets were deformed, and the parapodium was stretched out in the control group. Our previous studies have shown that a moderate adhesion of platelets is beneficial for the proliferation of endothelial cells.17 However, the aggregation and release reaction of platelets will lead to further platelet aggregation, fibrinogen will change to fibrin, and red blood cells and leukocytes will accumulate and result in thrombus. We further investigated the antithrombotic behavior of RGO-enzyme-coated slides with whole blood using the same method. After immersed in blood for 12 h, all slides were examined by SEM. Collagen coated control slides exhibited rough surfaces after blood contact, indicating obvious formation of blood clots. In contrast, the slides containing RGO-enzyme complexes showed relatively clean surfaces, clearly demonstrating excellent antiplatelet function (Figure 4d).

In Vitro Detection of Endothelial Cell Proliferation. Based on our previous research about the effect of adenosine on endothelial cells, we tested the effect of the RGO-enzyme complexes on the proliferation of ECs.31 EdU (5-Ethynyl-2’- deoxyuridine) is an analogue of thymidine and can replace thymidine and infiltrate into DNA during the cellular proliferative stage. Cell proliferation activity can be detected based on the specific reaction between EdU and Apollo, a type of fluorescent dye. Figure 5 indicates that endothelial cells on the RGO-enzyme-coated slides had higher proliferative activity, which could not be induced by RGO alone.


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In Vivo Animal Experiment. After the construction of RGO-enzyme complexes, they were coated on the surface of the collagen coated vascular matrix and TEBVs were obtained. TEBVs in each group were grafted into the common carotid artery of Sprague-Dawley rats (SD rats) (Figure 6a), followed by vascular anastomosis (Figure 6b). In general, ten SD rats were sacrificed for the preparation of the vascular matrix and TEBVs. Thirty-three SD rats underwent vascular transplantation, among which one died from an overdosed of anesthesia and two died after surgery probably due to excessive blood loss and hypothermia. Thirty rats were included in this study. Because our aim was to suppress the formation of thrombosis caused by early-stage platelet aggregation, we removed the TEBVs 7 days after transplantation. The hematoxylin-eosin (HE) staining results demonstrated that the RGO group and control group had thrombosis-induced vascular occlusion at post-grafting day 7 (Figure 6d); the patency rates were 20% and 30%, respectively, and the average blood flow in the patent TEBV were 2.9 and 3.1 mL/min at post-grafting day 7, respectively. The RGO-enzyme complexes group had a patency rate of 90% and an average blood flow in the patent TEBV of 6.3 mL/min at post-grafting day 7 (Figure 6c). Microcomputed tomography angiography at 7 days also showed that only the RGO-enzyme-coated TEBVs remained open (Figure 6e).

SEM images show the internal surface near the vascular anastomosis (Figure 7). There is a high risk of thrombosis at the site of anastomosis, but the proliferation and migration of endothelial cells in the RGO-enzyme complexes group were significantly accelerated, and there were only a few scattered platelets. Spindle-shaped endothelial cells were arranged neatly and covered the inner surface of the TEBVs. In contrast, in the RGO group and


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collagen-coated control group, the internal surfaces of the TEBVs were full of thrombi caused by platelet aggregation, and there were no endothelial cells. These results demonstrate that RGO-enzyme complexes will prevent thrombosis and accelerate endothelialization, which will guarantee the long-term patency of TEBVs.

CONCLUSIONS We successfully constructed RGO-enzyme-coated TEBVs for anti-platelet function and endothelialization. These TEBVs reached a patency rate of 90% 7 days after grafting. Mechanistically, RGO functioned as a support with a large open surface area to integrate two enzymatic catalysts, apyrase and 5’-NT, which synergistically facilitate the cascading reactions under mild physiological conditions. RGO-enzyme complexes could catalyze ADP into AMP and AMP into adenosine successively, thus weakening or even reversing the platelet aggregation induced by ADP and inhibiting thrombosis. Adenosine can also accelerate the endothelialization of TEBVs by regulating cellular energy metabolism and optimizing the microenvironment, thus ensuring the antithrombotic function of TEBVs even after RGO-enzyme complexes loses its activity. Our study may provide a strategy for TEBV construction.

METHODS Construction of RGO-Enzyme Complexes and Raman Spectroscopy

RGO-enzyme complexes were constructed as follows. One mg of RGO (Sigma) and 5 mg of DSPE-PEG2000-COOH (Nanosoft) were weighed in a tube, and 5 mL of ultrapure water was added. The tube was sonicated in a bath sonicator for 60 min at room temperature,


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and the water in the water bath was changed every 20 min to avoid overheating. The excess DSPE-PEG2000-COOH in RGO suspension was removed in a 4-mL centrifugal filter device (Merck, MWCO = 100 kDa) by repeating the centrifuge/water adding steps. The RGO dispersion was observed by TEM. Then, we added MES buffer to the RGO dispersion, and the pH was adjusted to 6.0. Carbodiimide hydrochloride (EDC·HCl, J&K) and N-hydroxysuccinimide (NHS, J&K) were added to activate carboxyl groups and introduce unstable O-acylisourea structure, and the reaction was kept at room temperature (22℃) for 2 h. After removing the excess EDC·HCl and NHS by filtration (MWCO = 100 kDa), 100 µL of apyrase (Sigma) solution (containing 10 UN apyrase) and 5’-NT (Abcom) solution (containing 10 µg 5'-NT) was added, and the reaction was kept at 4 °C overnight. Amino groups in the enzymes could react with O-acylisourea and form amide bonds. After another filtration (MWCO = 100 kDa), the RGO-enzyme complexes were characterized by Raman spectra.

Determination of Enzyme Activity

After being combined with RGO as detailed above, the RGO-apyrase and RGO-5’-NT complexes were coated to collagen-coated slides separately induced by EDC. The activity of the immobilized apyrase or 5’-NT was measured by Pi determination using the Malachite Green Phosphate colorimetric assay. The working solution was a 3:1 mixture of MG (0.045% malachite green hydrochloride) and AM (4.2% ammonium molybdate in 4N HCl) solutions that were prepared at least 20 min before assay. To obtain the color reagent, 4 mL of 1.5% Tween-20 was mixed with 100 mL of working solution. To avoid interference from


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nonspecific hydrolysis after stopping the enzyme reaction, 24% sodium citrate was used. The reaction buffer contained 50 mM Tris-HCl and 5 mM CaCl2 (pH=7.2). ADP or AMP (Aladdin) was added to a final concentration of 1 mM. RGO-enzyme-coated slides or collagen-coated slides were added to the buffer and the reaction was kept at 37°C. Every 2 minutes, 300 µL of reaction solution was obtained. To 100 µL of sample, 800 µL of color reagent was added and mixed. After 1 min, 100 µL of the citrate solution was added and mixed. The absorbance of the solution was measured at 630 nm after 30 min. A KH2PO4 solution was utilized to obtain a standard curve.

Detection of the Ability to Resist Shear Forces.

A plate flow chamber experiment was used to detect the anti-shearing ability of the RGO-enzyme complexes and their distribution on the surface of the collagen. First, enzymes were labeled with FITC (Sigma) in NaHCO3 buffer solution and purified by centrifugal filters (MWCO = 30000, rpm = 4000, 5 min) using buffer solution 3 times. Then, the enzyme-FITC was linked to RGO, which had been modified by DSPE-PEG2000-COOH, induced by EDC and NHS. After incubation of the collagen on a glass slide for 24 h, enzyme-FITC-RGO was added, followed by 24 h of EDC induced cross-linking. To simulate the normal in vivo shear forces generated by blood flow, the solution in the chamber was driven to flow at a rate of 1.54 mL/s using a circulation pump. The flow shear stress (FSS) was approximately 19.56 dyn/cm2, which was physiologically relevant. After 48 h, the slide was removed and subjected to fluorescence observation under a fluorescence microscope. The average optical density of each slide was measured using ImageJ.


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ADP, AMP and Adenosine Content Determination

The contents of ADP, AMP and adenosine were measured using HPLC. Fresh citrate anticoagulant blood was obtained from an SD rat (9:1 volume of a blood: citrate solution). Then, PRP was prepared by the Landesberg Method and divided into two groups.32 The collagen-coated slide was added to group A, and the RGO-enzyme-coated slide was added to group B. All reactions were kept at 37°C. Every 5 min, 600 µL of PRP was collected into a 2-mL centrifugal tube containing 80 µL of a stopping solution consisting of three drugs: 60 µL of dipyridamole (0.025%) to inhibit adenosine reuptake into red blood cells, 10 µL of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, 0.1 mM) to block adenosine deaminase activity, and 40 mM ethylenediamine-tetraacetic acid (EDTA) to inhibit platelet aggregation and the release of adenosine from platelets. Samples were then placed into microcentrifuge tubes and centrifuged at 14,000×g for 5 min. Supernatant were then placed into separate tubes and deproteinated with 60 µL of 50% trichloroacetic acid. Samples were centrifuged again for 5 min, and the supernatant was neutralized with potassium hydroxide. Samples were kept at 4°C for HPLC analysis.

In Vitro Antiplatelet Test

Fresh citrate anticoagulant blood was obtained from an SD rat, and PRP was prepared. The RGO-enzyme-coated slides or collagen coated slides were placed into tubes, and fresh PRP was added. The tubes were placed on an incubator shaker at 22°C. All slides were removed and gently rinsed with PBS buffer after 2 h, followed by fixation with glutaraldehyde. Antithrombotic behavior of RGO-enzyme-coated slides was tested using


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whole blood. In short, RGO-enzyme-coated slides or collagen coated slides were immersed in fresh whole blood for 12 h at 22°C. Slides were then washed with PBS buffer, followed by fixation with glutaraldehyde. After being dehydrated using gradient ethanol and air drying, all slides were stored at 4°C for further SEM analysis.

Detection of Platelet Activation

The plasma CD62P level was detected with a flow cytometer to reflect the activation of the platelets.33 Fresh citrate anticoagulant blood was obtained from an SD rat. The experimental group consisted of the RGO-enzyme complexes coated slides, and the control group consisted of the collagen-coated slides. After 1 h, slides were taken out and CD62P-PE antibody (BioLegend) was added to each group, and IgG1-PE antibody was added to the isotype control group. After incubating for 15 min in the dark, the samples were fixed by 1% paraformaldehyde. The samples were subjected to flow cytometry to measure the mean fluorescence intensity within 12 h.

Proliferation of Endothelial Cells on RGO-Enzyme Complexes Interface

The proliferation of endothelial cells was determined using the EdU Apollo In Vitro Imaging Kit (RIBOBIO). The RGO-enzyme complexes coated slides, RGO coated slides or collagen-coated control slides were placed in 12-well plates. Endothelial cells were cultured in each well at an initial density of 1×105/mL. After cell adherence, the medium was discarded, and 300 µL of EdU medium and 1 mM ADP were added to each well. After 4 h of incubation, ECs were rinsed and fixed in 4% paraformaldehyde. The EdU was stained by


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Apollo, and DNA was stained by Hoechst33342. All slides were observed immediately under a fluorescence microscope.

Construction of RGO-Enzyme-Coated TEBV

Under sterile conditions, the common carotid artery was obtained from SD rats and rinsed with heparin saline to remove the blood. The obtained carotid artery was digested to rupture the native cells and partially extract the cytoplasmic elements and soluble extracellular matrix (ECM) as described previously.34 First, the artery was digested using 0.15% trypsin at 37°C for 40 min with 5% CO2. Then, RNase, DNase and lipase were used to remove nucleic acids and fatty acids from the sample. After this multistep process, the obtained vascular matrix materials did not contain any nucleic acids, lipids, cellular membranes, cytoplasmic components, and soluble matrix molecules but did contain preserved vascular collagen fibers and elastic fibers.35 The DNA content in the decellularized grafts was checked using TIANcombi DNA Lyse&Amp PCR Kit (TIANGEN) and NanoDrop 1000 (Thermo Scientific) (Table S1). Subsequently, the vascular matrix material was incubated with 4 mg/mL collagen (Kensey Nash) solution for 24 h, followed by three PBS washes. EDC·HCl and NHS were added to the collagen-coated vascular matrix to introduce active sites onto the surface. The solution was discarded after 2 h, and the prepared RGO-enzyme complexes dispersion was added. The reaction was kept at 4℃ for 24 h. The TEBVs were washed gently using PBS before transplant.

Animal Experiments.


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All animal experiments were performed in accordance with the regulations on animal experiments of the Third Military Medical University (Chongqing, China) and were approved by the Ethics Committee of the Third Military Medical University. Ten-week-old SD rats were anesthetized with 1% sodium pentobarbital, and the RGO-enzyme complexes TEBVs, RGO TEBVs and collagen-coated control TEBVs were grafted into the left common carotid arteries separately by vascular anastomosis. There were 10 SD rats in each group. Blood flow was measured using a Doppler flowmeter at post-grafting day 7. The grafted TEBVs were removed after 7 days. A portion of the TEBVs underwent frozen sectioning, and slices were stained with HE staining. Another portion of TEBVs were dehydrated by gradient ethanol and fixed with glutaraldehyde for SEM observation.

Micro-Computed Tomography.

Before the TEBVs were removed, rats were anesthetized with 1% sodium pentobarbital and intraperitoneally injected with 1 mL of heparin. After 5 min of heparinization, the rat's chest was opened to expose the heart for intravascular contrast agent (iopromide) injection. The rats were then sacrificed, and a micro-CT scanner was used to evaluate the patency of the grafts.

Statistical Methods.

A t-test of the data and the homogeneity of the variance test were performed using SPSS12.0 software. All experimental data were expressed as the means ± standard deviation (SD). A p-value less than 0.05 was considered a statistically significant difference.


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Acknowledgments. This work was supported by the National Science Fund for Distinguished Young Scholars (No. 31625011), the National Key Research and Development Program (No. 2016YFC1101100), the National Key Research and Development Plan Young Scientists Program (No. 2017YFA0106000), the Training Program of the Major Research Plan of the National Natural Science Foundation of China (No. 91439116), the National Natural Science Foundation of China (No. 31470046), and the Young Elite Scientists Sponsorship Program by CAST (No.YESS20160180).

Supporting Information Available: The optical density of PRP was detected to prove that primary platelet aggregates dispersed in the presence of RGO-enzyme complexes (Supplementary Figure 1). The platelet aggregation of PRP was observed with phase contrast microscope (Supplementary Figure 2). The residual DNA content of the obtained decellularized grafts was checked (Supplementary Table 1). Zeta potentials of RGO, RGO-apyrase

and

RGO-apyrase-5'-NT

(RGO-enzyme

complexes)

were

measured

(Supplementary Table 2). The specific surface area of RGO was measured (Supplementary Table 3). Percent coverage of RGO-enzyme complexes on the surface of the glass slides was calculated (Supplementary Figure 3). This material is available free of charge via the Internet at http://pubs.acs.org.


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Figure Legends Scheme 1. Construction of the RGO-enzyme-coated TEBV for suppressing platelet aggregation. After platelet adhesion and activation, high concentrations of ADP caused by the release reaction led to platelet aggregation. ADP was converted into AMP and adenosine under synergistic catalytic action of apyrase and 5’-NT, thus lowering the concentration of ADP. AMP and adenosine suppressed platelet aggregation and promoted the proliferation of ECs. Figure 1. RGO dispersion and binding of the enzyme. (a) Apyrase and 5’-NT were covalently linked to RGO through amide bonds with the assistance of DSPE-PEG2000-COOH. (b) Transmission electron microscope image of the RGO dispersion. (c) Raman spectra of the RGO-enzyme complexes, RGO and enzyme.

Figure 2. RGO enhanced the anti-shearing ability of the enzymes. (a) Schematic of the plate flow chamber system. (b) Collagen-coated slides were used to simulate the lumen of the vessels during in vitro experiments. (c) The collagen was cross-linked with FITC labelled RGO-enzyme complexes or FITC-labelled enzyme. A circulation pump drove PBS for 48 h, and the fluorescence was determined. Scale bar: 100 µm. (d) Average optical density (AOD) of each group was measured by ImageJ. AOD of RGO group was significantly higher than the control group after 48 h. *p

Construction of Antithrombotic Tissue-Engineered Blood Vessel via

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