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Construction of Antithrombotic TissueEngineered 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* Department of Anatomy, State Key Laboratory of Trauma, Burns, and Combined Injury, National and 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 S Supporting Information *
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, and 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 complex loses its activity. KEYWORDS: tissue-engineered blood vessels, reduced graphene oxide, ADP, adenosine, apyrase, 5′- nucleotidase
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Willebrand factor 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
s 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 operations in patients by avoiding autologous cell harvesting, tissue-engineered blood vessels (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 von © 2017 American Chemical Society
Received: July 10, 2017 Accepted: October 16, 2017 Published: October 16, 2017 10964
DOI: 10.1021/acsnano.7b04836 ACS Nano 2017, 11, 10964−10973
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Cite This: ACS Nano 2017, 11, 10964-10973
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ACS Nano Scheme 1. Construction of the RGO-Enzyme-Coated TEBV for Suppressing Platelet Aggregationa
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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.
reaction, and the formation of the fibrin network will 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 antithrombus research based on catalyzing ADP using apyrase (CD39). Hohmann fused CD39 to activated GP IIb/IIIaspecific 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 a 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 ADPinduced platelet aggregation. CD39 also shows an inflammatory response modulation effect.3 Ziegler used the above-mentioned targ-CD39 for the treatment of myocardial infarction and 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 to 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” 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 10965
DOI: 10.1021/acsnano.7b04836 ACS Nano 2017, 11, 10964−10973
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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 RGOenzyme complexes, RGO, and enzyme.
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 RGO has been fully dispersed to even a single layer; the size of each RGO flake ranged from 0.7 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-PEG2000COOH 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 indicate 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 CO stretching and N−H deformation vibration.
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 (Figures S1 and S2).14 Another study found that moderate platelet adhesion and activation 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 adenosineinduced 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. 10966
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Figure 2. RGO enhanced the antishearing 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-labeled RGO-enzyme complexes or FITC-labeled enzyme. A circulation pump drove PBS for 48 h, and the fluorescence was determined. Scale bar: 100 μm. (d) AOD of each group was measured by ImageJ. AOD of RGO group was significantly higher than the control group after 48 h. *p < 0.05 (n = 10). Values are the mean ± SD.
Figure 3. Determination of enzyme activity. (a−c) Determination of enzyme activity through measurement of the Pi level. (a) Standard curve of phosphate concentration and absorbance at 630 nm. (b) Changes of Pi content (μmol) with time in the apyrase reaction system. (c) Changes of Pi content (μmol) with time in the 5′-NT reaction system. Values are the mean ± SD. (d−f) Concentration of ADP, AMP, and adenosine was measured by HPLC. (d) The concentration of ADP in the plasma with the RGO-enzyme complexes decreased gradually, and the concentration of AMP and adenosine gradually increased. (e) The concentration of ADP in the plasma without enzymes increased, whereas the concentrations of AMP and adenosine decreased. (f) Comparison of ADP, AMP, and adenosine content between the experimental group and control group at 20 min, *p < 0.05 (n = 10). Values are the mean ± SD.
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Figure 4. Activation and aggregation of platelets were detected. (a) Platelet surface CD62P expression was measured by flow cytometry. The red curve represents the isotype control group, the blue curve represents the collagen-coated control group, and the orange curve represents the RGO-enzyme complexes experimental group. (b) MFI of three groups; *p < 0.05 (n = 10) compared with the control group. Values are the mean ± SD. (c) The aggregation of platelets was observed by SEM. There were only a few platelets dispersedly distributed in the RGOenzyme complexes group, but there were more platelets and aggregations in the control group; *p < 0.05 (n = 10) compared with the control group. (d) Antithrombotic behavior of RGO-enzyme-coated slides. Control group exhibited obvious adhesion of aggregations, while the RGO-enzyme complexes group kept smooth; *p < 0.05 (n = 10) compared with the control group.
into anticoagulant factors in stress microenvironments to prevent clotting and thrombus formation. In Vitro Test of the Antishearing Ability of RGOEnzyme 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 antishearing ability of the RGO-enzyme complexes, and collagen-coated slides were used to simulate the lumen of the blood vessel (Figure 2a,b). After exposure to liquid shear stress for 48 h, few unmodified enzymes remained in the control group, but considerably more enzymes remained in the RGOenzyme 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
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 (
