Preparation, characterization and biocompatibility of heparinized single-walled carbon nanotubes

doi:10.3969/j.issn.2095-4344.2014.21.007

Abstract

Abstract:

BACKGROUND: With unique structure and physicochemical property, carbon nanotubes have promising application prospects in the fields of drug delivery, biosensor and biomaterials. However, carbon nanotubes are highly hydrophobic and trend to aggregate, and thus carbon nanotubes are hard to be dispersed in solution. Furthermore, carbon nanotubes induce blood coagulation and have cytotoxicity, which greatly limit the application of carbon nanotubes.
OBJECTIVE: To prepare heparinized single-walled carbon nanotubes and to study the effects of heparin-immobilization on the water solubility, stability as well as biocompatibility of carbon nanotubes.
METHODS: By the method of covalent grafting, heparinized single-walled carbon nanotubes was fabricated and characterized by Fourier transform infrared spectroscopy and carbazole assay. Transmission electron microscopy was used to investigate the dispersing performance and suspension stability of heparinized single-walled carbon nanotubes in aqueous solution. Anti-Xa activity and activated partial thromboplastin time assays were used to measure the anticoagulation activity of heparinized single-walled carbon nanotubes. MTT assay was used to evaluate the cytocompatibility of heparinized single-walled carbon nanotubes.
RESULTS AND CONCLUSION: Heparin was covalently linked to the surface of single-walled carbon nanotubes successfully. The amount of heparin on single-walled carbon nanotubes was measured to be 257.53 mg/g. Heparinized single-walled carbon nanotubes were well dispersed and stable in an aqueous solution without aggregation. The anti-Xa activity of heparinized single-walled carbon nanotubes was measured to be 36.53 U/mg, suggesting a significant anticoagulant activity. Further study of activated partial thromboplastin time assay found that the anticoagulant effect of heparinized single-walled carbon nanotubes could be prolonged. MTT assay revealed that heparinized single-walled carbon nanotubes had no cytotoxicity and showed good cytocompatibility. Taken together, the immobilization of heparin on single-walled carbon nanotubes will not only improve its solubility and stability in water, but also endow it with excellent biocompatibility.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biocompatible materials, nanotubes, heparin, carbon

CLC Number:

R318

Peng Yi-qing, Chen Jing-xiao, Teng Li-ping, Zhao Shan-cheng, Cheng Yong-mei, Deng Chao, Chen Jing-hua . Preparation, characterization and biocompatibility of heparinized single-walled carbon nanotubes[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(21): 3316-3321.

Figures/Tables 5

2.3 肝素化单壁碳纳米管表面肝素含量肝素经浓硫酸水解、氧化后，生成的糖醛酸(硫酸乙酰肝素中的艾杜糖醛酸或葡萄糖醛酸)与显色剂咔唑反应，生成紫红色溶液在530 nm处具有特异性吸收，具有良好的线性关系，以此测定肝素化碳纳米管表面肝素接枝量。以不同浓度肝素钠标准品的含量为横坐标，对应的530 nm处吸光度值为纵坐标，经过线性回归后得到标准工作曲线方程为y=28.191x-0.018 6，r2=0.996 5，通过肝素化单壁碳纳米管样品显色后在530 nm处的吸光度值计算，得到每毫克肝素化单壁碳纳米管含肝素多糖257.53 μg。 2.4 肝素化单壁碳纳米管的抗栓活性配制一系列不同梯度活力单位的肝素溶液，通过底物色法以肝素活力单位为横坐标，对应的405 nm处吸光值为纵坐标此测定肝素化单壁碳纳米管的抗Ⅹa因子活性。经过线性回归后得到标准工作曲线方程方程为y=-0.107 5x+1.012 9，r2=0.986 3，通过肝素化单壁碳纳米管样品显色后在530 nm处的吸光度值计算，测得其抗Ⅹa因子活性为36.53 U/mg，具有较好的抗栓活性。比对标准肝素活性，计算得到每毫克肝素化单壁碳纳米管中肝素多糖的含量为243.53 μg，与硫酸咔唑法检测肝素化单壁碳纳米管中肝素含量结果接近。 2.5 肝素化单壁碳纳米管的抗凝活性肝素钠活性常用活化部分凝血酶时间实验测试，活化部分凝血酶时间是指在血浆在钙离子和磷脂活化剂作用下凝固所需的时间，此实验一定程度上可以说明材料的血液相容性。绵羊血浆、羧基化单壁碳纳米管、酰氯化单壁碳纳米管、氨基化单壁碳纳米管及肝素化单壁碳纳米管溶液(均为0.02 g/L数据)活化部分凝血酶时间分别为(82.1±0.8)，(81.3±0.9)，(80.8±0.4)，(80.3±0.4)，(175.7±2.7) s。未经修饰羧基化单壁碳纳米管及修饰后酰氯化单壁碳纳米管、氨基化单壁碳纳米管对绵羊血浆活化部分凝血酶时间没有影响，与空白绵羊血浆活化部分凝血酶时间无明显差异；而加入0.02 g/L肝素化单壁碳纳米管后活化部分凝血酶时间延长至空白绵羊血浆的2倍左右，达到175.7 s。肝素钠带有强负电荷，它能与抗凝血酶Ⅲ结合，增强其对活化的Ⅱa、Ⅸa、Ⅹa、Ⅺa和Ⅻa凝血因子的抑制作用，阻止血小板凝集和破坏，妨碍凝血激活酶的形成；组织凝血酶原变为凝血酶；抑制凝血酶，妨碍纤维蛋白原变成纤维蛋白。由此可以认为，通过对单壁碳纳米管表面接枝肝素后，材料的抗凝活性得到极大的提升，也间接说明肝素成功接枝到了单壁碳纳米管上。从图5可知，羧基化单壁碳纳米管和氨基化单壁碳纳米管在不同浓度下均没有表现出任何抗凝能力，而肝素化单壁碳纳米管随着加入浓度加大，绵羊血浆活化部分凝血酶时间时间相应延长。表明碳纳米管经过共价接枝后，肝素成功固定到了单壁碳纳米管表面，且能发挥良好的抗凝作用，具有良好的生物活性，活化部分凝血酶时间的血液相容性得到了极大提升。"

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