武汉云克隆科技股份有限公司

Abstract: Multiwalled carbon nanotubes (MWCNTs) are cylindrical tubes of graphitic carbon with unique physical and electrical properties. MWCNTs are being explored for a variety of diagnostic and therapeutic applications. Successful biomedical application of MWCNTs will require compatibility with normal circulatory components, including constituents of the hemostatic cascades. In this manuscript, we compare the thrombotic activity of MWCNTs in vitro and in vivo. We also assess the influence of functionalization of MWCNTs on thrombotic activity. In vitro, MWCNT activate the intrinsic pathway of coagulation as measured by activated partial thromboplastin time (aPTT) assays. Functionalization by amidation or carboxylation enhances this procoagulant activity. Mechanistic studies demonstrate that MWCNTs enhance propagation of the intrinsic pathway via a non-classical mechanism strongly dependent on factor IX. MWCNTs preferentially associate with factor IXa and may provide a platform that enhances its enzymatic activity. In addition to their effects on the coagulation cascade, MWCNTs activate platelets in vitro, with amidated MWCNTs exhibiting greater platelet activation than carboxylated or pristine MWCNTs. However, contrasting trends are obtained in vivo, where functionalization tends to diminish rather than enhance procoagulant activity. Thus, following systemic injection of MWCNTs in mice, pristine MWCNTs decreased platelet counts, increased vWF, and increased D-dimers. In contrast, carboxylated MWCNTS exhibited little procoagulant tendency in vivo, eliciting only a mild and transient decrease in platelets. Amidated MWCNTs elicited no statistically significant change in platelet count. Further, neither carboxylated nor amidated MWCNTs increased vWF or D-dimers in mouse plasma. We conclude that the procoagulant tendencies of MWCNTs observed in vitro are not necessarily recapitulated in vivo. Further, functionalization can markedly attenuate the procoagulant activity of MWCNTs in vivo. This work will inform the rational development of biocompatible MWCNTs for systemic delivery.

摘要：多壁碳纳米管（MWCNTs）是石墨碳的圆柱管，具有独特的物理和电性能。多壁碳纳米管正在探索各种诊断和治疗应用。多壁碳纳米管的成功生物医学应用将需要与正常循环组分（包括止血级联成分）相容。在这篇文章中，我们比较了体外和体内多壁碳纳米管的血栓形成活性。我们还评估了多壁碳纳米管功能化对血栓形成活性的影响。在体外，通过活化部分促凝血酶原激酶时间（aPTT）测定，MWCNT激活凝固的内在途径。通过酰胺化或羧化作用的官能化增强了这种促凝血活性。机制研究表明，多壁碳纳米管通过强烈依赖于因子IX的非经典机制增强内源性途径的传播。多壁碳纳米管优先与凝血因子IXa结合，并可能提供一个增强其酶活性的平台。除了它们对凝血级联的影响之外，MWCNTs体外活化血小板，酰胺化MWCNT比羧化或原始MWCNT显示更大的血小板活化。然而，在体内获得对比的趋势，其中官能化倾向于减少而不是增强促凝血活性。因此，在小鼠中全身性注射MWCNT后，原始MWCNTs降低血小板计数，增加vWF和增加D-二聚体。相比之下，羧化MWCNTS在体内表现出很小的促凝血倾向，仅引起血小板的轻度和短暂降低。酰胺化的多壁碳纳米管引起血小板计数无统计学显着变化。此外，羧化或酰胺化的MWCNTs都不会在小鼠血浆中增加vWF或D-二聚体。我们得出结论：体外观察到的多壁碳纳米管的促凝血倾向不一定在体内重现。此外，功能化可显着减弱体内MWCNT的促凝血活性。这项工作将为生物相容性多壁碳纳米管的合理开发提供系统性的交付信息。

使用试剂原文信息：Mouse plasma was diluted 1:5 in phosphate buffered saline and D-dimer (E0506Mu) or mVWF (E0833Mu) levels were quantified by ELISA using a commercial assay kit (Uscn Life Science Inc.) according to the manufacturer’s instructions.