[论文解读] Reduction and simultaneous doping of graphene oxide to repel LDL in treatment of atherosclerosis disease

L M Rivera, A.F. Betancur+4 more|arXiv (Cornell University)|Feb 5, 2019

Graphene and Nanomaterials Applications参考文献 37被引用 4

一句话总结

本研究提出一种氮掺杂还原石墨烯（N-rGO）作为新型生物材料，通过静电排斥低密度脂蛋白（LDL）来预防动脉粥样硬化。通过同时还原与原位氮掺杂，该材料实现了高负表面电荷（通过EFM和接触角测量验证），导致LDL排斥（接触角95.7°–130.4°），并在HUVEC细胞中表现出高达100 μg/mL的优良生物相容性。

ABSTRACT

Atherosclerotic disease develops when there is endothelial dysfunction, making low density lipoproteins (LDL) which are cholesterol carriers traveling in the blood, very likely to penetrate the endothelium layer triggering the formation of the atheromatous plaque and leading to the obstruction of blood vessels. For some time now, devices called stents have been used to re-open the light of the plugged artery, allowing the restoration of blood flow. However, this treatment has disadvantages like developing intra-stent restenosis and progression of the atherosclerosis, clogging the blood vessel again. For these reasons it is necessary to develop new materials and strategies focused on the prevention and correction of this pathology and its subsequent complications. In the present work, a reduction and in situ nitrogen doping method in graphene oxide (N-rGO) was studied. By means of XPS a 9.57% at of nitrogen doping was demonstrated and was found that the pyrrole type nitrogen is predominant. Due to nitrogen doping and pyrrolic sites, the N-rGO surface reached high levels of negative electric charge, which was verified by Electrostatic Force Microscopy (EFM) and wettability tests between the LDL at different oxidation levels and the synthesized N-rGO; contact angles between 95.7° and 130.4° were obtained which indicates repulsion between both materials. The N-rGO was also characterized by SEM and TEM where a wrinkled morphology typical of this type of material was observed and the hexagonal atomic arrangement of GO is distinguished. Finally, cell viability tests using 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay were carried out on human umbilical cord endothelial cells (HUVEC) to analyze the cellular response at different concentrations of N-rGO, obtaining favorable behavior up to concentrations of 100 (μg/mL).

研究动机与目标

• 开发一种新型纳米材料，以防止血液血管中LDL积聚，这是动脉粥样硬化斑块形成的关键触发因素。
• 克服当前支架治疗的局限性，如再狭窄和疾病进展。
• 通过同时还原与氮掺杂，对氧化石墨烯进行工程化改造，以增强表面电荷和生物相互作用。
• 评估N-rGO与氧化LDL形式之间的静电相互作用，以评估其排斥潜力。
• 确认N-rGO在人内皮细胞中的生物相容性，以评估其在血管应用中的潜力。

提出的方法

• 在受控条件下使用水合肼对氧化石墨烯进行化学还原，制备还原石墨烯（rGO）。
• 在还原过程中使用氨气同时对rGO进行氮掺杂，实现原位氮掺杂。
• 利用X射线光电子能谱（XPS）表征氮含量和化学态，确认氮含量为9.57% at.，主要为吡咯型氮。
• 通过静电力显微镜（EFM）测量表面ζ电位和表面电荷密度，确认N-rGO具有高负电荷。
• 通过测量N-rGO与不同氧化程度LDL之间的接触角，量化疏水性和界面相互作用。
• 利用扫描电子显微镜（SEM）和透射电子显微镜（TEM）进行结构和形貌分析，确认其具有褶皱形貌和六方原子晶格。

实验结果

研究问题

• RQ1 能否通过同时还原与氮掺杂氧化石墨烯，生成具有足够负电荷的表面，以静电排斥LDL颗粒？
• RQ2 掺杂rGO中的主要氮化学态构型是什么，其如何影响表面电荷？
• RQ3 N-rGO的表面能与润湿性与不同氧化状态的LDL相比如何？
• RQ4 N-rGO在临床上相关浓度下对人脐静脉内皮细胞（HUVECs）的细胞毒性特征如何？
• RQ5 N-rGO材料在与血管应用相关的生理条件下，能否保持结构完整性和功能性表面特性？

主要发现

• XPS分析确认氮掺杂水平为9.57% at.，其中吡咯型氮为主要化学态。
• 静电力显微镜（EFM）和接触角测量表明N-rGO具有高度负电荷表面，与LDL相互作用时接触角范围为95.7°至130.4°。
• 较高的接触角范围表明N-rGO与LDL之间存在强烈的静电排斥，表明可有效防止LDL黏附。
• SEM和TEM成像证实rGO具有典型的褶皱形貌和六方原子晶格，表明还原成功且结构完整。
• MTT实验显示在HUVEC细胞中，N-rGO在浓度高达100 μg/mL时无显著细胞毒性，表明其在潜在血管应用中具有良好的生物相容性。
• 高负表面电荷与良好生物相容性的结合，使N-rGO成为预防支架涂层或血管植入物中LDL积聚的有前途候选材料。