单壁碳纳米管的毒性、功能化改性及靶向送药的研究
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摘要
碳纳米管(Carbon Nanotubes,CNTs)按结构可以分为单壁碳纳米管(Single-walled Carbon nanotubes,SWNTs)和多壁碳纳米管(Multi-walled Carbon nanotubes, MWNTs),由于具有优异的物理和化学性能,在传感器、场效应晶体管、储氢等诸多领域都有广阔的应用前景。CNTs用作纳米药物载体是近年来的新兴前沿方向,目前的研究热点主要集中在:一,CNTs的生物改性及毒性评价;二, CNTs基靶向送药系统的设计、制备和疗效评价。本文工作的特色在于用各种天然多糖对SWNTs进行功能化改性,定量的评价了CNTs的长度、直径、浓度和表面官能团对细胞毒性的影响,并制备了一类基于CNTs的靶向送药体系,分别在细胞水平和动物水平评估了该体系的疗效和副作用。具体为:
1)定量的研究了浓度、直径、长度和表面性能对CNTs细胞毒性的影响规律。研究结果发现CNTs的细胞毒性具有剂量和尺寸依赖性,CNTs浓度越大或直径越小,显示出的细胞毒性越大;经表面包覆改性后,表面官能团的类型和电荷性能会显著影响CNTs的细胞毒性,只含有羟基而且显电中性的直链淀粉包覆的CNTs具有更好的生物相容性;截短后的CNTs容易进入细胞并且主要分布在溶酶体中,多糖改性和直径变化对CNTs在细胞中的分布无明显影响。
2)利用壳聚糖(CHI)对SWNTs进行非共价修饰,并引入靶向分子叶酸(Folic acid,FA)和蒽环类抗癌药物阿霉素(Doxorubicin,DOX),制备出一种具有靶向和缓释效果的送药体系。该CNTs基送药体系具有很高的载药率,而且DOX的释放具有pH响应特性,在血液pH条件下很少释放,在细胞器酸性条件下才显著释放,结合其肿瘤靶向性能,比单独使用DOX能更有效的的杀死肿瘤细胞。
3)受上述实验结果鼓舞,我们进行了动物实验。把该靶向载药体系通过静脉注入荷瘤裸鼠体内并记录肿瘤体积和体重的变化,三周后杀死裸鼠测量血清化学参数并对肝脏、脾脏和肾进行病理分析。结果表明:在相同当量剂量时,该送药体系比单独使用DOX更能更有效抑制肿瘤的生长,同时其对肝、脾、肾等正常组织器官的毒副作用却比纯DOX小,具有高效低毒的优点。
Carbon nanotubes (CNTs) are seamless cylinders rolled up by grapheme sheets, which could be divided into single walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs) depending on the number of the grapheme layers. Due to their unique electrical, physical and chemical properties, CNTs have attracted tremendous interests in numerous fields such as sensors, field-effect-emitter and hydrogen storage. Most recently, CNTs have been intensively explored as nanovectors for targeted drug delivery and focused on 1) the biofunction alization and cytotoxicity of CNTs; 2) Design, preparation and evaluation of CNTs based drug delivery system. In this work, we used several natural polysaccharides to functionalize CNTs and quantitatively evaluated the effect of length, diameter, concentration and functional group of the polysacchrides on the cytotoxicity of CNTs. Furthermore, a targeted drug delivery system based on CNTs was prepared and investigated both in vitro and in vivo in terms of efficiency and side effects. The details are as follows:
1) Quantitatively investigating the effect of length, diameter, concentration and functional group on the cytotoxicity of CNTs. The experimental results reveal that 1) the cytotoxicity of CNTs are dose- and size-dependent. CNTs with larger concentration or smaller diameter are more toxic; 2) The cytotoxicity of CNTs was greatly effected by the types and charge properties of the functional groups. The CNTs gain better biocompatibility only when functionalized by amylose simultaneously bearing hydroxyl group and neutral surface charge. 3) Cut CNTs are easy to penetrate cell membranes and residue in lysosomals, which is independent of the diameters and polysaccharides functionalization.
2) Prepared a targeted drug delivery system (DDS) by attaching folic acid and doxorubicin to CHI wrapped SWNTs. It was found that the DOX was loaded at a high ratio and only released at acidic environment (e.g. pH= 5.5) while remain stable in the physiological envronment (pH= 7.4). The in vitro results show that the DDS was superior to free DOX due to its controlled release and targeted delivery of DOX.
3) The in vivo performance of the DDS was further investigated using nude mice bearing SMMC-7721 tumor as the animal model. The DDS was i.v. administrated into the mice which were sacrificed three weeks later for biochemical and histological examinations, during which the body weights and tumor volumes were recorded. It was demonstrated that, when at the equivalent DOX dose, the DDS was more efficient than free DOX in inhibiting the growth of tumors while caused less side effects.
1)定量的研究了浓度、直径、长度和表面性能对CNTs细胞毒性的影响规律。研究结果发现CNTs的细胞毒性具有剂量和尺寸依赖性,CNTs浓度越大或直径越小,显示出的细胞毒性越大;经表面包覆改性后,表面官能团的类型和电荷性能会显著影响CNTs的细胞毒性,只含有羟基而且显电中性的直链淀粉包覆的CNTs具有更好的生物相容性;截短后的CNTs容易进入细胞并且主要分布在溶酶体中,多糖改性和直径变化对CNTs在细胞中的分布无明显影响。
2)利用壳聚糖(CHI)对SWNTs进行非共价修饰,并引入靶向分子叶酸(Folic acid,FA)和蒽环类抗癌药物阿霉素(Doxorubicin,DOX),制备出一种具有靶向和缓释效果的送药体系。该CNTs基送药体系具有很高的载药率,而且DOX的释放具有pH响应特性,在血液pH条件下很少释放,在细胞器酸性条件下才显著释放,结合其肿瘤靶向性能,比单独使用DOX能更有效的的杀死肿瘤细胞。
3)受上述实验结果鼓舞,我们进行了动物实验。把该靶向载药体系通过静脉注入荷瘤裸鼠体内并记录肿瘤体积和体重的变化,三周后杀死裸鼠测量血清化学参数并对肝脏、脾脏和肾进行病理分析。结果表明:在相同当量剂量时,该送药体系比单独使用DOX更能更有效抑制肿瘤的生长,同时其对肝、脾、肾等正常组织器官的毒副作用却比纯DOX小,具有高效低毒的优点。
Carbon nanotubes (CNTs) are seamless cylinders rolled up by grapheme sheets, which could be divided into single walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs) depending on the number of the grapheme layers. Due to their unique electrical, physical and chemical properties, CNTs have attracted tremendous interests in numerous fields such as sensors, field-effect-emitter and hydrogen storage. Most recently, CNTs have been intensively explored as nanovectors for targeted drug delivery and focused on 1) the biofunction alization and cytotoxicity of CNTs; 2) Design, preparation and evaluation of CNTs based drug delivery system. In this work, we used several natural polysaccharides to functionalize CNTs and quantitatively evaluated the effect of length, diameter, concentration and functional group of the polysacchrides on the cytotoxicity of CNTs. Furthermore, a targeted drug delivery system based on CNTs was prepared and investigated both in vitro and in vivo in terms of efficiency and side effects. The details are as follows:
1) Quantitatively investigating the effect of length, diameter, concentration and functional group on the cytotoxicity of CNTs. The experimental results reveal that 1) the cytotoxicity of CNTs are dose- and size-dependent. CNTs with larger concentration or smaller diameter are more toxic; 2) The cytotoxicity of CNTs was greatly effected by the types and charge properties of the functional groups. The CNTs gain better biocompatibility only when functionalized by amylose simultaneously bearing hydroxyl group and neutral surface charge. 3) Cut CNTs are easy to penetrate cell membranes and residue in lysosomals, which is independent of the diameters and polysaccharides functionalization.
2) Prepared a targeted drug delivery system (DDS) by attaching folic acid and doxorubicin to CHI wrapped SWNTs. It was found that the DOX was loaded at a high ratio and only released at acidic environment (e.g. pH= 5.5) while remain stable in the physiological envronment (pH= 7.4). The in vitro results show that the DDS was superior to free DOX due to its controlled release and targeted delivery of DOX.
3) The in vivo performance of the DDS was further investigated using nude mice bearing SMMC-7721 tumor as the animal model. The DDS was i.v. administrated into the mice which were sacrificed three weeks later for biochemical and histological examinations, during which the body weights and tumor volumes were recorded. It was demonstrated that, when at the equivalent DOX dose, the DDS was more efficient than free DOX in inhibiting the growth of tumors while caused less side effects.
引文
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