Qi Lifeng, Xu Zirong, Chen Minli
Zhejiang University, Nano-biology Lab of Animal Science College, Hangzhou 310029, PR China.
Eur J Cancer. 2007 Jan;43(1):184-93. doi: 10.1016/j.ejca.2006.08.029. Epub 2006 Oct 17.
Chitosan nanoparticles (CNP), a kind of widely used drug carrier, have shown potent cytotoxic effects on various tumour cell lines in vitro and in vivo. This study sought to evaluate the antitumour effect of CNP on growth of human hepatocellular carcinoma (BEL7402) and the possible mechanisms involved. Cells were grown in the absence and presence of various concentrations of CNP with mean particle size of about 40nm. Cell viability, ultrastructural changes, surface charge, mitochondrial membrane potential, reactive oxygen species (ROS) generation, lipid peroxidation, DNA fragmentation and fatty acid composition were analysed by MTT assay, electron microscopy, zetasizer analysis, flow cytometry, spectrophotometric thiobarbituric (TBA) assays, DNA agarose gel electrophoresis and GC/MS respectively. For in vivo experiments, male BABL/c nude mice were implanted with BEL7402 cells subcutaneously to establish human hepatoma model. Chitosan, saline, and CNP with different mean particle size (40, 70 and 100nm) were administrated by oral administration (1mg/kg body weight). Tumour and body weight were measured, morphologic changes of tumour and liver tissues were studied under electron microscope. In vitro, CNP exhibited high antitumour activities with an IC(50) value of 15.01microg/ml, 6.19microg/ml and 0.94microg/ml after treatment for 24h, 48h and 72h respectively. CNP could induce cell necrosis observed by electron microscope and DNA fragmentation. The antitumour mechanism was mediated by neutralisation of cell surface charge, decrease of mitochondrial membrane potential and induction of lipid peroxidation. The tumour growth inhibitory rates on BEL7402 cells in nude mice treated with chitosan and CNP with different mean particle size (40, 70 and 100nm) were 24.07%, 61.69%, 58.98% and 34.91% respectively. Typical necrotic morphological changes of tumour tissues and no liver abnormalities were found under electron microscope. In this paper, results show a strong antitumour effect of CNP on human hepatoma cell line BEL7402 in vitro and in vivo. These findings suggest that CNP could be a kind of promising agent for further evaluations in the treatment of hepatocellular carcinoma.
壳聚糖纳米粒（CNP）是一种广泛应用的药物载体，已在体外和体内对多种肿瘤细胞系显示出强大的细胞毒性作用。本研究旨在评估CNP对人肝癌细胞（BEL7402）生长的抗肿瘤作用及其可能的作用机制。细胞在不存在和存在各种浓度（平均粒径约40nm）的CNP的情况下培养。分别通过MTT法、电子显微镜、Zetasizer分析、流式细胞术、分光光度法硫代巴比妥酸（TBA）测定、DNA琼脂糖凝胶电泳和气相色谱/质谱分析细胞活力、超微结构变化、表面电荷、线粒体膜电位、活性氧（ROS）生成、脂质过氧化、DNA片段化和脂肪酸组成。对于体内实验，将雄性BABL/c裸鼠皮下接种BEL7402细胞以建立人肝癌模型。通过口服给予壳聚糖、生理盐水和不同平均粒径（40、70和100nm）的CNP（1mg/kg体重）。测量肿瘤和体重，在电子显微镜下研究肿瘤和肝组织的形态学变化。在体外，CNP表现出高抗肿瘤活性，分别在处理24小时、48小时和72小时后的IC（50）值为15.01μg/ml、6.19μg/ml和0.94μg/ml。通过电子显微镜观察，CNP可诱导细胞坏死和DNA片段化。抗肿瘤机制是通过中和细胞表面电荷、降低线粒体膜电位和诱导脂质过氧化介导的。用壳聚糖和不同平均粒径（40、70和100nm）的CNP处理的裸鼠中，对BEL7402细胞的肿瘤生长抑制率分别为24.07%、61.69%、58.98%和34.91%。在电子显微镜下发现肿瘤组织有典型的坏死形态学变化，且肝脏无异常。本文结果表明，CNP在体外和体内对人肝癌细胞系BEL7402均有较强的抗肿瘤作用。这些发现表明，CNP可能是一种有前途的药物，有待在肝细胞癌治疗中进行进一步评估。
