SPIO示踪的MISO脂质体的构建及其肿瘤靶向放疗增敏作用的研究

Misonidazole (MISO), the first hypoxic cell radiosensitizer, has been in clinical trials for decades. MISO is more efficient than sodium glycididazole, i.e., its adiosensitization ratio (SER) is higher and it can produce a greater amount of sensitization for a higher dose. However, the dose of MISO is limited by the peripheral neurotoxicity, discouraging the phase III trials. It is well-known that the interstitial substance of solid tumor has a low pH value in 6.5. Our study design based on the data that the drug packed in the pH-sensitive liposome only could be released in a low pH environment. We presumed that the MISO can be concentrated in the tumor by the pH-sensitive liposome, which increasing SER and decreasing the peripheral neurotoxicity. As a tracer, the contrast agent without radioactive--superparamagnetic iron oxide nanoparticles (SPIO) will be used to follow MISO targeting liposome under magnetic resonance imaging. In our study, the preparation of MISO targeting liposome is as follows: the MISO and the SPIO will be packed in the hydrophobic and hydrophilic layers of pH- sensitive liposome, respectively. In our initial studies, these highly pH-sensitive liposomal vesicles can be specially targeted to tumor in bearing mice. In next study, we aim to optimize the prescription and improve the preparation process of the MISO targeting liposome. The nude mice bearing nasopharyngeal carcinoma, esophageal cancer or lung cancer will be used to measure SER and the peripheral neurotoxicity of MISO targeting liposome. This study will provide theoretical evidence for MISO targeting liposome in clinical treatment.

米索硝唑(MISO)是第一个经临床广泛研究的乏氧细胞放射增敏剂，其放射增敏比（SER）优于甘氨双唑钠，且随剂量增加可进一步提高，因其对外周神经组织的毒性限制了使用剂量，使其Ⅲ期临床试验失败。本课题采用脂质体技术将MISO包裹在 pH敏感脂质体的疏水层，将无放射性的显影剂--超顺磁氧化铁纳米粒（SPIO）包裹在亲水层，制得MISO靶向脂质体。利用pH敏感脂质体仅在低pH环境下释药，及实体瘤间质处的pH值（6.5）低的特点，以提高肿瘤组织中MISO的药物浓度，从而进一步增加SER、降低外周神经组织毒性，并通过SPIO于磁共振下显像示踪证实肿瘤靶向性。预试验表明该脂质体具有高度的pH敏感性及荷瘤鼠肿瘤内的靶向性。本课题拟进一步优化MISO靶向脂质体的处方、克服制备工艺的难题，并在荷鼻咽癌、食管癌、肺癌的荷瘤鼠体内验证进一步提高的SER及降低的外周神经组织毒性，为MISO再次进入临床打下基础。

本课题采用pH敏感脂质体为靶向载体，包裹米索硝唑使其在肿瘤内浓聚，进一步增加放疗增敏比、降低外周神经组织毒性，并包裹超顺磁氧化铁纳米粒（SPION）于磁共振下显像示踪证实肿瘤靶向性。首先建立HPLC结合金属离子沉淀法测定脂质体中MISO的含量，建立邻菲罗啉分光光度法测定脂质体内的SPIO含量。采用逆相蒸发法结合冻融法制得SPIO示踪的MISO pH敏感脂质体（SPION-MISO-SpHLs），并以包封率及释放度为双指标，采用正交试验对其制备工艺及处方进行优化。结果显示MISO及SPIONs的最大包封率分别为30.2%和 23.7%, 在 pH 5.5的释放介质中，MISO及SPIONs的最大释放度是在 pH 7.4中的2.49倍 和2.47倍。理化性质考察显示SPION-MISO-SpHLs外观为类球形，平均粒径约为 950 nm，SPIONs包裹在脂质体内部并均匀分散，zeta 点位分别是36.3 mV （pH 5.5）和 -58.9 mV （pH 7.4），并保留了SPION原有超顺磁的特性。在以CoCl2化学诱导法建立的肺癌A549乏氧细胞模型中，证实SPION-MISO-SpHLs具有明显pH敏感性，且与MISO+SPION+空白脂质体比较也具有显著差异。在肺癌A549细胞荷瘤裸鼠模型中，MRI扫描结果显示SPION-MISO-SpHLs具有靶向示踪性。肿瘤外照射试验结果显示SPION-MISO-SpHLs具有放疗增敏作用，且效果优于MISO。肌电图检测及胫神经病理结果显示SPION-MISO-SpHLs具有降低MISO外周神经毒性的作用。上述结果为MISO再次进入临床打下基础。项目资助发表SCI文章2篇，中文文章4篇，获国家授权发明专利1项，培养硕士生1名，并取得硕士学位，获优秀硕士论文称号。项目投入经费16.5万元，支出10.812万元，各项支出基本与预算相符。剩余经费5.688万元，剩余经费计划用于本项目研究后续支出。

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