超临界反溶剂技术构建叶酸偶联葡聚糖包载羟基喜树碱多组分肿瘤靶向纳米粒及其对肿瘤细胞作用的研究

Nanoparticles targeting to tumor cells is a new type of drug delivery system. Its major preparation methods include spray drying, chemical deposition and emulsification-homogenization. However, these methods exist some drawbacks such as low entrapment efficiency and complicated process. The nanoparticles prepared by supercritical method has advantages on high drug loading efficiency,small particle size and strong stability. Nevertheless, the systematic study on targeting preparation containing multicomponent material system has not yet been reported. The project made a kind of anticancer drug, hydroxycamptothecine(HCPT) a model compound and tried to prepare folate-conjugated HCPT-loaded dextran nanoparticles using supercritical method. Next, the assembly process and behavior of HCPT, folate, dextran during supercritical antisolvent process was revealed through phasigram. Moreover, relationships between entrapment efficiency ,the amount of surface folate of folate-conjugated HCPT-loaded dextran nanoparticles and major quantity of state including operating temperature, operating pressure, the ratio of feed rate and CO2 flow rate was established. Also the relationship between entrapment structure and drug release characteristics was probed and the effect from targeting nanoparticles on tumor cells was preliminarily validated. All above research results supply the theoretical basis for the preparation of multicomponent tumor targeting nanoparticles with high entrapment efficiency using supercritical antisolvent method.

肿瘤靶向纳米粒是一种新型的药物传递形式，常用制备方法主要为喷雾干燥法、化学沉积法、乳化/匀质法等，但存在包封率低、工序复杂的缺点。超临界法制备纳米粒具有载药高、粒径小、稳定性强的优势，而其在含有多组分物料体系的靶向制剂制备方面还未有系统的研究。本项目以抗肿瘤药物羟基喜树碱为模型化合物，尝试在超临界体系下构建叶酸偶联葡聚糖包载羟基喜树碱纳米粒，通过相图揭示含有羟基喜树碱、叶酸、葡聚糖多组分物料体系在超临界反溶剂过程中形成纳米粒的组装过程与行为，建立叶酸偶联葡聚糖包载羟基喜树碱纳米粒的包封率和表面叶酸量与操作温度、操作压力、进料速度与CO2速度之比等主要状态参量的关系，探索纳米粒的包封结构与药物释放特征的关系，并对获得的靶向纳米粒子进行肿瘤细胞效应的初步验证，为超临界反溶剂技术制备高包封率的多组分肿瘤靶向纳米粒子提供理论基础。

本项目以抗肿瘤药物羟基喜树碱为模型化合物，尝试在超临界体系下构建叶酸偶联葡聚糖包载羟基喜树碱纳米粒，通过相图揭示含有羟基喜树碱、叶酸、葡聚糖多组分物料体系在超临界反溶剂过程中形成纳米粒的组装过程与行为，建立叶酸偶联葡聚糖包载羟基喜树碱纳米粒（Fa-DEX-HCPTNPs）的包封率和表面叶酸量与操作温度、操作压力、进料速度与CO2速度之比等主要状态参量的关系，探索纳米粒的包封结构与药物释放特征的关系，并对获得的靶向纳米粒子进行肿瘤细胞效应的初步验证，为超临界反溶剂技术制备高包封率的多组分肿瘤靶向纳米粒子提供理论基础。.本研究的主要内容及获得研究结果如下：.1. 合成了叶酸偶联葡聚糖载体，其中叶酸的偶联度为2.97%；.2. 采用析因设计法优化了各操作因素即温度、压力、喷嘴孔径、流速、叶酸偶联葡聚糖浓度、羟基喜树碱浓度对Fa-DEX-HCPTNPs纳米粒的粒径及得率的影响。获得最佳制备条件为：温度40 °C、压强20 MPa、喷嘴直径300 μm、流速13.3 ml/min、叶酸偶联葡聚糖浓度5 mg/ml、羟基喜树碱浓度5 mg/ml时。此条件获得的平均粒径大小约为145.1 nm、包封率为57.6%、载药量为50.2%，且粒径分布均匀。.3. 采用SEM观察了不同操作条件所得Fa-DEX-HCPTNPs纳米粒的形貌，并结合FTIR、XRD、DSC法分析Fa-DEX-HCPTNPs纳米粒组装过程中的化学结构、晶体结果的变化情况。结果表明：Fa-DEX-HCPTNPs同羟基喜树碱与葡聚糖混合物各特征吸收峰一致，说明SAS过程未导致羟基喜树碱的降解，化学结构未发生改变。羟基喜树碱原粉为晶体态，叶酸偶联葡聚糖为无定形态，经过SAS过程后获得的Fa-DEX-HCPTNPs与叶酸偶联葡聚糖相似，仅在2θ=5.74°处有一小衍射峰，其结晶度大大降低。.4. 体外药物释放结果表明Fa-DEX-HCPTNPs纳米粒子具有明显的缓释性；体外活性评价表明，随着Fa-DEX-HCPTNPs纳米粒子剂量的增加，对SMMC-7721癌细胞抑制率呈上升趋势，FA提高了HCPT对SMMC-7721肿瘤细胞的定向攻击能力，并提高了对细胞的致死作用。细胞靶向性测试结果表明，Fa-DEX-HCPTNPs纳米粒子对肿瘤细胞的靶向性显著增强。