难溶性抗肿瘤中药成分智能靶点定位释药纳米载体的构建及研究

Many natural antitumor drugs extracted from plants have shown significant inhibition on the growth of cancer cells with low hematopoietic and immune system damage, thereby being the attracting more and more attention. However, serious drawbacks hamper their clinical utilizations, such as poor water solubility and more importantly, the serious side effects and decreased therapeutic efficiency induced by lacking of selectivity to tumor cells or to the drug release sites. The design of targeted drug delivery system with the ability of efficient drug loading and controlled drug release in targeted sites is of high demand for advancing clinical application of natural medicine. Given the significant difference in redox potential between intracellular and extracellular environments, this project will develop a novel redox-sensitive micelles based on amphipathic O, N-hydroxyethyl chitosan-octylamine conjugate (HECS-ss-OA) containing 3, 3'-dithiodipropionic acid (DPA) as bioreducible linkages. Then, oligosaccharide hyaluronic acid (OHA) which possesses strong affinity to tumor cell-specific surface markers will be coated on the surface of the cationic HECS-ss-OA micelles through electrostatic interaction to get OHA(GC-ss-AC) tumor targeted micellar complexes. The designed OHA(GC-ss-AC) micellar complexes with high drug loading of natural antitumor drugs are stability in bloodstream and could be selectively taken up to tumor cells via OHA-receptors mediated endocytosis and then achieve rapid disassembly once internalized into tumor cells, thereby improving intracellular drug release and increasing the antitumor efficacy. In this study, gambogic acid (GA) which is the active ingredient of traditional Chinese medicine will be chosen as the model drug encapsulated in the redox-sensitive micelles. Various OHA(GC-ss-AC) micelles with different molecular weight of chitosan, the substitution degree of hydrophobic moieties, the type of chemical linker will be prepared and their effects on the self-assembly abilities, drug loading capacities and triggered release sensitivity will be studied in detail. Moreover, the in vitro cellular pharmacokinetic and the in vivo biodistribution and tumor targeting behavior of OHA(GC-ss-AC) micelles will be investigated to explore the mechanisms of the cellular uptake of the redox-sensitive micelles and the triggered drug release in the intracellular reducing environment. It is expected that OHA(GC-ss-AC) reduction-sensitive micelles based on the combination of redox-sensitive, active targeting and micellar self-assembly techniques would be an efficient and promising carrier for intracellular natural medicine delivery.

许多天然抗肿瘤药物具有高效杀肿瘤细胞能力，而其低水溶性、无肿瘤靶向性、制剂释药部位无选择性限制了其临床应用。鉴于肿瘤细胞内强还原环境，本项目首次通过还原敏感连接臂（-ss-），构建羟乙基壳聚糖（GC）-疏水烷基（AC）两亲性偶联物（GC-ss-AC），并以具有肿瘤细胞靶向性的透明质酸寡聚糖（OHA）表面修饰其自组装胶束OHA(GC-ss-AC)，该体系可实现天然抗肿瘤药物高效负载、稳定转运、肿瘤细胞特异性摄取及靶细胞内触发定位释药目的。本项目将构建多种OHA(GC-ss-AC)，以中药有效成分藤黄酸（GA）为模型药物，系统评价其自组装和载药能力、肿瘤靶向性、还原敏感性；并结合胞内微动力学及体内肿瘤转运和抑瘤活性，探讨胞内触发释药机制。本项目首次结合还原敏感、主动靶向及胶束自组装技术，构建具靶点定位释药功能的递药载体，为推进中药有效成分抗肿瘤临床应用提供了一种新思路和方法。

化疗是目前临床治疗肿瘤的主要方法之一，但临床上多数抗肿瘤化疗药物为难溶性小分子化合物，体内代谢快、生物利用度低，无专一性，影响疗效。本项目以天然高分子材料壳聚糖为基本骨架，在骨架上引入亲水基团羟乙基，成为亲水的羟乙基壳聚糖（GC），通过二硫键（-ss-）连接臂介入疏水基团正辛胺（AC），合成两亲性壳聚糖衍生物（GC-ss-AC），此衍生物作为载体负载抗肿瘤药物藤黄酸（GA），外层包覆对肿瘤细胞具有高亲和力的透明质酸（HA），形成可响应肿瘤还原环境触发释药的靶向载药体系GA-HA(GC-ss-AC)。GC-ss-AC与非还原敏感GC-cc-AC的临界胶束浓度较低，预示着优良的载药性能。选择透析法载药，DSC结果表明GA以分子形式存在于载体骨架中。HA与GA-GC-ss-AC质量最佳比为3，粒径在200.0 nm左右，电位为-24.53 mV，载药量达18.44 %，包封率达81.54%。TEM观察载药胶束呈规则类球状结构，粒径分布较均匀。溶血实验结果表明载体具有一定的静脉注射安全性。体外还原敏感性结果显示，还原敏感胶束对还原条件的响应呈现时间和浓度依赖性。细胞摄取结果表明，A549细胞对载药胶束的吞噬呈现时间依赖性，具有主动转运的特征。经游离HA预处理后，摄取显著抑制，表明吞噬通过HA受体介导。胞内释放结果表明，还原敏感胶束可在胞质中快速释放药物，游离药物分子在脂质小体中进行蓄积，而非还原敏感胶束释放缓慢。MTT和凋亡结果表明，还原敏感制剂具有更强的细胞毒性，并通过HA受体介导内吞。组织分布结果证实胶束在A549荷瘤裸鼠体内具有较强的肿瘤靶向性。药效学结果表明，游离 GA和各制剂组均能显著抑制肿瘤生长， GA-HA(GC-ss-AC)组表现出最佳抑瘤效果。肿瘤组织H&E病理切片染色以及TUNEL组织凋亡检测也证实GA-HA(GC-ss-AC)的最佳抑瘤效果。在体毒性评价中，GA-HA(GC-ss-AC)组裸鼠体重无显著下降，肝肾毒性比游离GA低，证明该制剂能显著降低药物毒副作用。本项目将两亲性自组装技术、靶向技术、以及还原敏感技术结合，建立了一种高效负载、稳定转运、肿瘤细胞特异性摄取并定位释药的纳米靶向载体技术，提升药物疗效，降低毒副作用，应用前景良好。