双重响应的基因/化疗药物共载时序释放纳米递送体系靶向抗肝癌研究

miRNA-21 overexpression is closely related to cancer development, invasion, metastasis, angiogenesis and drug resistance, thus it can be used as a multi-effect therapeutic target for cancer treatments. Our previous study has found the combined therapy of antisense oligonucleotide (ASO) of miRNA-21 and adriamycin can effectively inhibit the growth and migration of liver cancer cells, and has significant chemosensitization effects. Therefore, this project constructs a novel dual-responsive nanoparticle carrier for targeting delivery and successive release of gene and chemotherapeutic drug. This nanoparticle carrier has a shell-core structure. The hydrophobic core is formed by complexation of ASO-miRNA-21 with poly β-aminoester that containing disulfide bonds in monomer units (SS-PBAE). The hydrophilic shell is formed by anchoring of β-carboxyl acid amidized pullulan that has charge-reversal property. Chemotherapeutic drug is conjugated to the polysaccharide backbone of pullulan via chemical bond. This nanoparticle drug carrier can be accumulated in the liver through the positive targeting effect of pullulan. After entring into tumor cells, ASO-miRNA-21 can be rapidly released by corresponding to the acidic endosomal pH and the high intracellular glutathione concentration, orderly. The release of therapeutic drug is controlled by the chemical bond breaking, thus shows a slower release rate compare to ASO-miRNA-21. Thus, two therapeutic agents can be released in an orderly manner and exert the combined effects to treat liver cancer.

miR-21的高表达与肿瘤的生长、转移、血管生成及耐药性等密切相关，作为肿瘤治疗靶标具有单击多靶效应。课题组前期研究发现miR-21反义核苷酸(ASO)联合阿霉素时序给药能有效抑制肝癌细胞的生长与迁移，并具有显著的化疗增敏作用。因此，本项目构建了一种双重响应的ASO-miR-21/化疗药物纳米共载体系，以期实现体内靶向及时序性药物递送。纳米体系具有核壳结构，内核由ASO-miR-21与含有二硫键的β-聚氨酯（SS-PBAE）复合而成；具有电荷翻转性质的β-羧基酰胺化普鲁兰多糖与化疗药物通过化学键偶联，以高分子前药形式锚定包裹疏水核组装形成亲水外壳。共载体系通过普鲁兰多糖的主动靶向作用蓄积于肝脏；通过对肿瘤细胞内涵体弱酸性及胞内高浓度谷胱甘肽的双重响应实现ASO-miR-21的快速释放；化疗药物则通过化学键的断裂调控释放，相对缓慢，从而完成两种治疗剂的靶向递送、有序释放及协同作用。

肝细胞癌（hepatocellular carcinoma, HCC）是人类最常见的恶性肿瘤之一，具有恶性程度高、易侵袭转移、预后差等特点，化疗是目前中晚期HCC临床治疗的重要手段。由于HCC的发生、发展是一个多步骤、多阶段、多基因参与的复杂过程，单一的化疗不能有效抑制其转移与恶化，因此将化疗与其它治疗手段联合是HCC治疗领域的重要研究课题。利用纳米载体技术在药物、基因的靶向递送以及整合多种治疗方法等方面的独特优势，本项目构建了系列靶向性、智能型纳米载体系统，用于抗肿瘤药物/基因或它治疗剂的高效共载、靶向递送、时序释放及其协同作用，取得了较好的科研成果，详述如下：一、以具有肝癌靶向作用的普鲁兰多糖及具有pH-/或氧化还原响应性的聚（β-氨基酯）为材料，构建多重刺激响应性纳米载体，共载化疗药物（阿霉素、紫杉醇）/基因（模型基因、miRNA-21反义核苷酸、miRNA-122）或其它治疗药物（新生血管抑制剂combretastatin A-4），并从分子、细胞与动物水平验证了这些纳米共载体系靶向递送与时序释放药物的能力及其协同增效的抗肝癌作用。二、以磷脂、普鲁兰多糖与Pluronic F68为载体材料，设计构建了共载阿霉素/光敏剂（IR780）或声敏剂（血卟啉）的纳米治疗体系，并系统地考察了这些纳米治疗体系联合化疗与光热/光动力或声动力治疗对肝癌的综合作用，包括对肿瘤细胞的杀伤、肿瘤干细胞耐药性的逆转、血管生成的抑制以及肿瘤微环境的重建等作用。三、合成了系列普鲁兰多糖衍生物，考察了其对人浆细胞样类树突状细胞CAL-1的激活效应，评价了将其用作抗肿瘤免疫佐剂的可行性，并将化疗药物顺铂与普鲁兰多糖衍生物偶联合成高分子前药，通过体内外实验证明了前药的抗肝癌作用。综上可见，该项目研究工作的开展为解决临床肝癌治疗难题提供了新的方法与策略。项目执行期间在Biomaterials, J Control Release, Acta Biomaterialia, ACS Appl Mater Interfaces, Polym Chem, Int J Nanomedicine等国际期刊上发表SCI论文共计17篇，待发表论文3篇；申请专利4项，获得授权2项；参加国际国内学术会议20人次，发表会议论文15篇；培养博士研究生3名，硕士研究生4名。