A6多肽导向多功能囊泡甲硫哒嗪药物用于乳腺癌干细胞的高效靶向化疗

The initiation, development and invasion of breast cancer are intimately associated with breast cancer stem cells (bCSCs). As a matter of fact, bCSCs are a main reason for the recurrence and metastasis of breast cancers. Their intrinsic drug resistance, however, makes common chemical drugs ineffective to bCSCs. To effectively kill bCSCs is essential for breast cancer treatment. In this project, we will design and develop A6 peptide-directed, reduction-responsive, reversibly crosslinked multifunctional polymersomal thioridazine (THZ) for targeted chemotherapy of CD44-overexpressed bCSCs. THZ is a potent inhibitor for cancer stem cells (CSCs), but it meets many drawbacks such as low selectivity and high systemic toxicity. The multifunctional polymersomes designed in this project can be easily prepared, can efficiently load THZ into their lumen by pH-gradient method, and effectively reduce drug side effects. THZ-loaded polymersomes have a high in vivo stability, can selectively bind to CD44, can be efficiently internalized by bCSCs, and quickly release THZ into the cytoplasm in response to intracellular reductive environment, leading to highly efficient and specific killing of bCSCs. Furthermore, these multifunctional polymersomes will load and release both THZ and common anti-breast cancer chemotherapeutics, doxorubicin hydrochloride, into both bCSCs and breast cancer cells, resulting in effective killing of both bCSCs and breast cancer cells, and thereby completely eliminating breast cancers in patients.

乳腺癌的发生发展、侵袭和转移与乳腺癌干细胞（bCSCs）密切相关，bCSCs是造成乳腺癌复发和转移的重要原因。bCSCs的强耐药性导致常规化疗药对其杀伤效果较差。如何高效杀死bCSCs是乳腺癌治疗的关键科学问题。本项目将设计制备A6多肽导向、还原响应、可逆交联的多功能聚合物囊泡甲硫哒嗪（THZ）药物用于CD44高表达bCSCs的高效靶向化疗。THZ是筛选出的高效肿瘤干细胞抑制剂，但其具有选择性差、系统毒性大等问题。项目设计的囊泡制备简单，可用pH梯度法高效包载THZ到内腔,有效降低THZ的毒副作用。载药囊泡在体内稳定性高，可望特异性结合CD44，实现bCSCs的高效内吞，并在胞内快速释放THZ，可望实现bCSCs的高效特异性杀伤。进一步地，我们将采用该囊泡同时装载THZ和乳腺癌化疗药物阿霉素盐酸盐，将药物快速释放到bCSCs和乳腺癌细胞中，同时杀死两种细胞，可望实现乳腺癌的彻底治愈。

项目针对如何高效杀死肿瘤干细胞，构建了两种主动靶向、还原响应、可逆交联的多功能聚碳酸酯囊泡用于高效肿瘤干细胞抑制剂甲硫哒嗪（THZ）的高效稳定包载和定向可控递送，并评估了THZ囊泡在富含血液肿瘤干细胞的小鼠原位模型中的治疗效果。主要的研究结果如下：（1）我们成功建立了富含白血病干细胞的MA9-GFP原代鼠源急性髓系白血病模型。建立模型采用的MA9-GFP细胞含有约30%的血液肿瘤干细胞（LSCs）亚群，该原代白血病细胞可体外培养、冻存，也可移植小鼠后建立稳定的白血病动物模型。（2）构建的A6多肽导向、还原响应、可逆交联的多功能聚合物囊泡（A6-Ps）可将特异性靶向肿瘤干细胞药物THZ递送至CD44+的富含LSCs的MA9-GFP白血病细胞，有效降低了白血病干细胞的含量，明显缓解小鼠白血病负担，减缓了发病进程。（3）开发的骨靶向功能化囊泡（Ald-Ps）将具有抗白血病和抗LSCs活性的THZ递送到骨髓微环境中，提高了THZ在骨组织中的浓度，但在MA9-GFP原代白血病小鼠模型中评估治疗效果时发现骨靶向THZ囊泡会引起小鼠体重严重下降，对肿瘤细胞及肿瘤干细胞的特异性杀伤差，未能达到预期效果。（4）A6多肽导向、还原响应、可逆交联的多功能聚合物THZ囊泡联用白血病临床化疗药物阿糖胞苷（Arac）在MA9-GFP原代白血病小鼠模型的治疗中显示出最佳的抗LSCs的效果，取得了最佳疗效。我们在项目开发的聚合物的基础上，还构建了NRP-1靶向、还原响应、载有NAC-1 siRNA的聚合物囊泡联合贝伐单抗用于转移性三阴乳腺癌的治疗；开发了CD44靶向、可逆核交联性质的多功能生物可降解卡非佐米（CFZ）胶束用于皮下和原位荷LP-1多发性骨髓瘤小鼠的主动靶向化疗。本项目构建的纳米药物递送体系功能性强、具有自主知识产权、临床转化潜力大，在抗肿瘤干细胞、抑制肿瘤转移、复发和耐药等方面有广阔的应用前景。