顺铂是一种广泛用于抗肿瘤治疗的一线化疗药物，通过与DNA结合阻断细胞复制导致细胞死亡。然而，顺铂的毒副作用较强且易产生耐药性的特点，限制了其在临床上的应用。近年来兴起的纳米药物载体可以实现肿瘤靶向的顺铂递送，同时有助于降低顺铂的全身毒性。但纳米药物在肿瘤靶向递送时，通常由内吞进入溶酶体途径，容易导致递送的药物被溶酶体降解或外排，不利于药物在胞内释放和发挥治疗效应。为了克服这一问题，促进药物的溶酶体逃逸已成为纳米药物递送的研究热点。天然多糖透明质酸具有优异的生物相容性和水溶性，能够特异结合肿瘤细胞表面高表达的CD44蛋白，在顺铂的肿瘤靶向递送研究中应用广泛。其中，基于顺铂-透明质酸的自组装纳米凝胶，可高效介导顺铂的肿瘤靶向摄取，但胞内溶酶体的“囚禁”也极大地限制了该纳米凝胶介导的顺铂胞内递送与最终的肿瘤治疗效果。本研究设计了两种基于顺铂-透明质酸的自组装纳米凝胶，分别共载不同的能够破坏溶酶体膜稳定性的药物分子，以促进顺铂在胞内递送时的溶酶体逃逸，以期降低顺铂的毒副作用并增强对肿瘤细胞的靶向性。

       首先，本文利用顺铂、氯喹和透明质酸之间的非共价相互作用，通过自组装策略制备了载双药的透明质酸纳米凝胶，在体内外系统地探究了其对三阴性乳腺癌的抗肿瘤效果和机理。在静电相互作用、氢键、π-π堆积等分子间弱相互作用下，该纳米凝胶具有良好的形貌和稳定性。在MDA-MB-231细胞实验中，当纳米凝胶进入溶酶体后，氯喹会破坏溶酶体膜稳定性，同时阻碍溶酶体自噬，促进顺铂的溶酶体逃逸，最终增加了顺铂与细胞核DNA结合，成功杀伤癌细胞。在MDA-MB-231荷瘤裸鼠模型抑瘤实验中，纳米凝胶能够有效增加药物在肿瘤部位的积累并减少毒性，表明该策略不仅促进了顺铂的胞内递送效率，并且可以有效降低顺铂的毒性。

      其次，在氯喹研究基础上，选用溶酶体损伤的阳性对照化合物LLOMe，探究其作为治疗性药物的可能性。利用顺铂、LLOMe和透明质酸之间的自组装，成功制备了共载顺铂和LLOMe的纳米凝胶。对该纳米凝胶进行系统表征后，我们发现其可以有效降低LLOMe对血液细胞的毒性。细胞水平实验采用人源宫颈癌细胞Hela细胞系，结果表明共载双药的纳米凝胶表现出很强的杀伤作用和促溶酶体逃逸作用。利用鼠源宫颈癌细胞系U14进行建模，体内实验进一步表明，LLOMe在纳米凝胶中的共载，可以显著提升顺铂的肿瘤靶向递送和治疗效果。机制研究证明，共载LLOMe可以有效诱导溶酶体的损伤，促进顺铂的溶酶体逃逸，并引起胞内ROS累积以及线粒体膜电位的异常。此外，初步发现了 LLOMe的加入可以促进肿瘤细胞发生免疫原性死亡，因此该纳米凝胶能够从多种角度实现对肿瘤的治疗。

      综合而言，本研究基于药物分子（包括顺铂）与透明质酸之间的非共价相互作用，能快速简易地制备纳米凝胶用于顺铂的肿瘤靶向递送。在透明质酸介导的CD44靶向摄取的基础上，充分利用氯喹与LLOMe的溶酶体损伤效应，促使顺铂从溶酶体中逃逸，从而增强肿瘤的治疗效果。对相关胞内递送效应机制的揭示，也将为纳米药物的设计和应用提供新的思路和参考。

Cisplatin is a widely used first-line drug in cancer therapy. It blocks replication by binding with DNA, leading to cell death. However, its strong toxic side effects and the development of resistance significantly limit its clinical application. The emergence of nanomedicine delivery systems in recent years offers the potential for targeted delivery of cisplatin to tumors while reducing systemic toxicity. Yet, during tumor-targeted delivery, nanomedicine often enters the lysosomes through endocytosis, leading to drug degradation or efflux from lysosomes, and hinders intracellular drug release and therapeutic effect. To overcome this problem, promoting the lysosome escape of drugs has become a research hotspot in nanomedicine delivery. The natural polysaccharide hyaluronic acid, with excellent biocompatibility and water solubility, can specifically bind to CD44 proteins overexpressed on tumor cell surfaces, making it widely used in tumor-targeted delivery of cisplatin. However, their therapeutic efficacy is greatly limited by the entrapment of cisplatin within lysosomes, hindering its intracellular delivery and ultimate therapeutic effects to tumors. In this study, two types of self-assembled nanogels based on cisplatin-hyaluronic acid were designed to co-load different drug molecules capable of destabilizing lysosome membranes, aiming to promote cisplatin escape from lysosomes during intracellular delivery, thereby reducing cisplatin toxicity and enhancing tumor targeting.

Firstly, this article utilized the non-covalent interaction between cisplatin, chloroquine, and hyaluronic acid to synthesize a double-drug-loaded hyaluronic acid nanogel through a self-assembly strategy, and systematically explored its anti-tumor effect on triple-negative breast cancer in vitro and in vivo. Under weak intermolecular interactions such as electrostatic interactions, hydrogen bonding, and π-π stacking, the nanogels exhibited good morphology and stability. In MDA-MB-231 cell experiments, when the nanogel entered the lysosomes, chloroquine would disrupt the stability of the lysosome membranes, inhibit lysophagy, and promote cisplatin escape from lysosomes, thereby increasing the binding of cisplatin to nuclear DNA and successfully killing cancer cells. In MDA-MB-231 xenograft models, the nanogel effectively increased drug accumulation at tumor sites and reduces toxicity, indicating that this strategy not only enhanced the efficiency of cisplatin intracellular delivery but also effectively reduced cisplatin toxicity.

Secondly, based on the research on chloroquine, the positive control compound LLOMe for lysosome damage was selected to explore its possibility as a therapeutic drug. By utilizing the self-assembly of cisplatin, LLOMe, and hyaluronic acid, nanogels co-loaded with cisplatin and LLOMe were successfully prepared. Systematic characterization of this nanogel revealed the ability to significantly reduce the hematotoxicity of LLOMe. In vitro experiments were conducted using the human cervical cancer cell line, Hela cells. The results indicated that the nanogel exhibited strong cytotoxicity and lysosomal escape effects. The mouse cervical cancer cell line U14 was then utilized to establish a mouse tumor model, and in vivo experiments further showed that co-loading of LLOMe in the nanogel clearly enhanced tumor delivery and therapeutic efficacy of cisplatin. Mechanistic studies demonstrated that co-loaded LLOMe effectively could induce lysosome damage, promote cisplatin escape from lysosomes, induce intracellular ROS accumulation and mitochondrial membrane potential abnormalities. Additionally, preliminary findings suggested that the addition of LLOMe could promote immunogenic cell death in tumor cells, enabling the nanogel to achieve tumor treatment from multiple perspectives.

In summary, this study utilized the non-covalent interaction between drug molecules (including cisplatin) and hyaluronic acid to obtain nanogel for tumor-targeted delivery of cisplatin rapidly and easily. Building upon the CD44-targeted uptake mediated by hyaluronic acid, the full utilization of the lysosome damage effects of chloroquine and LLOMe enabled the promotion of cisplatin escape from lysosomes, thereby enhancing the therapeutic efficacy against tumors. The elucidation of relevant intracellular delivery mechanisms will also provide new insights and references for the design and application of nanomedicines.