具双重靶向和穿膜性能的多功能纳米凝胶用于癌症的高效蛋白药物治疗

Anticancer protein therapeutics showing striking antitumor activity in certain advanced tumors have emerged as one of the most effective strategies for the treatment of cancers in the clinics. However, protein drugs with intracellular targets have not been introduced into the clinical settings due to their poor cellular uptake and inferior intracellular trafficking process. The development of efficient vehicles that are able to effectively deliver and release proteins into the target cells is the key to their clinical applications. In this project, we will design and develop multifunctional nanogels with controllable size, reduction-sensitivity, dual-targeting, and cell-penetrating capability for efficient cancer protein therapy. The nanogels can be readily formed from two hyaluronic acid derivatives, i.e. hyaluronic acid-tetrazole/tumor-homing cell-penetrating peptide (CPP) and hyaluronic acid-cystamine-methacrylate, by combing microfluidics and “tetrazole-alkene” photoclick reaction. These multifunctional nanogels have several unique features: (i) they have controllable size, high protein loading capacity, and high reproducibility; (ii) photoclick reaction offers high efficiency and bioorthogonality, which circumvents cross-reaction with and denature of therapeutic proteins; (iii) hyaluronic acid can selectively target to CD44+ cancer cells like lung cancer and leukemia cells; (iv) tumor-selective CPP can further improve their tumor-targetability, tumor penetration and endosomal escape; (v) they can rapidly release encapsulated proteins under the cytoplasmic reductive conditions, resulting in potent antitumor activity. These intelligent nanogel systems have a great potential for efficient and safe cancer protein therapy.

蛋白质抗癌药物可治愈部分肿瘤，成为最有效的肿瘤临床治疗手段之一。然而，胞内起作用的蛋白药物因胞内运输效率低，在临床尚无应用，如何在体内将其高效运送到细胞内靶点是关键问题。本项目拟构建尺寸可控、还原响应、具双重靶向和穿膜性能的多功能纳米凝胶用于癌症的高效蛋白药物治疗。该纳米凝胶基于透明质酸-四唑/肿瘤选择性穿膜肽（CPP）和透明质酸-胱胺甲基丙烯酸酰胺衍生物，通过结合微流控技术和“四唑-烯”光点击化学交联制得。该蛋白纳米药物具以下优异性能：1）粒径可控、载蛋白效率高、重复性好；2）光点击化学交联具高效专一性，可有效保持蛋白药物活性；3）透明质酸可选择性靶向到CD44受体过表达的肺癌和白血病等癌细胞；4）选择性CPP将进一步增强其肿瘤靶向性能，并有效增强其穿膜和内涵体逃逸能力；5）在细胞质还原环境下能快速释放蛋白药物，产生高抗癌活性。该智能纳米凝胶体系可望实现安全高效的癌症蛋白药物治疗。

蛋白药物相对于小分子药物具有高活性、高特异性和低毒性等优点，在肿瘤治疗中有巨大的应用潜力。但蛋白药物在临床转化和应用中还面临着诸多细胞外和细胞内的挑战，需要克服体内稳定性差、癌细胞内吞效率不足、难于逃离内涵体、胞内易降解等多重屏障。本项目创新性地构建了多功能微纳米载体用于不同蛋白药物的高效稳定包载和定向可控递送，实现了对多种癌症的安全高效治疗。主要结果如下：（1）联用微流控技术和无催化剂的光点击化学反应制备了小粒径、可示踪、能逃离内涵体和具有生物响应性的透明质酸纳米凝胶（NGs）用于蛋白药物的CD44介导的细胞质递送；（2）构建了EGFR和CD44双重靶向的生物响应性自发荧光的透明质酸纳米凝胶（EGFR/CD44-NGs）用来增强治疗性蛋白质药物（果粒酶B,皂草毒素等）对肿瘤的选择性和内吞进入细胞的能力，进而实现对乳腺癌生长和转移的高效抑制；（3）开发了尺寸小、制备简单的内膜为负电的具有不对称膜结构的聚脂肽囊泡纳米平台，用于蛋白药物的高效装载和靶向递送，构建的蛋白纳米药物能完全抑制原位A549肺癌的生长、延长小鼠存活时间，且毒副作用低；（4）开发了表面修饰有特异靶向性穿膜肽（CPP33）的、内腔带正电荷的不对称聚脂肽囊泡（CPP33-CLP）用于核酸药物的高效包载和系统递送，该载体不仅能特异性靶向进入到肿瘤细胞，还能帮助核酸药物逃离内涵体进入到细胞质，发挥序列特异性基因沉默作用，实现对肿瘤选择性的高效安全抑制；（5）联用微流控法和“四唑-烯”光点击化学交联法制备了单分散的酶可降解的透明质酸微凝胶（HMGs），实现了赫赛汀（Herceptin）蛋白质药物在皮下肿瘤的局部长效释放，进而高效抑制了卵巢癌的生长。项目构建的多功能微纳米递送体系为蛋白药物的稳定包载、定向递送和可控释放提供了重要的平台，可望用于多种肿瘤的安全高效治疗。

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