Click交联的逐级靶向智能纳米凝胶调节TAM极化方向用于肿瘤化学免疫协同治疗

As the first-line drug for hepatic carcinoma treatment, sorafenib showed undesirable tumor-associated immunology effect inhibition which was expected to be reversed by polarizing M2 phenotype of tumor-associated macrophages (TAM) to M1 phenotype in this study. Based on the new treatment concept of chemo-immunotherapy, the aim of this study is to firstly synthesize novel programmable targeting intelligent nanogel to realize co-delivery of sorafenib and IKKβ-siRNA which was found to be efficient in polarizing M2 phenotype to M1 phenotype. A novel skeleton material cyclodextrin modified azided carboxymethyl chitosan (CD-CMCS-N3) and a novel acid sensitive crosslinker adipic acid dihydrazide (yne-H-ADH-H-yne) were synthesized, respectively. The CD-CMCS-N3 would be crosslinked with yne-H-ADH-H-yne based on “click chemistry” mechanism, forming the porous intelligent nanogel. The sorafenib lipid nanosuspension (sorafenib-LNS) and mannose-modified-PEI condensed IKKβ-siRNA nanoparticles (M-PEI/IKKβ-siRNA) would be then embedded to get the co-loaded nanogel. Ad-PEG-NGR would be inserted into CD-CMCS-N3 to get the NGR modified nanogel. The intelligent nanogel will be programmable targeting, of which the initial procedure is to target to tumor site by NGR receptors followed by acid-stimuli impulse degradation. The following procedure contains the hepatoma cells targeting by Sorafenib-LNS and TAM targeting by M-PEI/IKKβ-siRNA, which enhance the M1 phenotype polarization from M2 phenotype and eventually reverse the tumor-associated immunology inhibition. By virtue of the synergistic anti-tumor effects of chemotherapeutics and immunotherapeutic siRNA, this study will make great contributes to the tumor chemo-immunotherapy strategy for clinical applications.

索拉非尼是肝癌治疗一线药物，但易引起免疫抑制，调节肿瘤相关巨噬细胞(TAM)由M2型向肿瘤杀伤性M1型极化可逆转肿瘤免疫抑制，增强肝癌治疗效果。本课题基于化疗联合免疫治疗新理念，首次合成纳米凝胶骨架新材料环糊精修饰叠氮化羧甲基壳聚糖(CD-CMCS-N3)和新型酸敏感交联剂炔基修饰的己二酸二酰肼衍生物(yne-H-ADH-H-yne)，利用click反应交联形成多孔智能纳米凝胶，嵌入索拉非尼纳米脂质混悬剂和甘露糖修饰载IKKβ-siRNA纳米粒；纳米凝胶外壳修饰NGR获得逐级靶向智能纳米凝胶。智能纳米凝胶利用NGR靶向肿瘤组织，在肿瘤微环境中pH敏感响应脉冲解聚，释放索拉非尼纳米脂质混悬剂靶向肿瘤细胞发挥化疗作用；释放载IKKβ-siRNA纳米粒靶向M2型TAM，使TAM向M1型极化，逆转肿瘤免疫抑制，发挥免疫化疗协同作用。构建逐级靶向智能载体，为临床开展化疗联合免疫治疗奠定研究基础。

原发性肝细胞癌（Hepatocellular Carcinoma，HCC）的治疗策略与药物研发是我国重大疾病治疗的重点研究领域之一。基于肿瘤相关巨噬细胞（TAMs）的肿瘤免疫与化疗联合治疗是近年来HCC治疗的研究热点。本课题旨在设计合理的纳米载体实现肿瘤细胞靶向的化疗药物与M2型巨噬细胞靶向的免疫药物异靶点共递送，提高HCC化学免疫联合治疗的效果。课题按研究计划顺利进行，完成了预期目标。主要完成的研究包括两部分：1）成功制备同质化异靶点杂合纳米粒共给药递送索拉非尼（SF）与IMD-0354用于肝癌化学与TAMs相关免疫联合治疗：首先分别制备载SF（CMCS/SF-CLN）和IMD-0354的纳米粒（CMCS/M-IMD-CLN），再分别利用肿瘤微环境pH敏感材料CMCS修饰，获得同质化核壳结构纳米粒。细胞摄取和体内组织分布实验表明，CMCS/SF-CLN和CMCS/M-IMD-CLN具有相似的体内分布且能够被分别递送到不同细胞。体内抗肿瘤效率和肿瘤组织中TAMs表型分析结果表明，CMCS/SF-CLN和CMCS/M-IMD-CLN在Hepa1-6荷瘤小鼠体内具有较好的协同抗肿瘤作用和M2型TAMs极化能力。2）成功制备异靶点逐级靶向核壳结构纳米粒共递送多柔比星（DOX）与促进TAMs极化的siRNA（CDMPR）用于肝癌化学免疫联合治疗。siRNA载于内核，DOX载于外壳，CDMPR在肿瘤微环境中pH响应性解离，载DOX的外壳靶向肿瘤细胞，载siRNA的内核靶向M2型巨噬细胞。体外Transwell实验和荷瘤小鼠免疫荧光实验表明，CDMPR在肿瘤组织中表现出pH敏感降解能力，IKKβ-siRNA被精确地传递到M2型TAMs，DOX被内化到肿瘤细胞中。对M2型TAMS极化能力研究表明，CDMPR在体外和体内均可将M2型TAMs极化为M1型。在Hepa1-6荷瘤小鼠模型抗肿瘤实验表明CDMPR具更高抗肿瘤效率。本研究为基于TAMs的化学免疫联合治疗纳米载体奠定了坚实的基础；相关研究内容发表SCI文章10篇，中文核心期刊文章5篇，待发表文章1篇，其中影响因子大于5分4篇。培养博士生3名，培养硕士生5名。超额完成任务书目标。项目投入直接经费60万元，目前支出57.5316万元，各项支出与预算相符。剩余经费计划用于本项目研究后续支出。