酶响应的树枝状大分子键合物在体外肿瘤模型中的渗透行为研究  

Evaluation of in Vitro Tumor Penetration Behavior of Enzyme-responsive Dendrimer-drug Conjugate

作　　者：陈思亲 周泉[1,2] 相佳佳 周珠贤 唐建斌[1] 申有青 Si-qin Chen;Quan Zhou;Jia-jia Xiang;Zhu-xian Zhou;Jian-bin Tang;You-qing Shen(Center for Bionanoengineering,Key Laboratory of Biomass Chemical Engineering of the Ministry of Education,College of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027;Hangzhou Global Scientific and Technological Innovation Center,Zhejiang University,Hangzhou 311200)

机构地区：[1]浙江大学化学工程与生物工程学院,生物纳米工程中心生物质化工教育部重点实验室,杭州310027 [2]浙江大学杭州国际科创中心,杭州311200

出　　处：《高分子学报》2021年第5期477-488,共12页Acta Polymerica Sinica

基　　金：国家自然科学基金(基金号51833008,51522304,21704090);浙江省重点研发计划(项目号2020C01123);中国博士后科学基金(基金号2019M662059)资助项目.

摘　　要：实体肿瘤组织中固有的高渗透压、高细胞密度和乏血供等生物屏障导致纳米药物难以在肿瘤组织中浸润,从而难以渗透到肿瘤内部发挥治疗作用.为了克服上述纳米药物的被动扩散瓶颈,提升其在肿瘤组织中的渗透效果,本文设计了一种基于主动转胞吞作用来实现跨细胞传递和肿瘤渗透的纳米载药系统.利用γ-谷氨酰胺转移酶(GGT)响应的两性离子基团(BGA)修饰了以喜树碱(CPT)为核心的第四代树枝状大分子(G4/CPT),制备了一种具有精准结构和肿瘤特异性酶响应电荷反转的药物-树枝状大分子键合物(G4/CPT-BGA),其分子量为20 kDa,粒径约为5 nm,表面电势约为−2 mV.研究发现G4/CPT-BGA能够被GGT催化产生由负到正的电荷反转,并且能够水解释放出所携载的化疗药物喜树碱,从而有效杀伤肿瘤细胞.通过流式细胞术实验和激光共聚焦显微镜证明了G4/CPT-BGA能通过小窝蛋白介导的细胞内吞被肿瘤细胞摄取,随后通过高尔基体介导的细胞外排途径被释放出细胞,由此通过这种迭代不断的“内吞-外排”作用(转胞吞)实现跨细胞传递.最后,通过激光共聚焦显微镜观察G4/CPT-BGA在三维肿瘤球中的浸润效果,证明了G4/CPT-BGA能够基于主动转胞吞作用实现在肿瘤球中的高效渗透作用.因此,用GGT响应的两性离子基团对树枝状大分子的端基进行修饰,能赋予该载体主动转胞吞作用的功能,从而克服被动转运过程中的扩散障碍,为设计其他具有肿瘤增强渗透能力的抗癌纳米药物递送系统提供了可能.The limited distribution of anticancer nanodrugs remains a bottle-neck for their therapeutic effect.Several strategies,such as surface modification,photothermal activation,and microenvironment modulation,have been studied to improve the penetration of anticancer drugs in solid tumours.However,the inherent high osmotic pressure,high cell density,lack of blood supply,and other biological barriers in solid tumors make it difficult for nanomedicine to infiltrate in the tumors,thus inaccessible to the distal cells to exert an effective therapeutic effect.Therefore,the way to deliver sufficient drugs to infiltrate in a tumour is a key problem that should be solved quickly.Herein,we constructed a molecularly precise polylysine-dendrimer-drug conjugate withγ-glutamyl transpeptidase(GGT)-sensitive termini and obtained a zwitterionicγ-glutamyl functionalized dendrimer-drug conjugate,i.e.,G4/CPT-BGA.The results showed the molecular weight of G4/CPT-BGA dendrimer was 20 kDa with a small size of 5 nm and a moderate surface charge of−2 mV.The G4/CPT-BGA could undergo rapid GGTtriggered charge-reversal from zwitterionic to cationic,thereby quickly endocytosed by tumor cells,releasing the conjugated drug(CPT)to exert effective cytotoxicity.The in vitro endocytosis and exocytosis experiments showed that G4/CPT-BGA was able to traffic in cells through a caveolae-mediated pathway,then traffic out of cells via Golgi-apparatus,thus achieving active transcytosis for transcellular delivery.The tumor penetration ability of G4/CPT-BGA was investigated using three-dimensional multicellular spheroids,which showed G4/CPT-BGA could evenly distribute throughout the spheroids via transcytosis-mediated active tumour infiltration.Therefore,a simple modification of the cationic dendrimer with GGT responsive zwitterionicγ-glutamine enables the dendrimer to actively transcytosis across cells,thereby avoiding the paracellular diffusion obstacles,which may also be applicable for designing anticancer nanomedicine systems with enhanced penetration

关 键 词：纳米药物 树枝状大分子 肿瘤渗透 主动转胞吞作用 γ-谷氨酰胺转移酶 

分 类 号：R943[医药卫生—药剂学]