“隐形”的癌细胞、线粒体逐级靶向纳米载药系统用于治疗耐药胰腺癌的研究

Chemoresistance is the major impediment for treating pancreatic cancer (PC). Targeting anti-cancer drugs to mitochondria may overcome the resistance of cancer. The mitochondrion possesses a transmembrane potential of up to 200 mV (negative inside), which make lysine highly targeting mitochondria. However, lysine can not work as carrier because of its rapidly elimination in vivo..Herb-derived compound triptolide (TP) can inhibit proliferation of chemoresistant pancreatic cancer cell lines through multiple mechanisms including the loss of mitochondrial membrane potential. However, its hydrophobicity and side effect hindered its translation to the clinic. Nano-carrier can overcome the foregoing dilemmas and targeting TP to tumor tissue based on the rationale of enhanced permeability and retention (EPR) effect..Recently,the applicants have established the platform for aptamer-based nanocarrier for targeted therapy and successfully achieved targeting osteoblast for osteogenic siRNA. In addition, the applicants developed nucleolin-specific aptamer-poly(ethylene glycol)–block–poly(D,L-lactide) (PEG-PDLLA) loading TP nanomicelle（nucleolin overexpressed on the surface of PC cells）which could facilitate TP targeting PC cells..Nevertheless, nanocarrier are synthetic material, which can be recognized by the innate immune system and rapidly cleared from the bloodstream leading to limited therapeutic effect. Even the attachment of targeting ligands to a nanopaticle does not alter its biodistribution, but only increases its internalization by target cells..Carrier erythrocytes (RBC), as endogenous cells with little or no antigenic response, can protect encapsulated cargos while allowing them to circulate in the blood stream for a long time and deliver drugs to a specific site in the body..Taken together, we will contrive a dual-target nano-micelle loading TP (D-TP-PM) using AS1411-PEG-PDLLA, Trilysine-PEG-PDLLA and MPEG-PDLLA polymeric material, then it was encapsulated in RBC (RBC-D-TP-PM). We have the following hypothesis that RBC-D-TP-PM could escape from MPS uptake and target to tumor sites through EPR effect, then AS1411 and trilysine stepwise mediation and enter the tumor, the mitochondria for releasing TP to work..To test the hypothesis, the following specific aims should be achieved. (1) To synthesize and characterize the AS1411-PEG-PDLLA and Trilysine-PEG-PDLLA copolymer. (2) To prepare and characterize D-TP-PM and RBC-D-TP-PM: (3) To validate the tissues, cellular and organelle distribution of D-TP-PM and RBC-D-TP-PM. .If the hypothesis could be supported, it would serve as the basis to further test the efficiency and safety of RBC-D-TP-PM in inhibiting chemoresistance.

癌细胞胞膜和胞内的多种抗药机制是导致胰腺癌化疗失败的重要原因。现有递药系统只能克服细胞膜介导的抗药机制。本课题将研制逐级靶向癌细胞内线粒体、载有增强线粒体通透性的雷甲素纳米递药系统。该递药系统表面被红细胞囊体包裹，内层带有癌细胞、线粒体逐级靶向的配体。红细胞囊体可使载药系统有效避开巨噬细胞，进入肿瘤组织，随着红细胞膜脱落，其癌细胞靶向配体使纳米药物高效靶向癌细胞并进入胞内，在癌细胞的溶酶体中，线粒体定位载体起效，靶向进入线粒体，而后释放雷甲素，通过雷甲素改变线粒体功能诱导癌细胞凋亡。本项目将系统研究该载药系统的制备、隐蔽性，癌细胞、线粒体逐级靶向定位过程及其体内外抗癌性能，以得到高效纳米递药系统用于肿瘤的化疗，为纳米载药系统的设计及功能化、克服肿瘤抗药性等提供新的策略。

癌细胞胞膜和胞内的多种抗药机制是导致胰腺癌化疗失败的重要原因。现有递药系统只能克服细胞膜介导的抗药机制。雷甲素Triptolide, TP，传统中草药雷公藤的主要活性成份之一，可通过多重机制改变线粒体功能从而诱导癌细胞凋亡，体外研究显示其抑制耐药胰腺癌效果优于吉西他滨。本课题确证了雷公藤甲素体内的显著抗肿瘤效果，同时所构建的以核仁素（胰腺癌细胞表面高表达)特异性适配子(AS1411)为“靶头”，雷甲素为“火药”的特异靶向胰腺癌细胞的“智能”纳米药，通过体外细胞选择性、体内活体成像技术，确证了以AS1411适配子介导的主动靶向纳米药（AS-PPT）对癌细胞具有靶向特异性，主动靶向AS-PPT与被动靶向TP-PM在抑瘤效果上，从生存期、肿瘤体积上有明显优势，进一步验证了其在动物肿瘤模型上的抗癌效果。项目资助发表SCI论文7篇，待发表两篇，培养研究生5名，其中3名已经取得硕士学位，2名在读。项目投入经费20万元，支出19.9939万元，各项支出基本与预算相符。