氧化响应的甲壳素基靶向共输送体系的化疗与免疫治疗的协同抗肝癌作用

Because of the very weak immunogenicity of most tumors, many hepatocellular carcinoma patients will still have tumor metastasis and their conditions become worsening after chemotherapy. In this proposal, we will design and synthesize a series of ROS-responsive chitin nanogels (GRC), whose surfaces bonded with tumor targeting peptide of GRP78P, as targeting co-delivery system of doxorubicin (DOX) and IL-12 for chemotherapy/immunotherapy synergistic anti-hepatoma of inhibiting tumor growth and metastasis. By using 13C-NMR, 1H-NMR, Fourier-transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), particle size and zeta analyzer, fluorescence spectrometer, multifunctional microplate, confocal laser scanning microscope and flow cytometry, the influence of grafting ratio of tumor targeting peptide, the density of ROS-responsive bonds, and encapsulation ratio of DOX/IL-12 on the morphology, stability, drug loading content, in vitro release behaviors and anti-tumor effect of the co-delivery system will be studied. Then, according to the “structure-activity” relationship, combined with western blot, HE staining, immunohistochemistry and immunofluorescence to clarify the synergistic anti-hepatoma of GRC/DOX/IL-12 by animal experiments. This work may provide new ideas for anti-hepatoma therapies, through building novel targeting chitin-based co-delivery system of drug and cytokine.

因为肿瘤的免疫原性很弱，很多肝癌患者经过化疗后仍然会发生肿瘤转移、病情恶化。为此，本项目拟设计合成一系列表面含肿瘤靶向多肽GRP78P、对肿瘤细胞内高丰度ROS具有响应性的甲壳素基纳米凝胶(GRC)，作为盐酸表柔比星(DOX)和细胞因子IL-12的共输送载体，用于抑制肝肿瘤生长和转移的化学与免疫的协同治疗。利用核磁氢/碳谱、红外、透射电镜、粒径电位仪、荧光光谱仪、多功能酶标仪、激光共聚焦、流式细胞仪等手段，研究共输送体系中靶向肽的接枝率、ROS响应键的密度和DOX/IL-12包封比例的不同，对其形貌、稳定性、载药量、体外释药行为和抗肿瘤效果的影响，获得其“构-效”关系的定量结果；再深入动物体内，结合Western blot、HE染色、免疫组化和免疫荧光等技术，弄清GRC/DOX/IL-12的协同抗肝癌作用。本项目拟建立的新型甲壳素基药物/细胞因子的共输送体系，可为肝癌的治疗提供新思路。

基于临床治疗中的化疗药物存在毒副作用大、溶解性差、利用率低和选择性差等问题，并且，患者经过化疗后，药效迅速下降、产生耐药性，免疫功能又遭到严重破坏、作用丧失殆尽，容易发生肿瘤转移，病情恶化后直接危害生命。因此，本项目通过简单的方法制备一系列生物相容性好，表面含肿瘤靶向多肽GRP78P、对肿瘤细胞内低pH和高丰度H2O2具有响应性的甲壳素基纳米凝胶(GRC)，作为盐酸表柔比星(DOX)，细胞因子IL-12和α-生育酚(α-TOS)的共输送载体，研究其抑制肿瘤生长的化学与免疫协同治疗效果。利用核磁氢谱、红外表征GRC的结构，通过对特征峰地观察，证明成功制备了甲壳素基纳米凝胶。透射电镜和粒径电位仪的测试结果说明GRC-α-TOS/DOX/IL-12为结构规整的类球形，平均粒径为275 nm，电位为+6.8 mV，包封IL-12，DOX和α-TOS的载药量分别为2.04 × 10-4, 4.01 × 10-2和7.12 × 10-2。α-TOS能增加细胞内H2O2的浓度，提高纳米凝胶的氧化释放能力。GRC对4T1细胞和巨噬细胞RAW264.7均展现低细胞毒性。同条件下，GRC-α-TOS/DOX/IL-12对4T1细胞的毒性明显高于GRC-α-TOS/DOX和GRC-α-TOS/IL-12，在与细胞共孵育4 h后，纳米凝胶释放的DOX和IL-12分布于细胞质和细胞核中。并且，GRC-α-TOS/DOX/IL-12对4T1细胞构建的BALB/c小鼠皮下肿瘤模型具有最佳的抗肿瘤效果，这归因于GRC-α-TOS/DOX/IL-12的肿瘤靶向性，定位于肿瘤的pH和H2O2的刺激响应释放性，促进巨噬细胞向抑制肿瘤生长的M1型极化以及提高的肿瘤组织免疫力。本项目建立的新型甲壳素基药物/细胞因子的共输送体系，可为癌症的治疗提供新思路。