目的：肿瘤治疗对人类来说依然是巨大的挑战。相对于直接清除或杀伤肿瘤细胞的手术、化疗和放疗等传统的治疗方式，通过激活机体自身的免疫系统来杀伤肿瘤细胞的免疫疗法开启了新的时代。其中，应用免疫检查点抑制剂PD-1单抗和CTLA-4单抗解除肿瘤免疫耐受进而激活机体的免疫系统已是一种常规的肿瘤免疫治疗手段，而且将两者联合使用在多种肿瘤治疗中可以产生叠加或协同效应。相比将两者分别给药，设计将两个免疫检查点抑制剂均同时作用于同一个细胞，观察能否产生更大的协同抗肿瘤免疫效应。本研究以两亲性高分子材料PLGA-S-S-PEG为自组装基元，嵌入阳离子脂质DOTAP制备纳米颗粒递送载体，在颗粒表面负载CTLA-4 Aptamer和PD-1 siRNA构建还原响应性杂化球形核苷酸纳米体系，并探索CTLA-4 和 PD-1 两免疫检查点抑制剂联合作用于同一个细胞是否产生更强的免疫协同作用及可能的作用机制。

方法：首先合成两亲性高分子PLGA-S-S-PEG并对其进行表征，包括凝胶渗透色谱（GPC）测合成产物的分子量，DTT测氧化还原性。之后采用薄膜水化超声法制备PLGA-S-S-PEG/DOTAP纳米颗粒（PDN），通过静电吸附作用结合PD-1 siRNA与CTLA-4 Aptamer，并对制备的载药纳米颗粒进行表征，包括粒径、Zeta电位、形态、载药量及稳定性。体外观察T细胞对CTLA-4 Aptamer和PD-1 siRNA的摄取量以及它们对T细胞的增殖作用。体内建立小鼠MC38结肠癌肿瘤模型，给药后通过酶联免疫吸附实验（ELISA）检测血清中的TNF-α等细胞因子的含量；采用流式细胞仪检测外周血、脾脏、肿瘤侧淋巴结、肿瘤局部CD3⁺CD4⁺和CD3⁺CD8⁺ T淋巴细胞的比例变化及对CD4 Teff/Treg和CD8 Teff/Treg的影响；并最终通过监测肿瘤体积和肿瘤重量观察药物对小鼠肿瘤的抑制效果。

结果：成功合成了两亲性高分子PLGA-S-S-PEG，并以此为基元制备了负载CTLA-4 Aptamer和PD-1 siRNA的杂化球形核苷酸纳米颗粒。纳米颗粒的表征结果显示空白纳米颗粒PDN的粒径为76.03±6.2 nm，Zeta电位为39.8±0.4 mV，载药纳米颗粒PDNp、PDNc、PDNpc的粒径分别为136.1±7.0 nm、149.5±5.4 nm、166.3±5.8 nm，Zeta电位分别为23.8±0.8 mV、22.4±0.6 mV，21.6±0.4 mV。所制得的纳米粒粒径均一，稳定性良好，TEM显示纳米粒呈相对均匀的球形结构。体外实验证实该纳米颗粒有效地促进了T细胞对siRNA的摄取，纳米颗粒表面负载CTLA-4 Aptamer可增加T细胞对siRNA的摄取量；T细胞增殖实验显示纳米载体负载CTLA-4 Aptamer和/或PD-1 siRNA后，可显著促进T细胞的增殖，两者联用的促T细胞增殖效果强于单独一种抑制剂的效果，且两者负载于同一个纳米球上作用于同一细胞后产生的T细胞增殖强度，高于两者分别负载于不同纳米球上联合应用产生的增殖效果。体内实验进一步证实：载药纳米粒组的肿瘤体积和重量明显小于PBS组，两检查点抑制剂联用的抑瘤效果增强，且两者负载于同一个纳米球作用于同一细胞后产生的抑瘤效果好于负载于不同纳米球联合治疗产生的抑瘤效果；进一步的流式结果证实：在提高外周血、脾脏、肿瘤侧淋巴结和肿瘤浸润CD3⁺CD4⁺和CD3⁺CD8⁺ T淋巴细胞所占比例及提高CD4 Teff/Treg和CD8 Teff/Treg比值方面，两检查点抑制剂联用都优于PD-1 siRNA和CTLA-4 Aptamer单药治疗组，并且两者负载在同一杂化球形核苷酸纳米颗粒产生的作用显著优于两检查点抑制剂分别给药联合治疗组；血清中细胞因子TNF-α的分泌水平也显示相似的趋势。

结论：成功制备了负载PD-1 siRNA和CTLA-4 Aptamer的PLGA-S-S-PEG/DOTAP杂化球形核苷酸纳米颗粒。该纳米颗粒在体外可显著刺激T细胞的增殖。将其通过尾静脉注入小鼠体内后，进一步证实该纳米颗粒可强烈刺激小鼠外周血、脾脏、肿瘤侧淋巴结以及肿瘤组织CD3⁺ CD4⁺ T细胞和CD3⁺ CD8⁺ T细胞的增殖，产生强抗肿瘤效应。进一步的机制实验分析，引起该协同效应产生的至少部分原因是由于该纳米颗粒在CTLA-4 Aptamer介导下可靶向进入调节性T细胞（Treg），同时阻断CTLA-4 和PD-1抑制途径，引起级联瀑布反应，大大减弱Treg的免疫抑制作用，进而产生协同抗肿瘤免疫反应。

ive: the treatment of cancer is still a great challenge for human beings. compared with traditional therapies such as surgery, chemotherapy and radiotherapy which directly clear or kill tumor cells, immunotherapy which mobilizes the immune system to kill tumor cells has ushered in a new era. the application of checkpoint inhibitors especially anti-pd-1 monoclonal antibody and anti-ctla-4 monoclonal antibody to release tumor immune tolerance and activate immune system has been an important means of tumor treatment and the combined application of the two can produce superposed or synergistic effect in a variety of tumor treatments. making the two immune checkpoints act on one same cell was designed to see if it can induce more synergistic anti-tumor effect compared to administration of them respectively. in this study, nanocarrier was prepared via self-assembly of the unit plga-s-s-peg and insertion of cationic lipid dotap. ctla-4 aptamer and pd-1 sirna were adsorbed on the surface of the nanocarrier and the hybrid spherical nucleotide nano delivery system was constructed. whether the combination use of the two immune checkpoints acting on one same cell could produce stronger synergistic anti-tumor effect was explored as well as the possible mechanism. 

methods: the polymer plga-s-s-peg was synthesized and characterized, including gel permeation chromatography(gpc) testing the weight average molecular weight of the reaction product and dtt testing redox activity. after that, plga-s-s-peg/dotap nanoparticle carrier was prepared by thin film hydration ultrasound method. pd-1 sirna and ctla-4 apt were absorbed onto the nanocarrier via electrostatic adherence. and the prepared drug-loaded nanoparticles were  characterized including particle size, zeta potential, morphology, drug loading and stability. the uptake of ctla-4 aptamer and pd-1 sirna by t cells and the proliferation induced by ctla -4 aptamer and pd -1 sirna were observed in vitro. mouse mc38 colon cancer tumor model was established in vivo. the level of serum cytokine tnf-α was detected by enzyme-linked immunosorbent assay（elisa）. the percentages of cd3⁺cd4⁺ and cd3⁺cd8⁺ t cells in the peripheral blood, spleen, tumor draining lymph node and tumor were measured by flow cytometry and the ratio of cd4 teff/treg and cd8 teff/treg were calculated. observe tumor inhibition effects by the change of tumor volume and the final tumor weight.

results: plga-s-s-peg was successfully synthesized and drug-loaded nanoparticles were successfully prepared. the characterization results showed that the particle size of blank nanoparticles was 76.03±6.2 nm and the zeta potential was 39.8±0.4 mv. for drug-loaded nanoparticles, the size of pdnp, pdnc and pdnpc were 136.1±7.0 nm, 149.5±5.4 nm and 166.3±5.8 nm, respectively and the zeta potential were 23.8±0.8 mv, 22.4±0.6 mv and 21.6±0.4 mv, respectively. the stability of the nanoparticles was good and tem figure showed the nanoparticles were spherical in structure. in vitro experiments, the nanocarrier effectively promoted the uptake of sirna by t cells and ctla-4 apt played a targeted role, further promoting the uptake of sirna by t cells. t cell proliferation test exhibited drugs when they were loaded onto nanocarrier could efficiently enhance t cell proliferation. when pd-1 sirna and ctla-4 aptamer were used together, the effect was better. also, the proliferation effect induced by the two loading on one nanosphere to a greater extent functioning on one same cell were superior to the two loading on different nanospheres. the results of in vivo experiments showed that the tumor volume and weight of the drug-loaded nanoparticles group were significantly smaller than that of the pbs group. pd-1 and ctla-4 combination blockade induced more efficient anti-tumor effect and the anti-tumor effect induced by nanoparticles that co-loaded pd-1 sirna and ctla-4 aptamer was better than the nanoparticles that pd-1 sirna and ctla-4 loaded separately. in terms of improving the proportion of cd3⁺cd4⁺ and cd3⁺cd8⁺ t lymphocytes and the value of cd4 teff/treg and cd8 teff/treg in peripheral blood , spleen, tumor-draining lymph node and tumor, the combined use of two checkpoint inhibitors that mostly acted on one same cell behaved better than that mostly acted on different cells, but both of them had a better performance than the single drug treatment group. the level of tnf-α showed a similar trend.

conclusion: pd-1 sirna and ctla-4 aptamer co-loaded plga-s-s-peg/dotap hybrid spherical nucleotide nanoparticles were successfully prepared. the drug-loaded nanoparticles could efficiently enhance t cell proliferation in vitro. moreover, they could strongly burst the proliferation of cd3⁺cd4⁺ and cd3⁺cd8⁺ t cells in the peripheral blood, spleen, tumor draining lymph node and tumor and inhibit tumor growth. at least part of the reason for this synergistic effect was that the nanoparticles could be targeted into treg under the mediation of ctla-4 aptamer, and at the same time block the ctla-4 and pd-1 inhibition pathways, leading to cascade reaction, which significantly weakened the immunosuppressive effect of treg and thus produced a synergistic anti-tumor immune response.