针对中晚期癌症的光热免疫治疗纳米系统的构建与作用机制研究

It is difficulty to treat advanced cancer due to the spread and metastasis of cancer cells. In this work, we establish a multifunctional nanoparticles by encapsulating hollow gold nanoparticles (HAuNS, with strong near-infrared photothermal conversion ability) and peptide drug (AUNP12, with the function of blocking PD-1/PD-L1 pathway between the cancer cells and immune cells) into biodegradable polylactic acid-glycolic acid (PLGA) nanoparticles. The surface of the nanoparticles is further conjugated with Apatmer AS1411 having specific binding ability for Nucleolin. Then the nanoparticles shows multifunctions of tumor-specific targeting delivery, controlled release of the peptide drug, and immune recognition to cancer cells via mediation of the NIR light. NIR mediated photothermal therapy often plays the role of short-term treatment, while a long period treatment is needed for immunotherapy in order to get a good efficacy. After systemic administration, the nanoparticles can selectively accumulate in tumor sites, and presents a short-term photothermal therapy under NIR light irradiation to eliminate the primary tumor rapidly, blocking the spread of cancer cells from the primary tumor. At the same time, the irradiation results in the triggered release of some AUNP12 from the nanoparticles, inducing the rapid increase of AUNP12 concentration in vivo. Subsequently, the high concentration of AUNP12 can be kept by its constant release due to the gradual biodegradation of PLGA, which induces a long-term immunotherapy to kill the diffused and metastasis cancer cells. The combined photothermal immunotherapy will be an efficient approach to treat advanced cancer.

针对中晚期癌症易发生以主体肿瘤为中心的癌细胞扩散转移而导致难以有效治愈难题，拟将具有强近红外光热转化性能的中空金纳米粒，和具有阻断癌细胞与免疫细胞之间PD-1/PD-L1通路能力的多肽药物AUNP12，共同包封于生物可降解聚PLGA纳米粒中；用能与Nucleolin特异性结合的Apatmer AS1411修饰表面，构建一种新型特异性靶向肿瘤的、光热治疗、药物控制释放、以及重启癌细胞免疫识别而行使免疫治疗的多功能纳米粒。依据光热治疗短期性以及免疫治疗长期性特点，该纳米粒经系统给药后，能选择性累积到肿瘤主体；在近红外光介导下，发挥短期光热治疗，迅速消除肿瘤主体，切断癌细胞扩散源，同时触发部分药物释放，迅速提升其体内浓度，随后借助材料生物降解性，恒速释放药物，较长时间维持有效浓度，实现长效免疫治疗，有效消灭弥散和远端扩散癌细胞群。两者治疗功效长短结合，协同起效，有望显著提升中晚期癌症治愈效果。

中晚期癌症的一个主要特点是癌症细胞从原发部位向周围组织发生浸润弥散，或通过循环系统等向远端组织发生扩散转移。这导致我们无法对癌症病患实施有效的手术治疗；而放化疗对机体的毒副作用与诱导肿瘤产生的耐药性，也使得传统治疗手段难以发挥预期的效果。因此，探索一种针对中晚期癌症特点的有效治疗手段是非常有必要。我们选用了具有较强的表面等离子共振特性的中空金纳米粒（Hollow gold nanospheres, HAuNS）利用HAuNS的近红外光热转换效应，实现对主体肿瘤绝大部分细胞的光热消融（Photothermal ablation，PTA）。同时应用一种抗PD-1受体的小分子多肽（Anti-PD-1 peptide，APP）阻断T淋巴细胞表面的PD-1受体减少其免疫逃逸现象。我们将HAuNS和APP共同包载于聚乳酸-羟基乙酸共聚物( (poly(lactic-co-glycolic acid, PLGA)中。通过对HAuNS和APP进行疏水化修饰，采用单溶剂乳化蒸发法制备得到共载HAuNS和APP的PLGA纳米粒（HAuNS and APP co-loaded nanoparticles，AA@PN）。考察了纳米粒体中 APP 的体外释放行为，发现在波段为808 nm（Nearinfrared light, NIR）的介导下，实现了纳米粒中的APP的脉冲式释放。瘤内注射该纳米粒并施予 NIR 照射后，HAuNS可发挥PTA治疗作用，迅速杀死小鼠体内绝大部分原发肿瘤癌细胞，同时触发纳米粒中的部分多肽药物的释放，迅速提高体内药物浓度水平，随后借助于PLGA材料的缓慢降解的特点，恒速释放药物，能够在较长时间内持续维持小鼠体内APP有效浓度，实现长效免疫检查点阻断效应，重启机体的固有免疫机能，有效的清除主体肿瘤中残存的少量癌细胞和远端肿瘤细胞。进一步引入Toll样受体激动剂CpG佐剂的使用。该治疗策略结合了光热，免疫佐剂和PD-1抑制剂的联合使用。通过建立小鼠结肠癌CT26 和乳腺癌4T1的肺转移模型，考察了AA@PN在CpG的辅助作用下，有效的抑制CT26及4T1肺转移的治疗效果。同时通过建立肿瘤术后复发模型和术后再接瘤模型，发现该纳米粒具有肿瘤疫苗样作用，可以诱导机体产生更多长效记忆性免疫细胞，有效的抑制肿瘤的术后复发。

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