实体肿瘤作为恶性肿瘤中最重要的组成部分，由于肿瘤的异质性、复杂的免疫抑制微环境和致密的物理屏障导致目前其治疗仍面临诸多挑战。免疫检查点阻断治疗可通过激活抗肿瘤免疫应答杀伤肿瘤，是肿瘤免疫治疗领域的重大突破，但其在患者中的响应率较低，严重限制了其临床应用。将抗肿瘤免疫治疗与放疗、化疗、光杀伤治疗等其他疗法相结合，可以产生协同作用，改善抗肿瘤治疗效果，具有良好的临床应用潜力。因此，本课题基于“实体肿瘤渗透增强和重塑肿瘤微环境”的设计策略，构建光激活型多功能纳米药物，实现光杀伤治疗、化疗和肿瘤免疫治疗协同抗肿瘤。

本课题以人血清白蛋白（Human serum albumin, HSA）为载体，设计构建光激活型光敏剂吲哚菁绿（Indocyanine Green, ICG）、化疗药物阿霉素（Doxorubicin，DOX）、血管紧张素受体阻滞剂缬沙坦（Valsartan，VAL）共负载多功能纳米药物（VAL&DOX&ICG@HSA NPs），经细胞膜穿透肽12聚-L-精氨酸（Poly-L-arginine, R12）和聚乙二醇亲水链（Polyethylene glycol，PEG）修饰后得到纳米药物PEG-VAL&DOX&ICG@R12/HSA NPs。在近红外激光的照射下，PEG-VAL&DOX&ICG@R12/HSA NPs表面的PEG亲水层断裂，释放出VAL，可下调肿瘤相关成纤维细胞（Cancer-associated fibroblasts，CAFs）的a平滑肌肌动蛋白(a-smooth muscle actin，a-SMA)表达水平，使活化的CAFs逆转为静息状态；同时，纳米药物表面暴露出的细胞膜穿透肽R12可促进其通过转胞吞途径深入实体肿瘤组织，从而进一步增强实体肿瘤渗透。体外细胞实验研究结果表明，PEG-VAL&DOX&ICG@R12/HSA NPs具有良好的光化疗协同杀伤作用，可以有效的杀伤肿瘤细胞；多细胞球体模型研究结果证实，在近红外激光激活作用下，PEG-VAL&DOX&ICG@R12/HSA NPs通过转胞吞作用可实现多细胞球体内的深层渗透。荷瘤小鼠体内抗肿瘤实验结果表明，PEG-VAL&DOX&ICG@R12/HSA NPs介导的光-化-免疫联合治疗策略可诱导肿瘤细胞发生免疫原性细胞死亡，促进肿瘤引流淋巴结中的树突状细胞成熟，下调CAFs的a-SMA表达水平，激活T细胞介导的抗肿瘤免疫反应，有效抑制肿瘤生长。此外，PEG-VAL&DOX&ICG@R12/HSA NPs联用PD-1免疫检查点阻断治疗，可诱导小鼠产生免疫记忆效应，从而有效地防止荷瘤小鼠局部肿瘤的转移和复发。

综上所述，本研究成功构建了一种光激活型精氨酸多肽修饰纳米药物，实现了实体肿瘤渗透增强和光杀伤治疗、化疗和肿瘤免疫治疗的协同抗肿瘤效果：首先，纳米药物表面修饰的细胞膜穿透肽R12可通过转胞吞途径介导纳米药物的实体肿瘤渗透增强；光杀伤-化疗杀伤协同作用可引起肿瘤细胞免疫原性死亡，启动机体抗肿瘤免疫应答；可下调肿瘤相关成纤维细胞的a-平滑肌肌动蛋白表达水平，重塑肿瘤免疫微环境，激活T细胞介导的抗肿瘤免疫反应，有效抑制肿瘤生长。

Although there have been significant advances in the management of primary malignancies, solid tumors are frequently metastatic at presentation and often fail to respond to conventional cancer therapy. Poor permeation of therapeutic agents and “immune-cold” tumor microenvironment in solid tumors are the two major challenges that lead to the inefficient therapeutic efficacy for cancer treatment. Thus, developing smart multifunctional drug-delivery strategies to simultaneously achieve deep tumor penetration and remodulate the immunosuppressive tumor microenvironment will be hopeful for effectively improving the therapeutic index of solid tumor treatment.

Here, a light-activated penetrable nanoplatform (PEG-VAL&DOX&ICG@ R12/HSA NPs) for co-delivery of the chemotherapeutic drug doxorubicin (DOX), the photosensitizer agent indocyanine green (ICG) and the angiotensin receptor blockers valsartan (VAL) was developed to achieve deep drug penentration and synergistic photo-chemo-immunotherapy. Studies showed that when exposed to the first wave of near infrared laser irradiation, the PEG hydrophilic layer as a “inert” surface could detach from the nanoplatform, exposing the arginine-rich peptide modified-cores that can facilitate deep drug penetration into solid tumor tissues via a transcytosis pathway. In vitro cellular studies showed that all nanoparticles treatment exhibited an improved cell inhibitory efficiency under laser irradiation as compared in the absence of irradiation, demonstrating that the combination of phototherapy and chemotherapy leaded to the optimized therapeutic efficiency. We further evaluated the penetration of the prepared nanoparticles using multicellular spheroids as an in vitro three-dimensional (3D) model mimicking 4T1 tumor tissue. The result suggested that the deep tumor penetration of PEG-VAL&DOX&ICG@R12/HSA NPs under laser irradiation occurred mainly through a transcytosis process. As expected, in 4T1 tumor-bearing mice, PEG-VAL&DOX&ICG@R12/HSA NPs treatment could effectively induce immunogenic cell death of tumor cells,  facilitate DCs maturation in the draing lymphnodes and down-regulate a-smooth muscle actin expression level of cancer-associated fibroblasts cells (CAFs) in tumors, thus promoting T cell-mediated antitumor immune responses to inhibit the growth of tumors. Moreover, combination therapy by PEG-VAL&DOX&ICG@R12/HSA NPs and anti-PD-1 monoclonal antibody can elicit the memory immune response to prevent tumor recurrence and metastasis in vivo. 

The presented delivery system bears several features as follows. First, the PEG derivatives used as a “inert” surface of the nanoparticles can mask the cell-penetrating functionality of R12 in the circulation, which will gradually dissociate from the nanoparticles in tumor sites under the first wave of laser irradiation, and exposing the R12-rich core to enhance penetrable ability of the nanoparticles. Second, PEG-VAL&DOX&ICG@R12/HSA NPs can exhibit the light-inducible VAL release behavior in tumor sites, while avoiding systemic adverse effects and reprogramming CAFs. Third, co-encapsulation of three different drugs (DOX, ICG and VAL) into the one nanocarrier can be achieved for the synergistical photo-chemo-immunotherapy. This work provides a promising delivery strategy to overcome the current limitations of nanomedicine for achieving more effective therapeutic index of solid tumor treatment.