癌症作为目前威胁人类公共健康的疾病，是亟待解决的难题，传统的治疗手段对晚期癌症的治疗的效果不佳且给患者带来剧烈痛苦。为了弥补传统癌症治疗方法的局限性，出现了新兴的治疗策略，如免疫疗法、基因疗法和光热疗法。但是单一疗法疗效差，存在难以有效清除肿瘤以及易发生转移的问题。因此，联合治疗成为一种趋势，纳米技术在癌症治疗中的应用受到广泛关注，纳米平台将多种抗癌药物结合起来从而变单一疗法为多重疗法，开启了癌症治疗的新纪元，在很大程度上提高了治疗效果，同时减少了不良反应。

光热疗法可以通过热消融抑制原发肿瘤，但是通常不能完全根除，也不能很好地预防转移和复发；光动力疗法通过产生活性氧来破坏肿瘤组织，但是由于肿瘤微环境中低氧环境以及ROS产生效率低不能有效地杀伤肿瘤；免疫疗法虽然能够克服肿瘤转移和复发，但是对实体瘤尤其体积大的原发肿瘤无法有效治疗。因此光热-光动力联合免疫疗法越来越受到关注，一方面通过光热疗法消除原发肿瘤和通过光动力疗法减少肿瘤缺氧的微环境，另一方面通过免疫疗法抑制肿瘤的复发和转移。在各种免疫疗法中，肿瘤疫苗通过抗原提呈细胞将肿瘤抗原递送到淋巴结中，激活T细胞引发细胞免疫和体液免疫反应，既可以抑制原发性肿瘤，又可以通过诱导机体产生长期记忆效果来预防肿瘤复发和转移，正成为研究的热点。然而常规的肿瘤疫苗是将抗原以不同形式导入机体内以诱导免疫应答，在肿瘤抗原特异性差、免疫原性弱的缺点，对于引起持续高强度的免疫效应有一定的难度。如果能在肿瘤部位产生肿瘤特异性抗原后，采取策略实现原位自组装肿瘤特异性疫苗，以促进抗原提呈细胞对抗原的摄取和加工处理，并有效呈递给T细胞，就能巧妙避开抗原差异性的难题，并通过协同免疫佐剂增强其免疫原性进一步启动增强免疫反应而实现肿瘤的高效个性化治疗。

鉴于此，我们构建了包载光敏剂ICG和IDO抑制剂NLG919的聚合物囊泡NIPS，以及包载TLR7/8激动剂IMQ、TLR4激动剂MPLA、并在外壳修饰阳离子脂质体DOTAP、马来酰亚胺功能化磷脂DSPE-PEG2K-Mal、甘露糖靶向磷脂DSPE-PEG5K-Mannose的聚合物囊泡mal-dot-PS。一方面通过聚合物囊泡NIPS在808 nm近红外激光器照射下产生光热效应杀伤肿瘤细胞并产生肿瘤相关抗原，并用IDO抑制剂NLG919抑制IDO的酶活性，从而抑制免疫微环境；另一方面通过聚合物囊泡mal-dot-PS上的捕获基团DOTAP和Mal捕获肿瘤相关抗原形成个性化疫苗，将抗原和2种免疫激动剂递送到DCs，有效促进DCs成熟、抗原交叉提呈及淋巴结迁移，激活T细胞而促进机体的细胞免疫和体液免疫反应，实现光热-光动力联合免疫疗法抑制肿瘤的生长、复发和转移，达到良好的抗肿瘤效应。具体研究内容如下：

多功能聚合物囊泡NIPS和mal-dot-PS的构建及相关理化性质表征：我们用PCL-PEG-PCL作为材料，用薄膜水化超声法分别将光敏剂ICG和IDO抑制剂NLG919包载在聚合物囊泡疏水层，将NaHCO₃包载在亲水内腔形成NIPS；同时将TLR7/8激动剂IMQ包载在聚合物囊泡的疏水层，将TLR4激动剂MPLA、DOTAP、Mal和Man嵌入磷脂层以及将NaHCO₃包载在亲水内腔形成mal-dot-PS。并对两者粒径及其分布、zeta电位和药物包封率进行了表征，也对NIPS的体内外光热效果、体内外细胞活性氧产生、体外细胞摄取、细胞毒、细胞凋亡和死活以及ATP的分泌、体内肿瘤凋亡情况进行了研究。结果表明该多功能聚合物囊泡稳定性较好，ICG、NLG919和IMQ包封率和载药量较高，也具备较好的pH响应性和光热性能以及良好的释药性能。同时在808 nm激光器照射下，聚合物囊泡NIPS具有较高的细胞毒性、ATP分泌、细胞摄取和产生活性氧的能力。体内外光热研究结果表明聚合物囊泡NIPS具有较好的光热性能，能长时间的驻留在肿瘤组织内，可通过PTT达到很好的杀伤肿瘤细胞的作用。

可在体自组装个性化疫苗的聚合物囊泡用于光热-光动力联合免疫抗肿瘤的效果及机制研究：探究了聚合物囊泡mal-dot-PS对IDO酶的作用、体外抗原捕获能力、对BMDCs的促成熟作用、及对抗原的交叉提呈能力；体内研究包括NIPS + Laser@mal-dot-PS对黑色素瘤的抑制、复发、转移情况以及体内免疫机制如促DCs成熟和抗原交叉提呈能力、CD4⁺和CD8⁺ T细胞产生、巨噬细胞极化、T淋巴细胞激活和增殖、IFN-γ和TNF-α分泌情况以及记忆效果情况进行了研究。体外研究表明mal-dot-PS对DCs细胞具有较好的生物相容性，可抑制IDO的酶活性，具有较好的抗原捕获能力，并在捕获抗原之后能够有效的促DCs成熟和抗原交叉提呈。体内实验表明在808 nm的激光器照射下，NIPS能够很好的发挥光热效果，杀伤肿瘤并产生肿瘤相关抗原，被mal-dot-PS捕获后自组装成共递送抗原和免疫激动剂的肿瘤个性化疫苗，实现DCs的高效摄取并促进DCs成熟、活化和淋巴结迁移，引起机体产生抗原特异性的CD8⁺和CD4⁺ T细胞免疫反应，分泌较多的细胞因子TNF-α和IFN-γ，同时导致M2型巨噬细胞减少和肿瘤的Tregs细胞减少，并产生良好的记忆效果和抑制肺转移能力。

Cancer, as a disease that threatens human public health, is an urgent problem to be solved. Traditional treatment methods are ineffective on advanced cancer and bring severe pain to patients.Therefore, novel treatment strategies such as immunotherapy, gene therapy, and photothermal therapy, have emerged to compensate for the limitations of traditional cancer treatments. However, the efficacy of monotherapy is poor, and it is difficult to effectively remove the primary tumor and prevent tumor metastases. Therefore, combination therapy has become a trend, and the application of nanotechnology in cancer treatment has received extensive attention. Nanoplatforms can combine multiple anticancer drugs to change monotherapy into combination therapy, opening a new era for cancer treatment. The combination theray can extensively improve anticancer therapeutic effect and reduce adverse effecs.

Photothermal therapy can suppress the primary tumor by thermal ablation, but is usually unable to completely eradicate residue tumors as well as prevent tumor metastasis and recurrence. Photodynamic therapy destroys tumor tissue by generating reactive oxygen species (ROS), but can’t effectively kill tumors due to the hypoxic tumor environment and the low efficiency of ROS generation. Although immunotherapy can overcome tumor metastasis and recurrence, it can’t effectively destroy solid tumors, especially large primary tumors. Therefore, photothermal-photodynamic therapy combined with immunotherapy has attracted more and more attention. On the one hand, the primary tumor is eliminated by photothermal-photodynamic therapy. On the other hand, tumor recurrence and metastasis can be supressed by immunotherapy. Among various immunotherapy strategies, tumor vaccine has become a research focus. It can deliver tumor antigens to lymph nodes through antigen-presenting cells, activate T cells to trigger cellular and humoral immune responses, which can both suppress primary tumors and induce long-term memory effects in the body. However, conventional tumor vaccines introduce antigens into the body in different forms to induce immune response, which has the disadvantages of poor tumor antigen specificity and weak immunogenicity, thus making it difficult to trigger sustained and high-intensity immune effect. If the tumor specific antigen produced at the tumor site can be captured by delivery system to formulate antigen-specific tumor vaccine, promote the uptake and processing of antigen by antigen-presenting cells, and realize effective antigen presentation to T cells, the problem of antigen difference can be skillfully avoided. Furthermore, the codelivery of both antigen and immune agonists can realize synergistic effect and induce robust immune responses to achieve efficient and personalized treatment of tumor.

In view of these, we constructed polymeric vesicle NIPS for the encapsulation of photosensitizer ICG and IDO inhibitor NLG919, as well as polymer vesicle mal-dot-PS for the incorporation of TLR7/8 agonist IMQ, TLR4 agonist MPLA, cationic liposome DOTAP, maleimide functionalized phospholipid DSPE-PEG2K-Mal, and mannose targeting phospholipid DSPE-PEG5K-Mannose into polymer vesicle mal-dot-PS. On the one hand, the polymer vesicle NIPS plus 808 nm laser can produce photothermal and photothermal effects to kill tumor cells and produce tumor-associated antigens. In addition, the IDO inhibitor NLG919 was released to inhibit the IDO enzyme activity. On the other hand, the tumor antigens were captured by mal-dot-PS via DOTAP and Mal to formulate personalized tumor vaccine. The targeted co-delivery of antigens and dual TLR agonists into DCs effectively promote the maturation of DCs, antigen cross-presentation, and lymph nodes migration. The activation of T cells promoted the cellular and humoral immune response, resulting in effective anticancer effect on the inhibition of tumor growth, recurrence and metastasis via photothermal-photodynamic therapy combined with immunotherapy. The specific research contents are as follows:

Construction and characterization of multifunctional polymeric vesicles NIPS and mal-dot-PS. Using PCL-PEG-PCL as the material, the photosensitizer ICG and the IDO inhibitor NLG919 were encapsulated in the hydrophobic layer of the polymer vesicles NIPS by thin film hydration ultrasound, while NaHCO₃ was encapsulated in the hydrophilic inner cavity to form NIPS. At the same time, polymeric vesicles mal-dot-PS were fabricated to encapsulate TLR7/8 agonist IMQ in the hydrophobic layer, NaHCO₃ in the hydrophilic lumen, as well as embed TLR4 agonists MPLA, DOTAP, Mal and Man within the phospholipid layer. The particle size and distribution, zeta potential and drug encapsulation efficiency of the NIPS and mal-dot-PS were characterized. In addition, the in vitro and in vivo photothermal effects, production of cellular reactive oxygen species (ROS), in vitro cellular uptake and cytotoxicity, cellular apoptosis cellular death and survival as well as ATP secretion and in vivo tumor apoptosis of NIPS were studied. The results showed that the multifunctional polymer vesicles had good stability, high encapsulation efficiency and drug loading capacity of ICG, NLG919 and IMQ, and also had good pH responsiveness, photothermal properties and good drug release properties. Under 808 nm laser irradiation, the NIPS exhibited higher cytotoxicity, more ATP secretion, and the higer ability of cellular uptake and ROS production. The in vitro and in vivo  photothermal studies showed that the polymeric vesicle NIPS had good photothermal properties, prolonged residence in tumor tissues for a long time, and achieved a good effect on eradicating primary tumors.

Study on the antitumor effect and mechanism of polymer vesicles that can self assemble in vivo personalized vaccine for photothermal-photodynamic therapy combined with immunotherapy. The effect of polymer vesicles mal-dot-PS on IDO enzyme activity, in vitro antigen capture ability, BMDCs maturation, and antigen cross presentation were investigated. In vivo studies included the inhibition effect of NIPS + Laser@mal-dot-PS on melanoma growth, recurrence and metastasis, as well as immune mechanisms, such as DCs maturation and antigen cross-presenting ability, CD4⁺ and CD8⁺ T cell production, macrophage polarization, T lymphocyte activation and proliferation, IFN-γ and TNF-α secretion, and memory effects. In vitro studies showed that mal-dot-PS had good biocompatibility to DCs cells and antigen capture ability, and can effectively promote DCs maturation and antigen cross-presentation after antigen capture. In vivo experiments showed that under the irradiation of 808 nm laser, NIPS could exert excellent photothermal effects to effectively kill primary tumors and produce tumor-associated antigens, which were captured by mal-dot-PS to self-assemble tumor personalized vaccine and realize codelivery of both antigen and dual immune agonists. Mechanism study demonstrated that the tumor personalized vaccine can be efficiently internalized by DCs to promote DCs maturation, activation and migration to the lymph nodes, resulting in the enhanced in vivo CD8⁺ and CD4⁺ T cell immune responses, increased secretion of cytokines TNF-α and IFN-γ, decreased M2 macrophages and tumor Tregs, and excellent memory effects to inhibit lung metastasis.