Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences , Shandong University , 44 Wenhuaxi Road , Jinan , Shandong Province 250012 , China.
School of Pharmacy and Medical Sciences , University of South Australia , Adelaide , South Australia 5001 , Australia.
ACS Appl Mater Interfaces. 2018 Nov 7;10(44):37797-37811. doi: 10.1021/acsami.8b09946. Epub 2018 Oct 25.
Enhancing cytosol delivery of exogenous antigens in antigen presenting cells can improve cross-presentation and CD8+ T cell-mediated immune response. The antigen cytosol delivery speed, which has great importance on the rate of MHC class I molecules (MHC I) antigen presentation pathway and cytotoxic T lymphocytes (CTLs) induction, has not been well studied. We hypothesized that micelle-tailored vaccine with multiple cascaded lysosomal responsive capabilities could accelerate lysosomal escape and enhance cancer immunotherapy. To test our hypothesis, we created a novel micellar cancer vaccine (ovalbumin-loaded pH/redox dual-sensitive micellar vaccine, OLM-D) by cleavable conjugation of an antigen with house-made amphiphilic poly(l-histidine)-poly(ethylene glycol) (PLH-PEG) in current study. OLM-D was supposed to achieve cascade cytosol delivery of ovalbumin through three steps in terms of (i) initial redox triggered ovalbumin release, (ii) promoted proton inflow and micelle disassembly, and (iii) speeded proton sponge effect and lysosome bulging/broke. Redox-sensitive antigen release and consequently accelerative OLM-D disassembly were confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), transmission electronic microscopy (TEM), particle sizes, zeta potentials, and in vitro Ova release evaluation. The speeded cytosol delivery of ovalbumin was visualized under a confocal laser scanning microscope (CLSM). The ability of OLM-D to increase the MHC I molecule combination rate and antigen cross-presentation efficiency was identified by antigen presentation assay and maturation assay in bone marrow-derived dendritic cells (BMDCs). In vivo, the capability of OLM-D to accumulate in draining lymph nodes (LNs) after injection was visualized by real-time near infrared fluorescence imaging (NIRF) and the distribution order in different LNs was first observed (a, d, c, b). Enhanced cancer immunity of OLM-D was confirmed by increased CD3+CD8+ T cell quantity, CD3+CD8+25D11.6+ T cells quantity, and IFN-γ, IL-2 secretion post subcutaneous or intraperitoneal injection ( p < 0.05). Taken together, our results indicated that OLM-D provided a promising cascade cytosol delivery strategy, which held great potential to guide further design of nano-particulate cancer vaccines for efficient cancer immunotherapy.
增强抗原呈递细胞中外源抗原的细胞质递送速度可以改善交叉呈递和 CD8+T 细胞介导的免疫反应。抗原细胞质递送速度对 MHC I 类分子（MHC I）抗原呈递途径和细胞毒性 T 淋巴细胞（CTL）诱导的速度有重要影响，但尚未得到很好的研究。我们假设具有多个级联溶酶体反应能力的胶束疫苗可以加速溶酶体逃逸并增强癌症免疫治疗。为了验证我们的假设，我们通过在当前研究中用自制的两亲性聚（L-组氨酸）-聚（乙二醇）（PLH-PEG）切割偶联物将抗原与一种新型胶束癌症疫苗（载卵清白蛋白的 pH/氧化还原双敏感胶束疫苗，OLM-D）。OLM-D 有望通过以下三个步骤实现卵清白蛋白的级联细胞质递送：（i）初始氧化还原触发的卵清白蛋白释放，（ii）促进质子流入和胶束分解，以及（iii）加速质子海绵效应和溶酶体肿胀/破裂。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、透射电子显微镜（TEM）、粒径、Zeta 电位和体外 Ova 释放评估证实了氧化还原敏感的抗原释放和随后 OLM-D 分解的加速。使用共聚焦激光扫描显微镜（CLSM）可视化卵清白蛋白的细胞质递送速度。通过骨髓来源的树突状细胞（BMDC）中的抗原呈递测定和成熟测定，确定了 OLM-D 增加 MHC I 分子结合率和抗原交叉呈递效率的能力。在体内，通过实时近红外荧光成像（NIRF）可视化注射后 OLM-D 在引流淋巴结（LN）中的积累能力，并首次观察到其在不同 LN 中的分布顺序（a、d、c、b）。通过皮下或腹腔注射后 CD3+CD8+T 细胞数量、CD3+CD8+25D11.6+T 细胞数量和 IFN-γ、IL-2 分泌的增加，证实了 OLM-D 增强的癌症免疫力（p<0.05）。总之，我们的结果表明，OLM-D 提供了一种有前途的级联细胞质递送策略，为进一步设计用于有效癌症免疫治疗的纳米颗粒癌症疫苗提供了指导。
