共载吲哚菁绿和Nrf2-siRNA的多功能纳米粒子对抗口腔鳞状细胞癌的体外作用研究

Study on Multifunctional Nanoparticles Co-loaded with Indocyanine Green and Nrf2-siRNA against Oral Squamous Cell Carcinoma In Vitro

目的:构建共载吲哚菁绿(indo cyanine green,ICG)和核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)-短干扰RNA(small interfering RNA,siRNA)的多功能纳米载药粒子,并探究其联合光热疗法与抗氧化抑制作用增效的光动力疗法协同对抗口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)的体外作用与机制。方法:利用超声乳化法和静电力吸附作用制备基于聚氨基酯/聚乳酸-羟基乙酸共聚物且共载ICG和Nrf2-siRNA的纳米粒子PPI-siRNA。表征PPI-siRNA纳米粒子的形貌、粒径大小和分布、表面电位及其载药情况;评价PPI-siRNA纳米粒子对ICG和Nrf2-siRNA的胞内递送以及Nrf2-siRNA逃逸溶酶体吞噬的性能;通过检测舌鳞状细胞癌细胞SCC-25随激光照射的升温效应、热休克蛋白60表达和活性氧生成水平考察PPI-siRNA纳米粒子的光热和光动力性能;考察细胞内Nrf2及其调控的下游基因谷氨酸-半胱氨酸连接酶修饰亚基和谷氨酸-半胱氨酸连接酶的表达水平,从而探究Nrf2-siRNA助力光动力效应的作用机制;运用噻唑蓝比色法考察PPI-siRNA联合激光照射对SCC-25的细胞毒性作用。结果:PPI-siRNA纳米粒子呈形貌规则的球状结构,粒径约为180 nm,并成功共载ICG与Nrf2-siRNA;PPI-siRNA纳米粒子可以高效递送ICG和Nrf2-siRNA入胞,且可以确保Nrf2-siRNA逃逸溶酶体吞噬,从而发挥基因沉默作用;PPI-siRNA纳米粒子具备良好的光热和光动力性能,Nrf2-siRNA可以通过下调抗氧化蛋白和基因的表达显著提升光学疗法的抗肿瘤效率。结论:PPI-siRNA纳米粒子可以联合光热疗法与基因沉默作用增效的光动力疗法并有效抑制SCC-25的体外生长,在OSCC的临床治疗方面具有广阔的应用前景。

Objective:To construct multifunctional nanoparticles co-loaded with indocyanine green(ICG)and nuclear factor erythroid 2-related factor 2-small interfering RNA(Nrf2-siRNA),and to explore the combined effects and mechanisms of photothermal and antioxidant inhibition amplified photodynamics against oral squamous cell carcinoma(OSCC)in vitro.Methods:Double emulsion method and electrostatic adsorption were used to prepare PPI-siRNA nanoparticles based on polyurethane(PBAE)/polylactic acid-glycolic acid copolymer(PLGA)co-loaded with ICG and Nrf2-iRNA.The morphology,size distribution,zeta potential,and drug loading efficiency of nanoparticles were characterized.The SCC-25 cellular uptake of ICG and the escaping lysosomal phagocytosis ability of Nrf2-siRNA were evaluated.The photothermal and photodynamic effects of PPI-siRNA nanoparticles were investigated by detecting the heating effect,expression of intracellular heat shock protein(HSP60),and the generation of reactive oxygen species(ROS).The expression levels of E2-related factor 2(Nrf2)and its downstream genes GCLM and GCLC regulated by the Nrf2 were investigated to explore the assisting photodynamic effects mechanism of Nrf2-siRNA.The cytotoxic effect of PPI-siRNA nanoparticles combined with laser irradiation was evaluated by MTT assay and live/dead cell assay.Results:The PPI-siRNA nanoparticles exhibited spherical structure with regular morphology and particle size of about 180 nm.PPI-siRNA nanoparticles could efficiently deliver ICG and Nrf2-siRNA into cells,and prevent Nrf2-siRNA from being engulfing from lysosome to exert gene silence effect.PPI-siRNA nanoparticles possessed notable photothermal and photodynamic properties.Nrf2-siRNA could significantly improve the anti-tumor efficiency of phototherapy by down-regulating the expression of antioxidant proteins and genes.Conclusion:PPI-siRNA nanoparticles can effectively inhibit the in vitro growth of OSCC by combining phototherapy and gene silencing amplified photodynamic efficacy,and have a broad application prospect in the clinical treatment of OSCC.