HPV VLPs-based Multifunctional Assembly for Synergistic BCL-2 Silencing and Mitochondrial Apoptosis in NSCLC Therapy

Fluorescence spectral comparison of the HPV VLP, FITC-SP5-2 and FITC-VLP-SP5-2 assemblies; ImageJ analysis on the fluorescence intensities of the assembly of FITC-VLP and FITC-VLP-SP5-2 (1:2); Agarose gel electrophoresis of the bi-assembly of siRNA_D-pep constructed from different molar ratios of P/N; Zeta-potentials of siRNA, D-pep and the siRNA_D-pep assembly; TEM images and corresponding size distribution of the assemblies of siRNA_D-pep and siRNA_D-pep@VLP-SP5-2; Zeta-potentials of siRNA_D-pep@VLP and siRNA_D-pep@VLP-SP5-2 assemblies; Fluorescence spectra of FITC-D-pep at different concentrations (3, 5, 7.5, 10, 15, 20 μg/ml) and that of D-pep@VLP-SP5-2 or siRNA_D-pep@VLP-SP5-2 assembly, respectively; In vitro apoptosis analysis in different cell lines; Combinatorial index (CI) values across fraction affected (Fa) in A549 cells; WB analysis on the expression level of BCL-2 in A549 cells after incubation with D-pep, siRNA_D-pep, siRNA@VLP, siRNA_D-pep@VLP, siRNA_D-pep@VLP-SP5-2, siRNA_D-pep@HmA; and siRNA, siRNA@VLP, siRNA@VLP-SP5-2, respectively; The proportion of mitochondrial depolarization in A549 cell lines being detected by flow cytometry after co-incubation with siRNA_D-pep, siRNA_D-pep@VLP, and siRNA_D-pep@VLP-SP5-2 for 12 h, respectively, as well as the curve of membrane potential over time; Detection of Cyt c in the extracted mitochondria by Raman spectroscopy and the illustration of relative band intensities; The proportion of caspase-3 activity being test using flow cytometry after co-incubation of A549 cells with siRNA_D-pep, siRNA_D-pep@VLP, and siRNA_D-pep@VLP-SP5-2 for 3 h, 6 h, and 12 h, respectively; WB analysis on expression levels of survivin in A549 cells after incubation with D-pep, siRNA_D-pep, siRNA@VLP, siRNA_D-pep@VLP, siRNA_D-pep@VLP-SP5-2, and siRNA_D-pep@HmA for 24 h, respectively; Therapeutic performance of the siRNA_D-pep@VLP-SP5-2 assembly in mouse model; Flow cytometric assay of VLP-SP5-2-FITC tri-assembly in different cell lines; CCK-8 of VLP-SP5-2 assembly; calibration curve of siRNA_D-pep bi-assembly based on the Zeta-potentials; encapsulation efficiency quantification of the tetra-functional assembly.人乳头瘤病毒病毒样颗粒（HPV VLP）、异硫氰酸荧光素（FITC）标记的SP5-2（FITC-SP5-2）以及FITC-VLP-SP5-2组装体的荧光光谱对比分析；采用ImageJ软件对摩尔比为1:2的FITC标记VLP与FITC-VLP-SP5-2组装体的荧光强度进行分析；不同P/N摩尔比构建的小干扰RNA（siRNA）与D型肽（D-pep）双组装体（siRNA_D-pep）的琼脂糖凝胶电泳（agarose gel electrophoresis）检测；siRNA、D-pep以及siRNA_D-pep组装体的Zeta电位（Zeta-potential）检测；siRNA_D-pep组装体与siRNA_D-pep@VLP-SP5-2组装体的透射电子显微镜（TEM）成像及对应粒径分布分析；siRNA_D-pep@VLP与siRNA_D-pep@VLP-SP5-2组装体的Zeta电位检测；不同浓度（3、5、7.5、10、15、20 μg/ml）的FITC标记D-pep，以及D-pep@VLP-SP5-2、siRNA_D-pep@VLP-SP5-2组装体的荧光光谱检测；不同细胞系的体外细胞凋亡分析；A549细胞系中不同效应分数（fraction affected，Fa）下的组合指数（combinatorial index，CI）值检测；分别采用D-pep、siRNA_D-pep、siRNA@VLP、siRNA_D-pep@VLP、siRNA_D-pep@VLP-SP5-2、siRNA_D-pep@HmA以及siRNA、siRNA@VLP、siRNA@VLP-SP5-2孵育A549细胞后，对细胞内B细胞淋巴瘤因子2（BCL-2）的表达水平进行蛋白质免疫印迹（WB）分析；分别采用siRNA_D-pep、siRNA_D-pep@VLP、siRNA_D-pep@VLP-SP5-2与A549细胞共孵育12小时后，通过流式细胞术（flow cytometry）检测细胞线粒体去极化比例，同时记录膜电位随时间变化的曲线；采用拉曼光谱法（Raman spectroscopy）检测提取的线粒体中的细胞色素c（Cyt c），并对相对条带强度进行标注说明；分别采用siRNA_D-pep、siRNA_D-pep@VLP、siRNA_D-pep@VLP-SP5-2与A549细胞共孵育3小时、6小时、12小时后，通过流式细胞术检测半胱氨酸天冬氨酸蛋白酶3（caspase-3）的活性比例；分别采用D-pep、siRNA_D-pep、siRNA@VLP、siRNA_D-pep@VLP、siRNA_D-pep@VLP-SP5-2及siRNA_D-pep@HmA孵育A549细胞24小时后，对细胞内生存素（survivin）的表达水平进行蛋白质免疫印迹分析；siRNA_D-pep@VLP-SP5-2组装体在小鼠模型中的治疗效果评估；不同细胞系中VLP-SP5-2-FITC三组装体的流式细胞术检测；VLP-SP5-2组装体的CCK-8细胞活性检测；基于Zeta电位的siRNA_D-pep双组装体校准曲线构建；四功能组装体的包封率定量检测。