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.