In Silico Design and Experimental Validation of siRNA-Functionalized DNA Origami Nanoliposomes Targeting Bcl-۲, XIAP, ADRM۱, and miR-۲۱ for Gastric Cancer Therapy

Background: Gastric cancer remains a leading cause of cancer-related mortality, with conventional therapies hampered by poor specificity and significant toxicity. MicroRNA-۲۱ (miR-۲۱) acts as an oncogene by promoting proliferation, inhibiting apoptosis, and suppressing anti-tumor immunity. Additionally, anti-apoptotic genes Bcl-۲ and XIAP contribute to chemoresistance, while ADRM۱ drives metastatic progression. DNA origami nanotechnology offers a versatile platform for targeted delivery of nucleic acid therapeutics. This study combined in silico design with experimental validation to develop a multi-targeted siRNA delivery system. Objective: To design, characterize, and evaluate the anticancer efficacy of cationic nanoliposomes encapsulating DNA origami nanostructures functionalized with siRNA targeting miR-۲۱, Bcl-۲, XIAP, and ADRM۱ in MKN-۴۵ gastric cancer cells. Methods: Bioinformatics approaches including NCBI BLAST, OligoWalk thermodynamic analysis, and miRBase database mining were employed for target selection and siRNA design. DNA origami scaffolds were assembled using an eight-oligonucleotide annealing protocol and characterized by agarose gel electrophoresis, dynamic light scattering (DLS), zeta potential measurements, transmission electron microscopy (TEM), and UV spectrophotometry. The constructs were encapsulated within cationic liposomes composed of DOSPA and DOPE. MKN-۴۵ cells were treated with various concentrations of the formulations. Cell viability was assessed by MTT assay at ۲۴, ۴۸, and ۷۲ hours. Apoptosis was quantified through Annexin V-FITC/propidium iodide staining followed by flow cytometry. Results: In silico analysis identified optimal siRNA sequences with favorable thermodynamic profiles (ΔG = -۶.۷ to -۸.۲ kcal/mol, Tm = ۳۲.۵–۳۵.۲°C). DNA origami structures demonstrated a mean size of ۱۰ nm, uniform distribution (PDI=۰.۱), and negative surface charge (-۱۲ mV). Following liposomal encapsulation, the complete nanocomplex exhibited a mean size of ۸۸ nm, satisfactory monodispersity (PDI=۰.۳), positive surface charge (+۲۸ mV), and loading efficiency of ۵۹%. MTT assays disclosed a concentration-dependent decline in cellular viability, with IC₅₀ values of ۴.۶۶ μg/mL at ۴۸ hours for viability. Flow cytometry revealed that the complete formulation substantially elevated the apoptotic cell population to ۳۵.۲۳% compared to ۱۸.۵۷% in untreated controls at ۱ μg/mL concentration. Conclusion: The integrated in silico-experimental approach successfully developed a DNA origami-based nanoplatform for multi-targeted siRNA delivery, demonstrating significant apoptosis induction and cell growth suppression in gastric cancer cells.

کلیدواژه ها:

DNA origami ، miR-۲۱ ، nanoliposome ، siRNA ، gastric cancer ، bioinformatics ، Bcl-۲ ، XIAP ، ADRM۱ ، apoptosis