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In Silico Design and Experimental Validation of siRNA-Functionalized DNA Origami Nanoliposomes Targeting Bcl-2, XIAP, ADRM1, and miR-21 for Gastric Cancer Therapy
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چکیده: (5 مشاهده) |
Background: Gastric cancer remains a leading cause of cancer-related mortality, with conventional therapies hampered by poor specificity and significant toxicity. MicroRNA-21 (miR-21) acts as an oncogene by promoting proliferation, inhibiting apoptosis, and suppressing anti-tumor immunity. Additionally, anti-apoptotic genes Bcl-2 and XIAP contribute to chemoresistance, while ADRM1 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-21, Bcl-2, XIAP, and ADRM1 in MKN-45 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-45 cells were treated with various concentrations of the formulations. Cell viability was assessed by MTT assay at 24, 48, and 72 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 = -6.7 to -8.2 kcal/mol, Tm = 32.5–35.2°C). DNA origami structures demonstrated a mean size of 10 nm, uniform distribution (PDI=0.1), and negative surface charge (-12 mV). Following liposomal encapsulation, the complete nanocomplex exhibited a mean size of 88 nm, satisfactory monodispersity (PDI=0.3), positive surface charge (+28 mV), and loading efficiency of 59%. MTT assays disclosed a concentration-dependent decline in cellular viability, with IC₅₀ values of 4.66 μg/mL at 48 hours for viability. Flow cytometry revealed that the complete formulation substantially elevated the apoptotic cell population to 35.23% compared to 18.57% in untreated controls at 1 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.
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متن کامل [PDF 573 kb]
(2 دریافت)
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نوع مطالعه: پژوهشي |
موضوع مقاله:
عمومى
دریافت: 1405/2/6 | پذیرش: 1405/4/11 | انتشار: 1404/8/28
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