Leveraging Selective Knockdown of Sost Gene by Polyethyleneimine–siRNA–Chitosan Reduced Gold Nanoparticles to Promote Osteogenesis in MC3T3-E1 & MEF Cells

Abstract

Aim: Osteoporosis is a systemic skeletal disorder characterized by reduced osteoblast differentiation, predominantly by overexpression of the Sost gene. A layer-by-layer approach enabled encapsulation of Sost siRNA to enhance the short half-life and poor transfection capacity of siRNA. Materials & methods: Polyethyleneimine and siRNA on chitosan-coated gold nanoparticles (PEI/siRNA/Cs–AuNPs) were engineered using chitosan-reduced gold nanoparticles. They were characterized by dynamic light scattering, scanning electron microscopy, transmission electron microscopy, Fourier transform infrared and gel-mobility assays. Detailed in vitro experiments, gene silencing and western blots were performed. Results: A total of 80% knockdown of the target sclerostin protein was observed by PEI/siRNA/Cs–AuNPs, q-PCR showed threefold downregulation of the Sost gene. Osteogenic markers RunX2 and Alp were significantly upregulated. Conclusion: We report a safe, biocompatible nanotherapeutic strategy to enhance siRNA protection and subsequent silencing to augment bone formation.

Keywords: :

• gene silencing
• gold–chitosan nanoparticles
• osteogenic biomarkers
• osteoporosis
• RT-PCR
• Sost siRNA

Supplementary data

Author contributions

AK Verma: Contributed to the conceptual and experimental design of all the experiments, the evaluation and complete analysis of the results and the manuscript writing. AK Verma also organized the financial grant from Department of Biotechnology, GOI and supervised this research project. K Niveria and Z Yab performed the experiments, wrote the main manuscript and analyzed the data. The authors have read and approved the final manuscript. L Biswas assisted with the experimentation throughout.

Acknowledgments

A special thanks to AK Panda and their lab (NII) for access to instrumentation and providing MC3T3 E-1 cells.

Financial disclosure

This work was primarily supported by funding from major research project-DBT (grant no: BT/PR 21645/NNT/28/1224/2017): ’Small molecule functionalized biopolymeric nanoparticles encapsulating siRNA for targeted delivery to osteoblasts for managing bone disorders’. K Niveria acknowledges CSIR for their SRF and L Biswas is thankful to UGC-SRF for their fellowship. Z Yab and A Mahtab acknowledge fellowship support from DBT. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.