Basic and Translational Sciences

Wednesday September 16, 2020 from

Room: E-Poster Hall

P-2.63 siRNA gene knockdown with functionalised porous silicon nanoparticles

Sebastian O Stead, Australia

University of Adelaide

Abstract

siRNA gene knockdown with functionalised porous silicon nanoparticles

Sebastian O. Stead1,2, Juewan Kim3, Francis D. Kette1,2, Griffith B. Perkins3, P. Toby H. Coates1,4.

1School of Medicine, University of Adelaide, Adelaide, Australia; 2College of Medicine and Public Health, Flinders University, Bedford Park, Australia; 3Department of Molecular & Cellular Biology, University of Adelaide, Adelaide, Australia; 4Central Northern Adelaide Renal and Transplantation Service (CNARTS), The Royal Adelaide Hospital, Adelaide, Australia

Dendritic cells (DCs) are a key player within the immune system, with the capacity to establish immunological tolerance within transplant recipients.
Aim: To conduct a proof of concept experiment confirming GAPDH siRNA loaded porous silicon nanoparticles (pSiNPs), functionalised with anti-CD11c antibodies can induce targeted gene knockdown.
Method: DCs were derived from human peripheral blood (CD34+CD14+CD11c-) monocytes and differentiated with IL-4 and GM-CSF into CD11c+ immature DCs. Semicarbazide functionalised pSiNPs were covalently bound to both anti-CD11c antibodies and GAPDH siRNA (siRNA-pSiNPs). Nanoparticles were co-cultured with human monocyte-derived DCs, with pSiNP uptake being assessed after 24h by flow cytometry. GAPDH gene expression of siRNA-pSiNP treated DCs was assessed via polymerase chain reaction.
Results: CD11c functionalised nanoparticles showed increased uptake by DCs in a time- and dose-dependent manner with anti-CD11-pSiNP uptake 48% greater, compared to non-targeting control pSiNPs in the first 30 minutes (p < 0.05). Unloaded NPs were not cytotoxic to DCs and were immunologically inert, with no increased expression of CD40, CD80 or CD86 seen. siRNA-pSiNPs showed successful GAPDH knockdown (p < 0.05) within DC without the need of a transfection reagent.
Conclusion: These proof of concept experiments showed that pSiNP can be used as a novel vector for the delivery of siRNA to DCs for gene knockdown, potentially replacing commonly used viral vectors or lipofection reagents. Future experiments will utilise functional CD80-siRNA for the induction of tolerogenic DCs.

Presentations by Sebastian O Stead

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