SIRNA GENE KNOCKDOWN WITH FUNCTIONALISED POROUS SILICON NANOPARTICLES

Abstract

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.