Silica nanocapsules as a nonviral delivery platform for iPSC-RPE

Abstract

Purpose : A growing number of genetic therapies are being tested for retinal degenerative disorders, but these therapeutics often face challenges with regard to their packaging and delivery. Furthermore, for therapeutics such as genome editors, transient expression is ideal to prevent any deleterious effects associated with prolonged and/or unregulated expression. To overcome these challenges, we tested silica nanocapsules (SNCs) as a nonviral delivery vehicle in induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE). The unique SNCs can deliver diverse biologics including nucleic acids and genome editors. Our objective was to identify SNC formulations that efficiently deliver an mRNA-based test article to iPSC-RPE.

Methods : Wildtype iPSC-RPE cells were differentiated, purified and plated on laminin-coated surfaces prior to being treated with SNCs harboring GFP mRNA. A water-in-oil microemulsion method was used to prepare SNCs that incorporated an imidazole-containing silica reagent to improve its endosomal escape capability. For quick release of cargo in the cytosol, SNCs were formulated to be glutathione-responsive. In addition to the baseline SNC formulation, modifications including PEGylation, peptide conjugation, and peptide coating were tested. All SNC formulations were diluted in RPE culture media at <10% (v/v) and added to iPSC-RPE. On day 4 post-treatment, cells were imaged via fluorescence microscopy and GFP+ cells were quantified using flow cytometry.

Results : Brightfield imaging of SNC-treated iPSC-RPE throughout the course of treatment showed no morphological evidence of acute toxicity. All SNC formulations resulted in robust GFP expression in iPSC-RPE as assessed via fluorescence imaging. Quantification using flow cytometry showed that transfection efficiencies ranged from 6% to 24%. Highest average transfection efficiencies (24±0.7%) were obtained using the baseline and peptide-conjugated SNC formulations, with lower efficiencies observed using the PEGylated (19±0.8%) and peptide-coated SNCs (6±0.7%) (p < 0.005 and p < 0.0001, respectively).

Conclusions : Our results support the use of SNCs as a versatile and tunable platform for the delivery of gene-based therapeutics of diverse sizes or composition to human RPE in an in vitro setting. Further testing and optimization of SNCs may solidify their use as an important tool for future clinical applications, particularly those involving genome editors.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.