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Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

ABSTRACT: Lipidoid nanoparticles (LNPs) are the delivery platform in Onpattro, the first FDA-approved siRNA drug. LNPs are also the carriers in the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines. While these applications have demonstrated that LNPs effectively deliver nucleic acids to hepatic and muscle cells, it is unclear if LNPs could be used for delivery of siRNA to neural cells, which are notoriously challenging delivery targets. Therefore, the purpose of this study was to determine if LNPs could efficiently deliver siRNA to neurons. Because of their potential delivery utility in either applications for the central nervous system and the peripheral nervous system, we used both cortical neurons and sensory neurons. We prepared siRNA-LNPs using C12-200, a benchmark ionizable cationic lipidoid along with helper lipids. We demonstrated using dynamic light scattering that the inclusion of both siRNA and PEG-lipid provided a stabilizing effect to the LNP particle diameters and polydispersity indices by minimizing aggregation. We found that siRNA-LNPs were safely tolerated by primary dorsal root ganglion neurons. Flow cytometry analysis revealed that Cy5 siRNA delivered via LNPs into rat primary cortical neurons showed uptake levels similar to Lipofectamine RNAiMAX-the gold standard commercial transfection agent. However, LNPs demonstrated a superior safety profile, whereas the Lipofectamine-mediated uptake was concomitant with significant toxicity. Fluorescence microscopy demonstrated a time-dependent increase in the uptake of LNP-delivered Cy5 siRNA in a human cortical neuron cell line. Overall, our results suggest that LNPs are a viable platform that can be optimized for delivery of therapeutic siRNAs to neural cells.

SUBMITTER: Khare P

PROVIDER: S-EPMC8648339 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RNA interference (RNAi) has an unprecedented potential as a therapeutic strategy for reversibly silencing the expression of any gene. Therapeutic delivery of the RNAi mediator, i.e., small interfering RNA (siRNA), can be used to address diseases characterized by gene overexpression, for example inflammatory conditions like chronic obstructive pulmonary disease (COPD). Macrophages play a key role in COPD pathogenesis and are recruited to the airways and lung parenchyma, where they release proinflammatory cytokines, e.g., tumor necrosis factor-alpha (TNF-α). Hence, targeting TNF-α with siRNA is a promising therapeutic approach for COPD management. However, a safe and effective delivery system is required for delivery of TNF-α siRNA into the cytosol of hard-to-transfect macrophages. The purpose of this study was to optimize the intracellular delivery of TNF-α siRNA to the lipopolysaccharide-activated murine macrophage cell line RAW 264.7 using lipidoid-polymer hybrid nanoparticles (LPNs) composed of the lipid-like transfection agent lipidoid 5 (L5) and the biodegradable polymer poly (D,L-lactide-co-glycolide). Applying a quality-by-design approach, the influence of critical formulation variables, i.e., the L5 content and the L5:siRNA ratio (w/w), on critical quality attributes (CQAs) was investigated systematically using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). The CQAs were identified based on the quality target product profile and included size, polydispersity index, zeta potential, encapsulation efficiency and loading for achieving efficient and safe TNF-α gene silencing in activated RAW 264.7 cells. Formulations inducing efficient gene silencing and low cytotoxicity were identified, and the optimal formulations displayed L5 contents of 15 and 20% (w/w), respectively, and an L5:siRNA weight ratio of 15:1. All tested formulations within the OOS mediated efficient and sequence-specific TNF-α gene silencing in RAW 264.7 cells at TNF-α-siRNA concentrations, which were significantly lower than the concentrations required of non-encapsulated TNF-α-siRNA, highlighting the benefit of the delivery system. The results also demonstrate that increasing the loading of siRNA into the delivery system does not necessarily imply enhanced gene silencing. This opens new avenues for further exploitation of LPNs as a robust platform technology for delivering TNF-α siRNA to macrophages, e.g., in the management of COPD.

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Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells.

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