Loading small interfering RNAs entrapped into grapefruit-derived extracellular vesicles using a microfluidic device

Small interfering RNAs (siRNAs) knockdown the expression of target genes by causing mRNA degradation and are a promising therapeutic modality. In clinical practice, lipid nanoparticles (LNPs) are used to deliver RNAs, such as siRNA and mRNA, into cells. However, these artificial nanoparticles are toxic and immunogenic. Josai University researchers focused on extracellular vesicles (EVs), natural drug delivery systems, for the delivery of nucleic acids. EVs deliver RNAs and proteins to specific tissues to regulate various physiological phenomena in vivo. The researchers have developed a novel method for the preparation siRNAs encapsulated in EVs using a microfluidic device (MD). MDs can be used to generate nanoparticles, such as LNPs, by controlling flow rate to the device, but the loading of siRNAs into EVs using MDs has not been reported previously. The researchers demonstrate a method for loading siRNAs into grapefruit-derived EVs (GEVs), which have gained attention in recent years for being plant-derived EVs developed using an MD. GEVs were collected from grapefruit juice using the one-step sucrose cushion method, and then GEVs-siRNA-GEVs were prepared using an MD device. The morphology of GEVs and siRNA-GEVs was observed using a cryogenic transmission electron microscope. Cellular uptake and intracellular trafficking of GEVs or siRNA-GEVs to human keratinocytes were evaluated by microscopy using HaCaT cells. The prepared siRNA-GEVs encapsulated 11% of siRNAs. Moreover, intracellular delivery of siRNA and gene suppression effects in HaCaT cells were achieved using these siRNA-GEVs. These findings suggested that MDs can be used to prepare siRNA-EV formulations.

Effect of pressure on particle size, PdI, and loading efficiency of
siRNA-GEVs prepared using a microfluidic method

Figure 3

The response surface method was applied to analyze data obtained using the design of experiment (DoE) approach. Response surface models of (a) mean particle size, (b) loading efficiency, and (c) PdI are shown. Pressures A and B are fluid pressures in the siRNA/PBS and GEVs/EtOH channels, respectively.

Itakura S, Shohji A, Amagai S et al. (2023) Gene knockdown in HaCaT cells by small interfering RNAs entrapped in grapefruit-derived extracellular vesicles using a microfluidic device. Sci Rep 13, 3102 (2023). [article]

Leave a Reply

Your email address will not be published. Required fields are marked *