Gene knockdown in HaCaT cells by small interfering RNAs entrapped in 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. Thus, we 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. Here, we propose 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. In this study, we demonstrated 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. Our findings suggested that MDs can be used to prepare siRNA-EV formulations.

Effect of Rubbing Application on the Skin Permeation of Active Ingredients from Lotion and Cream.

Effect of rubbing application on the skin permeation of a hydrophilic drug caffeine (CAF) and lipophilic drug rhododendrol (RD) from lotion and cream were investigated. Skin permeation of CAF was markedly increased by rubbing action independent of the formulation type. In addition, the skin penetration-enhancement effect was affected by the rubbing direction: rubbing application against the direction of hair growth showed the highest permeation compared with rubbing applications along the direction of hair growth and in a circular pattern on the skin. On the other hand, no enhancement effect was observed by the rubbing actions on the skin permeation of RD, regardless of formulation type. Change in the infundibula orifice size of hair follicles by the rubbing and following skin stretching may be related to the higher skin permeation for CAF. In contrast, high RD distribution into the stratum corneum may be a reason why no enhancement effect was observed by the rubbing action. These results can be helpful to predict safety and effectiveness of topically applied formulations.