Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication

Abstract

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.

Impact of cell culture parameters on production and vascularization bioactivity of mesenchymal stem cell‐derived extracellular vesicles

“…Tissue culture polystyrene flasks were coated with 0.1% gelatin at 37°C for 1 hr prior to seeding HDMECs and HUVECs. All cell types were cultured in the designated growth culture medium in the presence of EV‐depleted serum as previously described . Cell passage for MSCs was designated as P1 upon arrival from the manufacturer.…”

“…Conditioned media from all cells at appropriate seeding densities and passages were collected after 24 hr of culture unless otherwise stated. EVs were isolated by differential centrifugation with 100,000 × g as the final centrifugation step as previously described . Pelleted EVs were resuspended in 1X PBS and subsequently washed with 1X PBS using Nanosep 300 kDa MWCO spin columns (OD300C35; Pall).…”

Engineering Extracellular Vesicles as Nanotherapeutics for Regenerative Medicine

“…Protein [65] siRNA [82] Extrusion EVs and cargo molecules are loaded into a syringe and injected forcefully though a porous membrane. The mechanical force disrupts the EV membrane and entraps the cargo within the EV.…”

Extracellular Microvesicles as Game Changers in Better Understanding the Complexity of Cellular Interactions—From Bench to Clinical Applications

“…Thus, like liposomes, they could be loaded with bioactive compounds and drugs and employed in the clinic as vehicles for drug delivery [3, 17, 18]. In fact, there are already several reports on using this strategy to deliver therapeutic compounds to cells.…”

“…Importantly, this approach increased paclitaxel cytotoxicity [17]. In another report, ExMVs were effectively employed to deliver siRNA molecules targeting HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, to breast cancer cells [18]. The results of first clinical trials using this technology in several clinical settings are expected soon [1–6, 19–21].…”