Engineering Cardiac Small Extracellular Vesicle-Derived Vehicles with Thin-Film Hydration for Customized microRNA Loading

Abstract

Cell therapies for myocardial infarction, including cardiac ckit+ progenitor cell (CPC) therapies, have been promising, with clinical trials underway. Recently, paracrine signaling, specifically through small extracellular vesicle (sEV) release, was implicated in cell-based cardiac repair. sEVs carry cardioprotective cargo, including microRNA (miRNA), within a complex membrane and improve cardiac outcomes similar to that of their parent cells. However, miRNA loading efficiency is low, and sEV yield and cargo composition vary with parent cell conditions, minimizing sEV potency. Synthetic mimics allow for cargo-loading control but consist of much simpler membranes, often suffering from high immunogenicity and poor stability. Here, we aim to combine the benefits of sEVs and synthetic mimics to develop sEV-like vesicles (ELVs) with customized cargo loading. We developed a modified thin-film hydration (TFH) mechanism to engineer ELVs from CPC-derived sEVs with pro-angiogenic miR-126 encapsulated. Characterization shows miR-126+ ELVs are similar in size and structure to sEVs. Upon administration to cardiac endothelial cells (CECs), ELV uptake is similar to sEVs too. Further, when functionally validated with a CEC tube formation assay, ELVs significantly improve tube formation parameters compared to sEVs. This study shows TFH-ELVs synthesized from sEVs allow for select miRNA loading and can improve in vitro cardiac outcomes.

Knockdown of deleterious miRNA in progenitor cell–derived small extracellular vesicles enhances tissue repair in myocardial infarction

“…Thirty hours before sEV collection, cells were treated with the 1:1 mixture of Lipofectamine 2000 (Thermo Fisher Scientific) and MISSION Synthetic microRNA inhibitor (Sigma-Aldrich) for miRNA depletion under serum starvation in serum-free Ham’s F-12 medium. After 6 hours of miRNA inhibitor treatment, the medium was changed to fresh serum-free Ham’s F-12 medium and collected after 24 hours to isolate sEVs by sequential ultracentrifugation as previously described ( 17 ). All sEVs were resuspended in 300 μl of PBS and were quantified using nanoparticle tracking analysis on NanoSight NS300 (Malvern Panalytical, Malvern, UK).…”

“…In this manner, sEVs play a critical role in cell therapy by delivering molecular cargo and mediating intercellular or interorgan signaling (17). The beneficial effects of sEV have been largely attributed to cargo microRNA (miRNA): short noncoding RNA that binds mRNA and induces degradation (18).…”

“…During sEV biogenesis, cells load nucleic acids, proteins, and other molecules into the vesicle and release the vesicle into the extracellular space, where it may be internalized by neighboring cells. In this manner, sEVs play a critical role in cell therapy by delivering molecular cargo and mediating intercellular or interorgan signaling ( 17 ). The beneficial effects of sEV have been largely attributed to cargo microRNA (miRNA): short noncoding RNA that binds mRNA and induces degradation ( 18 ).…”

Customized Loading of microRNA-126 to Small Extracellular Vesicle-Derived Vehicles Improves Cardiac Function after Myocardial Infarction

“…We explored electroporation as a method for CPC-derived ELV synthesis to build upon our previous work using thin-film hydration. 18 Electroporation uses small voltage pulses to create temporary openings in the vesicle membrane and allows the miR cargo of choice to be encapsulated into the vesicle through a diffusion gradient (Figure 1A). We confirmed that the shape of the synthesized ELVs was similar to that of CPC sEVs by using transmission electron microscopy (Figure 1B).…”

“…Given that the ELVs in our prior published work formed by self-assembly, controlling the amount of cargo loaded was challenging. 18 To enhance the scalability and versatility of ELVs as customized cargo-carrying vehicles, we explored the potential tunability of miR loading with electroporation. First, we optimized the sEV inherent RNA cargo depletion to provide vesicles with "cargo-free" cavities from which to synthesize the ELVs.…”