Uptake of Small EVs by Skeletal Muscle Myoblasts In Vitro and Subsequent Myogenic Response to Cargo

Small extracellular vesicles (EVs) are mediators of intercellular communication. They transport bioactive cargo such as proteins and nucleic acids between cells, contributing to tissue function and repair. Small EVs released by skeletal muscle influence myogenesis in a cargo-dependent manner, with muscle-specific microRNAs (myomiRs) playing a central role in regulating the balance between myoblast proliferation and differentiation. Among these, miR-206 is a key regulator. Despite its therapeutic potential, miR-206 application is limited by RNA instability and challenges associated with RNA delivery methods. Small EVs provide a promising delivery platform due to their biocompatibility and ability to protect and efficiently deliver their cargo. This study hypothesised that loading miR-206 into small EVs would protect it from degradation and enable effective delivery to recipient cells. Small EVs were generated from proliferating C2C12 myoblasts cultured in EV-depleted media and isolated using differential ultracentrifugation. Nanoparticle tracking analysis confirmed that the majority of isolated particles were within the expected size range (mean diameters below 200 nm). Cell Mask Orange staining demonstrated that 74.45% of the isolated particles were membrane-bound and thus vesicular (n=3). Transmission electron microscopy revealed the characteristic cup-shaped morphology, with particles remaining below 200 nm. Western blot analysis further confirmed the presence of TSG101, Alix, and CD81, and the absence of the endoplasmic reticulum protein calnexin, indicating minimal cellular contamination. Endogenous miR-206 expression was assessed in proliferating C2C12 cells and in their released small EVs before cargo loading. Small RNA fractions were isolated and quality controlled using TapeStation and Nanodrop analysis. miR-206 was detected in all samples, with lower levels observed in small EVs compared to parent cells. miR-206 expression increased progressively in differentiating cells with advancing differentiation days. Small EVs were subsequently loaded with a fluorescent miR-206 mimic. Loading analysis revealed increasing enrichment of miR-206 in small EV pellets with increasing input concentrations. Small EVs loaded with 100 pmol miR-206 showed the highest enrichment (706-fold), followed by 50 pmol (358-fold) and 20 pmol (199-fold, all p<0.0001), relative to unloaded small EVs. The loading efficiency was best with 50 pmol (98.52%, p=0.0096), which was selected for downstream experiments. Control conditions demonstrated that the Exo-fect reagent was required for effective miR-206 loading. Confocal microscopy confirmed the uptake of miR-206-loaded small EVs by recipient C2C12 cells over a 16 h treatment period, with qPCR analysis demonstrating a marked increase in intracellular miR-206 levels (177-fold) compared to untreated cells. Treatment with unloaded small EVs resulted in a modest increase in miR-206 levels (1.5-fold), while miR-206 inhibitor-loaded small EVs reduced cellular miR-206 levels (0.25-fold). Functional assays demonstrated that miR-206-loaded small EVs reduced myoblasts and decreased Pax7 levels. During differentiation, miR-206 delivery promoted myogenic fusion, with treated cells displaying the highest number of nuclei in fused myotubes (35 per field of view, n=3). This was supported by significantly increased levels of MyoD and Myogenin. This study demonstrated that C2C12 myoblast-derived small EVs can be efficiently isolated, characterised, and loaded with miR-206 for delivery to recipient cells without degradation. This supports their potential as a delivery platform for therapeutic miRNA cargo.

Grantor dc:publisher

Stellenbosch : Stellenbosch University

Year dc:date.issued

2026

Author dc:creator

• Dlamini, Nonhle

Advisor dc:contributor.advisor

• Myburgh, Kathryn H.

Language dc:language.iso

en

Repository record dc:identifier.uri

https://scholar.sun.ac.za/handle/10019.1/135744

OAI identifier oai:identifier

oai:scholar.sun.ac.za:10019.1/135744