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Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.

ABSTRACT: Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63⁺, CD81⁺, or CD9⁺ EVs. Loading of GFP into individual vesicles was quantified by Single-Molecule Localization Microscopy. The combined results demonstrated TSPAN14, CD63 and CD63/CD81 fused to the PDGFRβ transmembrane domain as the most efficient EV-sorting proteins, accumulating on average 50-170 single GFP molecules per vesicle. In conclusion, we validated a set of complementary techniques suitable for high-resolution analysis of EV preparations that reliably capture their heterogeneity, and propose highly efficient EV-sorting proteins to be used in EV engineering applications.

SUBMITTER: Silva AM

PROVIDER: S-EPMC8329990 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Abstract Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named “Sonication and Extrusion‐assisted Active Loading” (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin‐loaded milk‐derived EVs (Dox‐mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10‐fold enhancement of drug encapsulation efficiency compared with passive loading. Single‐particle analysis by nano‐flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox‐mEVs which would otherwise be overlooked by bulk‐based approaches. Correlation analysis between doxorubicin auto‐fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid‐enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid‐probe‐ and immuno‐mediated magnetic isolation techniques to selectively remove the contaminants of non‐lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL‐prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as‐developed cargo‐loading approach and nano‐flow cytometry‐based characterization method will provide an instructive insight in the development of EV‐based delivery systems.

Project description:Extracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV-related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV-producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40-60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluorescent EVs to link single vesicle analysis with single molecule quantification.

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Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.

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