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. 2021 Jul 21;14(8):703.
doi: 10.3390/ph14080703.

Engineered EVs for Oxidative Stress Protection

Anna Maria Tolomeo  1   2 , Santina Quarta  2   3 , Alessandra Biasiolo  2   3 , Mariagrazia Ruvoletto  2   3 , Michela Pozzobon  1   4 , Giada De Lazzari  1   2 , Ricardo Malvicini  1   2   5 , Cristian Turato  6 , Giorgio Arrigoni  7   8 , Patrizia Pontisso  2   3 , Maurizio Muraca  1   2   4
Affiliations

Engineered EVs for Oxidative Stress Protection

Anna Maria Tolomeo et al. Pharmaceuticals (Basel). .

Abstract

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.

Keywords: SerpinB3; cytoprotection; extracellular vesicles; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Example of SerpinB3 expression. (A) Representative immunofluorescence analysis of SerpinB3 expression in a SerpinB3 transfected HepG2 cell clone (HepG2/SB3) and in an empty vector transfected HepG2 cell clone (HepG2/CTR) used for EV preparation. The cells were immunostained with anti-SerpinB3 antibody (red) and the nuclei were counterstained with DAPI (blue). (B) Analysis of SerpinB3 (SB3) transcripts in the corresponding clones by quantitative real-time PCR (Q-PCR). The relative expression of SB3 mRNA is expressed by calculating 2-ΔCt. The experiment was performed in triplicate.
Figure 2
Figure 2
Characterization of extracellular vesicles derived from HepG2s. Representative size distribution of naïve EVs (nEVs) (A) and of EVs-SB3 (B) analysed by Resistive Pulse Sensing. (C) Number of nEVs and of EV-SB3 secreted per cell. Result are mean ± standard error (n = 5 independent experiments), * p < 0.05. Transmission electron microscopy analysis of freshly nEVs (D) and of freshly EVs-SB3 (E); scale bar refers to 200 nm. (F) SerpinB3 quantification by ELISA assay. Result are mean ± standard error (n = 5 independent experiments), *** p < 0.001. (G) Western Blot of nEVs and EVs-SB3 for SerpinB3. (H) Distribution of surface biomarker expression in nEVs and in EV-SB3.
Figure 3
Figure 3
Bioinformatic analysis of proteins identified in EV preparations. (A) The enriched Gene Ontology (GO) terms that are associated to the proteins identified in EVs samples, as obtained by the gProfiler tool (p value > 0.01) and further processed with the Revigo software (n = 3 independent preparations). (B) STRING network of physical/functional interactions involving only the proteins that have a significantly different abundance in nEVs and in EVs–SB3.
Figure 4
Figure 4
Left panel: Fluorescence expression of DIL-EVs uptake in HepG2 cells over a 6–24 h timeframe. Original magnification 63×. Right panel: Graphic representation of DIL-EVs quantification in HepG2 cells. The negative controls were subtracted from the fluorescence intensity of nEVs and EVs-SB3 and final results were normalized against staining of the nuclei with Dapi. The experiment was performed in triplicate.
Figure 5
Figure 5
Protective activity of different EVs preparation and of recombinant SerpinB3. (A) Histograms represent the percent of gain of survival of HepG2 cells treated with 250 μM H2O2 for 72 h in presence of different EVs or of recombinant SerpinB3, compared to untreated cells. SB3 concentration was identical to that detected in the corresponding EVs-SB3 preparations. (B) Histograms represent the percent of gain survival in cardiomyocytes treated with 250 μM H2O2 in presence of different EVs preparations at 1.00 × 107/mL concentration or of recombinant SerpinB3, compared to untreated cells. Both experiments were performed in triplicate. (C) Fluorescence images of Live and Dead assay in primary cardiomyocyte treated for 2 h with 250 μM H2O2 in presence of different EVs preparations or of recombinant SerpinB3. Original magnification 63×.
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