Hidrogel injetável contendo vesículas extracelulares modificadas com péptido para potenciar a cardioproteção

The main objective of this project is the development of a therapeutic strategy to increase the therapeutic efficacy of sEVs in the heart after infarction. This strategy is based in three main observations: (i) it has been shown that Gal-3 is highly expressed in the left ventricle during the early stages following MI [10]; (ii) Peptides that target and inhibit Gal-3 are cardioprotective [13]; (iii) sEVs modified with Gal-3-targeting peptide are taken up by cells overexpressing Gal-3 (from our preliminary data). To accomplish the main objective, the surface of sEVs from mesenchymal stromal cells (MSCs) isolated from the Wharton jelly will be engineered with an optimal density of a Gal-3-targeting peptide and embedded in a hydrogel that will serve as a vehicle for their local delivery to the ischemic myocardium. The following specific aims will be pursued: Aim 1: To develop hydrogels loaded with engineered sEVs. We have previously reported the embedding of sEVs in hyaluronic acid hydrogels for skin regeneration[14]. Our unpublished preliminary results show that we can modify sEVs with a peptide targeting Gal-3 via click-chemistry (Fig. 1), leading to increased uptake and bioactivity in endothelial cells (Fig. 3). The objectives of this WP are: (i) to modify and characterize sEVs with different densities of peptides (low, medium and high) for maximal effect; (ii) to investigate the uptake and bioactivity of engineered sEVs in endothelial cells and cardiomyocytes; (iii) to demonstrate the anti-fibrotic effect of the peptide immobilized on the sEV surface; (iv) to embed engineered sEVs in hyaluronic acid hydrogels and study sEV stability, release profile, and demonstrate bioactivity over time. Aim 2: To evaluate the bioactivity of sEVs in ex vivo models of myocardial injury. Myocardial slices were reported as a useful model to study cellular, molecular, and functionality changes related to fibrosis [17]. We will also use myocardial slices from mice and explanted human hearts. We will have access to hearts of transplanted individuals with heart failure, giving us the advantage of testing our therapy in clinically relevant samples. Our unpublished preliminary results show that we can increase the expression of Gal-3 and decrease contractility in rat myocardial slices exposed to ischemia-reperfusion (Fig. 4). We also show that engineered sEVs accumulate more than native sEVs in ischemic slices (Fig. 6). The objectives of this WP are: (i) to evaluate the functional impact of sEVs on tissue contractility; (ii) to study in detail the uptake of native and engineered sEVs by different cells of the cardiac tissue and correlate with the expression of Gal-3 (published data indicates that Gal-3 is expressed both by cardiomyocytes and endothelial cells after MI[10]) and (iii) to demonstrate the anti-fibrotic effect of engineered sEVs. Aim 3: To demonstrate the cardioprotective effect of engineered sEVs in a mouse model of MI. The objectives of this WP are: (i) to evaluate the retention of sEVs in cardiac tissue; (ii) to assess the effect of sEVs on cardiac function; (iii) to evaluate vascular recovery and cell survival; (iv) to determine the impact of sEVs on the molecular program leading to fibrosis.