Characterization of the cardioprotective potential of discrete extracellular vesicles populations from second-trimester human amniotic fluid against Doxorubicin cardiotoxicity

Anthracyclines such as Doxorubicin (Dox) are currently broadly used in chemotherapy, representing gold-standard drugs in oncology. Nevertheless, their therapeutic use is limited by cumulative dose-dependent cardiotoxicity, which may evolve into heart failure and long-term morbidity among cancer survivors. In this context, extracellular vesicles (EVs) are emerging as promising cell-free therapeutic tools due to their ability to deliver bioactive molecules and modulate survival, redox balance, inflammation, and mitochondrial function. This PhD project investigated two EV populations: (i) EVs derived from human amniotic fluid stem cells (hAFSC-EVs), obtained through either ultracentrifugation (UC) or ultracentrifugation combined with size-exclusion chromatography (UC+SEC), and (ii) EVs directly isolated from acellular second-trimester human amniotic fluid (hAF-EVs). Three complementary approaches were pursued. Considering that previous work by the lab team, where I carried out my PhD, had shown that the total secretome of human amniotic fluid stem cells (namely the in vitro cell-conditioned medium, hAFSC-CM) had relevant cardioprotective effects against Dox-related cardiotoxicity in vitro and in vivo, I first validated that hAFSC-EVs can recapitulate the effect of the whole secretome by providing significant protection against Dox-induced apoptosis and DNA damage in a dose-dependent manner on neonatal mouse cardiomyocytes in vitro. Proof-of concept engineering with a cardiac homing peptide via copper-free click chemistry was also attempted to enhance myocardial tropism of hAFSC-EVs against such a scenario. Secondly, hAF-EVs were also profiled and their paracrine capacity tested on 3D human cardiac microtissues, where they counteracted oxidative stress and fibrosis, supported by proteomic and transcriptomic signatures consistent with anti-inflammatory and antioxidant functions. Third, hAFSC-EVs and hAF-EVs were then comparatively assessed in a more clinically relatable model of accelerated cardiac ageing as driven by oxidative stress on human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) subjected to Dox induced premature senescence. Both hAFSC- and hAF-EVs reduced oxidative stress, but hAFSC-EVs only maintained mitochondrial function and ATP production, impaired by Dox. Mechanistically, hAFSC-EVs exerted stronger metabolic effects, likely due to enrichment in mitochondrial complex proteins, while hAF-EVs offered an advantage in yield and scalability. Altogether, these findings establish amniotic fluid-derived EVs - whether progenitor cell derived or directly retrieved from the biological fluid - as robust cardioprotective candidates. While hAFSC-EVs provide superior bioenergetic support, hAF-EVs represent a practical, abundant, and readily scalable source, thus broadening their translational potential.