Towards implementing optimized methodologies and in-depth molecular profiling to assess the pharmacological potential of human lung fibroblast exosomes as bio-shuttles for cancer therapeutics

Despite their favorable biopharmaceutical properties, exosomes face standardization challenges in isolation and characterization, hindering the development of EV products suitable for clinical applications. The MRC-5 cell line poses a cellular model approved by regulatory affairs for drug development, facilitating the clinical translation of MRC-5 cell-derived nanotherapeutics. This study aims to: (a) identify the optimal methodology for isolating intact MRC-5 cell-derived exosomes with high purity and integrity suitable for drug delivery applications, (b) elucidate the global miRNA and proteomic profile of the isolated exosomes using high-throughput methodologies and bioinformatics, (c) investigate the exosome uptake capacity towards the "autologous" normal MRC-5, and "heterologous" malignant human lung adenocarcinoma A549 and tongue squamous HSC-3 cells, (d) evaluate the pharmacological profile of doxorubicin (DOX)- and curcumin (CUR)- loaded exosomes towards the normal and tumorigenic cells in vitro. The proposed isolation protocol combining ultrafiltration with membrane-based affinity binding, yielded non-aggregated exosomes with superior physicochemical properties. The miRNA profiling data revealed the enrichment of several tumor-suppressive miRNAs mainly involved in gene regulation pathways, whilst the proteomic profiling highlighted the role of exosomal protein in extracellular matrix remodeling. The drug delivery profiling indicated an "intrinsic" tropism of the exosomes towards the malignant A549 and HSC-3 cells. Exosomal CUR and exosomal DOX inhibited tumor cell proliferation more efficiently than free drugs, with synergistic effects upon co-administration. This study provides a comprehensive morphological, physicochemical and molecular characterization of MRC-5 cell-derived exosomes and validates the "intrinsic" tropism of CUR- and DOX- loaded exosomes towards tumorigenic cells, paving the way towards their further exploitation as drug delivery nanocarriers.