Exosomes as efficient platforms for delivering adenosine-tetra peptide conjugate to pancreatic cancer cells: An in vitro/in silico study

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Pancreatic cancer is a fatal malignancy due to poor response to chemotherapeutic treatments. Although adenosine has demonstrated promising cytotoxic effects, its low half-life has restricted its use in cancer treatment. The tetra peptide of Gly-Phe-Leu-Gly is a cathepsin B substrate, and its conjugation to adenosine may produce a prodrug with fewer adverse effects. This study aimed to prepare a tetra peptide prodrug of adenosine and encapsulate it in two types of cell-derived exosomes. A synthetic prodrug of adenosine was prepared with N-substitution of Gly-Phe-Leu-Gly. Exosomes were isolated from AsPC-1 or RAW 264.7 cell lines. The drug or prodrug loading into exosomes and their release profiles were assessed in PBS at pH 7.4. The exosomes' cellular uptake was analyzed by flow cytometry method. MTT assay and Annexin V/FITC kit was used to measure cytotoxicity and apoptotic profile on the pancreatic cancer cells, respectively. An in silico study by molecular docking simulations and ligand‐binding energy evaluation was also done to study binding affinity of adenosine-tetrapeptide prodrug towards Cathepsin B active site. Exosomes with particle sizes of 100–160 nm were shed more efficiently by RAW 264.7 cell line. Drug loading in exosomes was efficient (∼42 %), and the release profile was sustained for more than 24 h. Cellular uptake was higher in autologous exosomes. AsPC-1-exosomes encapsulating prodrug formulation showed higher cytotoxicity, higher cellular uptake, and more efficacious apoptosis. compared to the free prodrug. Gratifyingly, results of in silico analysis perfectly corroborated the potential binding affinity of adenosine-tetrapeptide prodrug towards Cathepsin B active site with improved energy indices compared to free tetra-peptide.