Development of biotechnological drugs with aptamer targeted exosome-mediated and lysosomotropic effective for the treatment of cervical cancer

Objective: Exosomes are natural extracellular vesicles found in biological fluids that carry many cheques from biological molecules to drugs. In recent years, aptamers consisting of DNA or RNA sequences have been essential in targeting drug delivery systems. Cisplatin (cis), Chloroquine (klor), and Raloxifene (ral) are in lysosomotropic structure, and new carrier systems and molecular targeting are needed to increase the biological activities of these drugs. This study aimed to load chloroquine, raloxifene, and cisplatin into exosomes and target the carrier system with aptamers. The developed formulations investigated the cytotoxic, lysosomotropic, apoptotic, and autophagic effects in HeLa cervical cancer cells. Material and Methods: Exosomes released from HeLa cells were isolated by ultracentrifugation and characterized by western blot, SEM, and zeta analysis. Chloroquine, raloxifene, and cisplatin were individually loaded into exosomes by electroporation, and aptamers specific to HeLa cells were bound to exosomes. Exo-apt-drug formulations (Exo-apt-cis, Exo-apt-klor, and Exo-apt-ral) were incubated with HeLa cells for 24 hours, MTT determined their cytotoxicity, and their lysosomotropic activity was determined by Lysotracker fluorescent staining, apoptotic (Bax, BCL- 2) and its autophagic effects (LC3AB, Beclin-1) were examined by western blot and qPCR. Results: The exosomes isolated from HeLa cells contained specific surface markers such as CD9, CD63, and CD81 and were 66±12 nm in size. In HeLa cells, Exo-apt-cis, Exo-apt-klor, and Exo-apt-ral have been shown to reduce cell proliferation at a lower concentration than Cisplatin, Chloroquine and Raloxifene. Exo-apt-cis, Exo-apt-klor, and Exo-apt-ral formulations bound explicitly to HeLa cells and facilitated the transport of drugs to lysosomes. Exo-apt-cis, Exo-apt-klor, and Exo-apt-ral formulations increased protein and gene expressions of Bax, LC3AB, and Beclin-1 and decreased protein and gene expressions of BCL-2. Conclusion: In the developed delivery system loaded with cisplatin, kloroquine, and raloxifene in the targeted exosome with aptamer, easy entry of lysosomotropic drugs into the cell was ensured, and their biological activity was increased. The findings of this study will shed light on new biotechnological drug development studies for cervical cancer treatment.