Preparation of chitosan nanoparticle containing tenofovir alafenamide (TAF) formulations, investigation of genotoxic and mitotoxic effects

Aim: TAF is a new nucleotide reverse transcriptase inhibitor group (NRTI) drug, approved for the treatment of chronic hepatitis B in 2016. In this study, it was aimed to prepare, characterize chitosan nanoparticle containing TAF drug and to investigate mitotoxic/genotoxic effects of TAF drug and its combinations. Material and Method: 45 µM TAF and chitosan nanoparticle were applied to HepG2 cells for six days. Possible genotoxic/cytotoxic effects of TAF and its combinations were evaluated by (Micronuclei) Mn, comet and apoptosis tests. Toxic effects on mitochondria were investigated by evaluating mtDNA, mitochondrial membrane potential (ΔΨm), superoxide anions, ROS levels, and RYR1, PCK1, ATF2 genes expression. Results: In terms of genotoxic tests, Mn and comet values were found higher in TAF and and chitosan nanoparticle containing TAF groups, and chitosan group was also determined for comet test (p<0.05). As an indicator of mtDNA level, COXII gene expression changes were found low in the TAF group, whereas chitosan and chitosan containing TAF groups were high (p<0.05). For ΔΨm, it was observed that chitosan containing TAF and TAF application groups decreased compared to the control (p<0.05). Cellular ROS level was found higher than control in all application groups (p<0.05). There was no difference between the groups in apoptosis test with Annexin V (p>0.05). Among the genes associated with the mitochondria-nucleus signal pathway, the level of gene expression of ATF2 has not changed. RYR1 gene expression increased in chitosan containing TAF group while decreased in chitosan and TAF groups. It was observed that PCK1 gene expression increased in TAF group and decreased in chitosan and chitosan containing TAF groups (p<0.05). Conclusion: TAF has been shown to have genotoxic and mitotoxic effects in HepG2 cells at the studied dose. TAF encapsulated with chitosan and chitosan has not been found to have genotoxic and mitotoxic effects in many parameters. Key Words: Chitosan encapsulation, genotoxicity, hepatitis B, HepG2, mitotoxicity, TAF.