Non-Clinical Investigation of Tuberculosis Drugs: Conjugated Norbornene- Based Nanocarriers Toxic Impacts on Zebrafish

Introduction: Rifampicin conjugated (R-CP) and rifampicin-isoniazid dual conjugated (RI-CP) norbornene-derived nanocarriers are newly designed for pH stimuli-responsive delivery of tuberculosis (TB) drugs. Its biosafety level is yet to be well established. Objectives: This study aimed to assess the impacts of the nanocarriers on liver cells using the zebrafish animal model and human liver cell line model (HepG2). Methods: Initially, lethal dose concentration for the norbornene-derived nanocarrier systems in zebrafish was determined. The toxic effects were analysed at the sub-lethal drug concentration by histopathological study, total GSH level, gene expression, and DNA damage in zebrafish liver cells. Fish erythrocyte nuclear abnormalities were also evaluated. Cell viability and oxidative stress level (ROS generation) after exposure to the nanoconjugates were determined using HepG2 cells in the in vitro study. Results: In vivo studies of both R-CP and RI-CP showed 100% mortality at 96 hours for exposure concentration >100mg/l and showed toxic changes in zebrafish liver histology, GSH, and DNA damage levels. Noticeably upregulated PXR, CYP3A, and cyp2p6 genes were observed in RI-CP exposure than in RIF or R-CP molecules. The in vitro study revealed a dose-dependent effect on cell viability and ROS generation for RIF, R-CP, and RI-CP exposures in HepG2 cells. Conclusion: The current study reports that the rifampicin conjugated (R-CP) and rifampicin-isoniazid conjugated (RI-CP) norbornene derived nanocarriers exhibit enhanced toxic responses in both adult zebrafish and HepG2 cells. The pH-sensitive norbornene-derived nanocarriers on conjugation with different drugs exhibited varied impacts on hepatic cells. Hence the present investigation recommends a complete metabolomics analysis and norbornene carrier-drug interaction study to be performed for each drug conjugated norbornene nanocarrier to ensure its biosafety.