Objective To investigate the subcellular distribution patterns of the combination of cantharidin (CTD) and baicalin (BA) based on cell pharmacokinetics and elucidate the synergistic mechanism against hepatocellular carcinoma. Methods Using human hepatoma HepG2 cells as the model, the dynamic concentrations of the drugs in whole cells and organelles (nucleus, mitochondria, endoplasmic reticulum, and lysosomes) over 12 h after administration of single drugs (CTD 6 μg/mL, BA 30 μg/mL) and their combination (CTD 6 μg/mL + BA 30 μg/mL) were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Pharmacokinetic parameters were calculated using a non-compartmental model with Phoenix WinNonlin software. Results At the whole-cell level, the combination increased the intracellular area under the curve (AUC_0—t) of CTD by 48.9% and decreased its clearance (P < 0.05), but did not significantly affect the pharmacokinetics of BA. At the subcellular level, the combination significantly increased the AUC_0—t of CTD and BA in nucleus, lysosome, mitochondria and endoplasmic reticulum by 93.5%, 46.4%, 38.3%, 52.3%, and 68.4%, 40.0%, 41.0%, and 46.7%, respectively (P < 0.05, 0.01). Furthermore, the time to reach peak concentration (t_max) for both drugs in mitochondria was earlier in the combination group, and the mean residence time (MRT_0—t) of CTD in the endoplasmic reticulum was significantly prolonged (P < 0.01), indicating a spatiotemporally synergistic drug delivery optimization. Conclusions The combination of CTD and BA could synergistically optimize the distribution of drugs in key subcellular structures such as nucleus, mitochondria, endoplasmic reticulum and lysosomes, and may enhance the anti-hepatocellular carcinoma effect by inducing DNA damage, accelerating mitochondria-mediated apoptosis and promoting endoplasmic reticulum stress, which provides a theoretical basis for the design of traditional Chinese medicine combinations based on organelle targeting.

cantharidin  /  baicalin  /  traditional Chinese medicine combination  /  anti-hepatocellular carcinoma  /  cellular pharmacokinetics  /  subcellular distribution