Mechanistic Insights into pH-Responsive Drug Release and Antitumor Effects of an Albumin-Pirarubicin Conjugate.

PURPOSE: This study provides mechanistic insight into the behavior of HSA-THP, a human serum albumin-pirarubicin conjugate linked via a pH-sensitive hydrazone bond. Building upon our previous synthesis, we aimed to elucidate its intracellular uptake, and pH-responsive drug release behavior under acidic intracellular and extracellular conditions in vitro, and to evaluate its in vivo pharmacological properties.

METHODS

Intracellular uptake of HSA-THP was evaluated in colon 26 monolayer cultures using fluorescence imaging and HPLC. Cytotoxic effects were compared in 2D and 3D spheroid cultures. Tissue distribution and antitumor efficacy were assessed in tumor‑bearing mice, while systemic toxicity was evaluated in healthy mice following intravenous administration.

RESULTS

Both intact HSA-THP and released THP were detected within tumor cells, with cleavage likely promoted by acidic intracellular compartments. The conjugate was also inferred to release THP under mildly acidic extracellular conditions, allowing dual-site drug liberation. In 3D spheroids, the cytotoxicity gap between HSA-THP and free THP seen in 2D cultures narrowed markedly, suggesting albumin-mediated tissue penetration and sustained intratumoral release. Pharmacokinetic analysis revealed prolonged plasma retention and preferential tumor accumulation, with higher levels of liberated THP in tumors but minimal amounts in normal tissues. This distribution pattern resulted in improved antitumor activity with reduced systemic toxicity.

CONCLUSIONS

HSA-THP couples albumin's natural trafficking with pH-triggered dual-site drug release, providing mechanistic insight into how albumin conjugation influences drug distribution and activity. These findings establish a fundamental basis for the rational design of albumin-based macromolecular drug conjugates.