Thermoreversible Chitosan/β-Glycerophosphate Depot for Sustained Localized Delivery of Doxorubicin in Breast Cancer

Background/Objectives: Direct intratumoral injection of doxorubicin (DOX) can increase local drug exposure, but rapid escape from the tumor limits retention and contributes to systemic toxicity. This study developed a thermoreversible chitosan/β-glycerophosphate (CS/β-GP) hydrogel designed to form an in situ depot and prolong local DOX delivery. Methods: A three-factor Box–Behnken design was used to optimize CS, β-GP, and DOX concentrations against gelation temperature, gelation time, viscosity, and 7-day cumulative release. The optimized formulation was characterized by rheology, injectability, degradation, swelling, porosity, and 4T1 cytotoxicity. Intratumoral retention, plasma and organ biodistribution, antitumor efficacy, body-weight changes, and cardiac toxicity were then evaluated in 4T1 tumor-bearing BALB/c mice. Results: The optimized hydrogel contained 1.684% w/v CS, 6.601% w/v β-GP, and 0.200% w/v DOX, gelled at 36.3 ± 0.55 °C within 1.65 ± 0.28 min, and released 84.66 ± 8.38% of DOX over 7 days. DOX-loaded hydrogel preserved concentration-dependent cytotoxicity. During in vivo studies, hydrogel delivery lowered the observed plasma DOX peak by approximately 55% and retained measurable intratumoral DOX for 168 h, whereas free DOX solution was no longer detectable. Tumor growth suppression was sustained, with tumor volumes of 205.62 ± 36.85 versus 701.69 ± 69.35 mm3 for free DOX solution on day 24. Repeated hydrogel treatment also markedly attenuated cardiac troponin-I (cTnI) and creatine kinase-MB (CK-MB) elevations relative to free DOX solution. Conclusions: Thermoreversible CS/β-GP delivery reshaped DOX exposure from a rapidly dispersing intratumoral dose into a sustained local depot, improving tumor control while reducing the early plasma peak and attenuating cardiac-injury biomarker elevations.