Abstract

To overcome the clinical limitations of amphotericin B (AmB), such as poor aqueous solubility, significant systemic toxicity, and dose-dependent nephrotoxicity, we developed a pH-responsive nanodelivery system (HH18@AmB) based on engineered human serum albumin (HSA) fused with 18 histidine residues (HH18). The histidine-rich design enables HH18 to undergo hydrophobicity-driven self-assembly, encapsulating AmB within uniform nanoparticles with an average size of ~150 nm and a high drug-loading capacity of 23.08 ± 2.93%. In vitro release studies confirmed a sustained, pH-dependent drug release profile under acidic conditions mimicking the microenvironment of fungal infections. Notably, the encapsulated AmB remains predominantly in the monomeric state, which is associated with reduced toxicity. Cytotoxicity assays demonstrated that HH18@AmB exhibited significantly enhanced biocompatibility against RAW 264.7 macrophages and HEK 293 T renal cells, maintaining cell viability above 65%, while retaining potent antifungal activity. The minimum inhibitory concentration against Candida albicans was determined to be 4 μg/mL, and at this concentration, HH18@AmB inhibited biofilm formation by 55%. In vivo pharmacokinetic evaluation revealed a prolonged circulation time, with the half-life (t₁/₂) extended by 2.12-fold and the area under the curve (AUC 0-t ) increased by 9.39-fold compared to free AmB. Moreover, HH18@AmB exhibited reduced renal accumulation, indicating alleviated nephrotoxicity. By integrating pH-responsive drug release, HSA-mediated stability, and markedly improved safety profile, HH18@AmB represents a promising nanotherapeutic strategy to enhance the clinical utility of AmB, particularly for the treatment of drug-resistant fungal infections.