Preparation and in vitro evaluation of amphotericin B-chondroitin sulfate polymer prodrug

Amphotericin B (AmB) remains the gold-standard antifungal agent, yet its clinical utility is severely limited by dose-dependent hemolytic and nephrotoxic side effects arising from its aggregation-prone nature and non-selective membrane binding. To address this challenge, we developed a polymer prodrug by conjugating AmB to oxidized chondroitin sulfate (CSox) via a Schiff-base linkage designed to be pH-labile, selected for CS’s CD44-targeting capability, intrinsic anti-inflammatory activity, and favorable polyelectrolyte properties. The resulting CSox-AmB conjugate self-assembled into uniform micelles (141.60 ± 4.03 nm, zeta potential −25.47 ± 0.59 mV) with a drug loading of 7.07 ± 0.13%, maintaining AmB predominantly in its monomeric state (A348/A409 = 0.865 < 1). The prodrug exhibited sustained release (∼68% at 24 h) while preserving antifungal activity comparable to free AmB against Candida albicans. Concurrently, it attenuated mammalian cell cytotoxicity by 2.3- to 2.5-fold and markedly attenuated hemolytic activity in vitro (<1% at 25 μg·mL−1). This integrated design-conjugation enabling self-assembly, assembly enabling monomeric stabilization, and controlled release enabling efficacy with markedly reduced toxicity-positions CSox-AmB as a promising prodrug strategy that uncouples the antifungal activity of AmB from its inherent toxicities, warranting further in vivo evaluation toward clinical translation.