Phosphatidylcholine-Based Nanoformulation of Ergosterol Peroxide Enhances Anticancer Efficacy in Cervical Cancer Models

Background/Objectives: As a fungus-sourced sterol endoperoxide, ergosterol peroxide possesses extensive antitumor properties. However, unfavorable aqueous solubility, rapid in vivo clearance, and poor accumulation within tumor sites constitute major obstacles restricting its progression toward clinical application. Methods: Herein, we constructed a novel phospholipid nanoformulation by supramolecular self-assembly of ergosterol peroxide with soybean phosphatidylcholine via a thin-film dispersion method. Results: The optimized ergosterol peroxide–soybean phosphatidylcholine nanoformulation exhibited a uniform spherical morphology (~98 nm), excellent colloidal stability in serum-containing medium, and pH-responsive drug release behavior. In vitro, the ergosterol peroxide–soybean phosphatidylcholine nanoformulation demonstrated significantly enhanced cellular uptake in HeLa cervical cancer cells compared with free ergosterol peroxide, achieving markedly superior antiproliferative and pro-apoptotic efficacy through amplified reactive oxygen species generation. Furthermore, the ergosterol peroxide–soybean phosphatidylcholine nanoformulation effectively induced immunogenic-cell-death-associated damage-associated molecular patterns. In TC-1 tumor-bearing mice, intravenous administration of the ergosterol peroxide–soybean phosphatidylcholine nanoformulation exhibited potent dose-dependent antitumor efficacy, with biodistribution analysis confirming enhanced intratumoral ergosterol peroxide accumulation via the enhanced permeability and retention effect. Of particular note, ergosterol peroxide–soybean phosphatidylcholine nanoformulation treatment led to a substantial increase in the abundance of splenic B cells, NK cells, CD4+ helper T-cells and CD8+ cytotoxic T-cells; hematology testing together with histopathological examination further demonstrated only marginal systemic toxicity after treatment. Conclusions: Collectively, this soybean phosphatidylcholine-based nanoplatform provides a feasible and promising pharmaceutical strategy for overcoming the inherent delivery limitations of ergosterol peroxide, offering a potential candidate for cervical cancer treatment.