Engineered Human Ferritin Nanocage Enhances Intracellular Vancomycin Delivery and Activity Against Gram-Positive...

The limited cellular penetration of vancomycin (Van) restricts its intracellular availability and represents a major challenge for treating infections caused by intracellular Gram-positive bacteria. We investigated whether the engineered ferritin nanocage HFn-D93G could improve intracellular Van delivery while preserving the intrinsic antibacterial activity of Van.

HFn-D93G-Van was characterized for drug loading, nanocage integrity, particle size, pH-dependent release, and biocompatibility; molecular docking examined Van-protein interactions. Cellular uptake, potential uptake pathways, and subcellular distribution were examined in RAW264.7 cells, while intracellular Van accumulation, spatial association with bacteria, antibacterial activity, and cytokine responses were assessed in infected macrophages. In vivo efficacy was evaluated in a murine S. aureus peritonitis-sepsis model (n = 6 mice per group) by assessing intracellular bacterial burden in peritoneal macrophages, major-organ bacterial burdens, serum cytokines, and histopathology.

HFn-D93G-Van achieved 28.71 ± 1.82% drug loading while retaining nanocage morphology and α-helical structure. At 48 h, Van release increased from 27.1% at pH 7.4 to 67.4% at pH 6.0 and 84.7% at pH 5.0. MICs remained comparable to free Van, whereas intracellular Van increased 7.34-fold (P < 0.0001). Uptake was energy dependent and involved multiple endocytic pathways, with CD71-associated involvement. HFn-D93G-Van showed greater spatial association with intracellular bacteria and reduced S. aureus, S. epidermidis, and E. faecium burdens by 2.00, 1.62, and 1.76 log₁₀ units, respectively, with lower cytokine levels. In vivo, peritoneal-macrophage intracellular S. aureus was reduced by 0.88 log₁₀ units versus free Van (P < 0.0001), while major-organ burdens were comparable between Van and HFn-D93G-Van. Histopathological alterations appeared less pronounced after both treatments.

HFn-D93G increased intracellular Van accumulation and reduced intracellular bacterial burdens, with the intracellular delivery advantage further observed in peritoneal macrophages in vivo. Further studies of pharmacokinetics, biodistribution, localized infection models, immunogenicity, and long-term safety are required to define its preclinical potential.