Triple-targeting nanosystems with synergistic effects on iron Trojan horse, fluoroquinolone antibiotics, and photodynamic therapy specifically kill intracellular MRSA.

Rationale: Iron is necessary for the survival of microorganisms. The uptake network is highly expressed by host cells and methicillin-resistant Staphylococcus aureus (MRSA) and competes with heme iron. Gallium porphyrin (GaPR), a heme mimetic compound, was synthesized to develop an innovative nanosystem as a triple-targeting agent for uptake network recognition. GaPR is also used as a dual therapeutic molecule of "iron trojan horse" and photosensitizer to achieve synergistic antibacterial effects with levofloxacin to eradicate intracellular MRSA-a problem that conventional therapeutic techniques cannot overcome due to limited drug penetration, antibiotic resistance, and off-target effects. Methods: A library of hemimetic compounds was synthesized. GaPR was selected as the optimal candidate owing to its antibacterial activities and competitive binding affinity for iron uptake receptors. The optimal GaPR and the photosensitizer tetrakis-(4-carboxyphenyl)-porphyrin (TCPP) were used to prepare a levofloxacin (Lev)-loaded zirconium-based organometallic scaffold (Lev-GaPR-PCN). Hyaluronic acid (HA) was linked to the Lev-GaPR-PCN surface via ROS-reactive thioketal bonds (TK). The triple-targeting performance and synergistic efficacy of HA-Lev-GaPR-PCN against intracellular MRSA were tested in vitro and in vivo. Results: GaPR showed strong bactericidal activity against MRSA by interfering with iron metabolism. GaPR-PCN exhibited excellent binding ability with host-derived heme-binding proteins (Hpx/LRP1 and Hpg/CD163) and the iron-regulated surface determinant (Isd) system of MRSA for infection site, infected cell, and intracellular targeting. HA coating enabled covert circulation and decreased nonspecific uptake by healthy cells (< 5% fluorescence intensity after 6 h) while promoting infection-induced release via hyaluronidase and ROS. In vitro, HA-Lev-GaPR-PCN achieved 3.42-fold greater colocalization with intracellular MRSA (Pearson correlation: 0.41 vs. 0.12 for PCN-224 controls) and decreased the extracellular/intracellular minimum inhibitory concentrations (MICs) of Lev under PDT from 8/64 μg/mL to 1/2 μg/mL. In vivo, it resulted in prolonged retention (72 h vs. 36 h) and a 1.5-2.5-fold greater fluorescence intensity at infection sites for non-HA nanosystems. Compared to Lev alone, it decreased the bacterial load by 501-fold (2.7 log) and abscesses (diameter: 0.6 cm vs. 3.3 cm) by combining chemical, metabolic, and physical antibacterial mechanisms without causing toxic effects. Conclusion: This study represents a paradigm shift in intracellular infection therapy for MRSA and other resistant bacteria using a hemimetic compound as a triple-targeting and dual therapeutic agent that provides a streamlined, clinically feasible solution with high efficacy and specificity.