Antimicrobial photodynamic activity of gallium-substituted haemoglobin on silver nanoparticles

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), a major scourge in skin and soft-tissue infections, expresses surface-bound haemoprotein receptors that can be exploited for the targeted delivery of photosensitizers. We have developed a nanosized agent for targeted antimicrobial photodynamic therapy (aPDT), comprised of GaPpIX (a hemin analog with potent photosensitizer activity) encapsulated in haemoglobin (GaHb), mounted on 10 nm Ag nanoparticles (AgNPs). The average GaHb-AgNP contains 28 GaPpIX units stabilized by Hb αβ-dimer units. Eradication (>6-log reduction) of S. aureus and MRSA can be achieved by a 10-second exposure to 405 nm irradiation from a light-emitting diode (LED) array (140 mW cm⁻²), with GaHb-AgNP loadings as low as 5.6 μg mL⁻¹ for S. aureus and 16.6 μg mL⁻¹ for MRSA, corresponding to nanomolar levels of GaPpIX. This reduction in bacterial count is several orders of magnitude greater than that of GaHb or free GaPpIX on a per mole basis. The GaHb–AgNP platform is also effective against persister MRSA and intracellular MRSA, and can provide comparable levels of aPDT with a 15-minute irradiation by an inexpensive compact fluorescent lightbulb. Collateral phototoxicity to keratinocytes (HaCaT cells) is low at the GaHb-AgNP concentrations and fluences used for aPDT. GaHb adsorbed on 10 nm AgNPs is much more potent than that on 40 nm AgNPs or 10 nm AuNPs, indicating that both size and plasmon-resonant coupling are important factors for enhanced aPDT. Electron microscopy analysis reveals that GaHb-AgNPs are not readily internalized by S. aureus but remain attached to the bacterial cell wall, the likely target of photo-oxidative damage.