Antimicrobial photodynamic activity of gallium-substituted haemoglobin on silver nanoparticles.

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 αß-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.

Rapid Uptake and Photodynamic Inactivation of Staphylococci by Ga(III)-Protoporphyrin IX.

Antimicrobial photodynamic therapy (aPDT) is a promising method for the topical treatment of drug-resistant staphylococcal infections and can be further improved by identifying mechanisms that increase the specificity of photosensitizer uptake by bacteria. Here we show that Ga(III)-protoporphyrin IX chloride (Ga-PpIX), a fluorescent hemin analog with previously undisclosed photosensitizing properties, can be taken up within seconds by Staphylococcus aureus including multidrug-resistant strains such as MRSA. The uptake of Ga-PpIX by staphylococci is likely diffusion-limited and is attributed to the expression of high-affinity cell-surface hemin receptors (CSHRs), namely iron-regulated surface determinant (Isd) proteins. A structure-activity study reveals the ionic character of both the heme center and propionyl groups to be important for uptake specificity. Ga-PpIX was evaluated as a photosensitizer against S. aureus and several clinical isolates of MRSA using a visible light source, with antimicrobial activity at 0.03 µM with 10 s of irradiation by a 405 nm diode array (1.4 J/cm2); antimicrobial activity could also be achieved within minutes using a compact fluorescent lightbulb. GaPpIX was not only many times more potent than PpIX, a standard photosensitizer featured in clinical aPDI, but also demonstrated low cytotoxicity against HEK293 cells and human keratinocytes. Ga-PpIX uptake was screened against a diverse panel of bacterial pathogens using a fluorescence-based imaging assay, which revealed rapid uptake by several Gram-positive species known to express CSHRs, suggesting future candidates for targeted aPDT.

Small-Molecule-Based Self-Assembled Ligands for G-Quadruplex DNA Surface Recognition.

Most drugs are small molecules because of their attractive pharmacokinetics, manageable development and manufacturing, and effective binding into the concave crevices of bio-macromolecules. Despite these features, they often fall short when it comes to effectively recognizing the surfaces of bio-macromolecules. One way to overcome the challenge of biomolecular surface recognition is to develop small molecules that become self-assembled ligands (SALs) prior to binding. Herein, we report SALs made from 8-aryl-2'-deoxyguanosine derivatives forming precise hydrophilic supramolecular G-quadruplexes (SGQs) with excellent size, shape, and charge complementarity to G-quadruplex DNA (QDNA). We show that only those compounds forming SGQs act as SALs, which in turn differentially stabilize QDNAs from selected oncogene promoters and the human telomeric regions. Fluorescence resonance energy-transfer melting assays are consistent with spectroscopic, calorimetric, and light scattering studies, showing the formation of a "sandwichlike" complex QDNA·SGQ·QDNA. These results open the door for the advent of SALs that recognize QDNAs and potentially the surfaces of other bio-macromolecules such as proteins.