Rational control of combined photothermal and photodynamic therapy for effective eradication of biofilms

New therapies are essential for eliminating antibiotic-resistant bacteria and their biofilms, which are a major global health threat, causing millions of deaths annually. Here, we demonstrate a combination of photodynamic therapy (PDT) and photothermal therapy (PTT) for the inhibition of biofilms of Pseudomonas aeruginosa and Staphylococcus epidermidis using aminolevulinic acid (ALA)-loaded polyacrylic acid-coated superparamagnetic iron oxide nanoparticles (PAA-SPIONs) at 200, 600 and 1000 μg mL^-1 Fe concentrations under 640 nm (0.75 W cm^-2), 808 nm (2.6 W cm^-2) and 640 + 808 nm (0.75 + 2.6 W cm^-2, 20 min) irradiation. PTT experiments indicate ALA/PAA-SPION concentration-dependent heating up to 10.2 °C for PAA-SPIONs and 9.3 °C for ALA/PAA-SPIONs under combined 640 + 808 nm laser excitation. Bacterial growth inhibition by ALA/PAA-SPIONs was investigated with and without laser irradiation for 10 min using 150 and 600 μg Fe per mL or 0.5 mM and 2 mM ALA on both bacterial types. These experiments indicate a 3 to 6-log reduction in P. aeruginosa compared to control samples (without nanoparticles or a laser) with increasing Fe and ALA concentrations. Growth was completely inhibited by ALA/PAA-SPIONs under 640 + 808 nm irradiation. ALA/PAA-SPIONs caused growth inhibition of S. epidermidis between 2-log and 4-log with increasing wavelengths, Fe and ALA doses. PAA-SPIONs and a laser together inhibited the biofilms of P. aeruginosa with 3 to 11-log reductions with increasing laser wavelengths. The reduction of the biofilm with ALA/PAA-SPIONs and a laser reaches 8-log for 640 nm and 13-log for 808 nm excitation. We accurately model the wavelength, time, and nanoparticle concentration dependence of PTT for the first time. These results pave the way for effective PDT/PTT elimination of biofilms of P. aeruginosa and S. epidermidis.