Antimicrobial photodynamic inactivation: a bright new technique to kill resistant microbes

Abstract

Photodynamic therapy (PDT) uses photosensitizers (non-toxic dyes) that are activated by absorption of visible light to form reactive oxygen species (including singlet oxygen) that can oxidize biomolecules and destroy cells. Antimicrobial photodynamic inactivation (aPDI) can treat localized infections. aPDI neither causes any resistance to develop in microbes, nor is affected by existing drug resistance status. We discuss some recent developments in aPDI. New photosensitizers including polycationic conjugates, stable synthetic bacteriochlorins and functionalized fullerenes are described. The microbial killing by aPDI can be synergistically potentiated (several logs) by harmless inorganic salts via photochemistry. Genetically engineered bioluminescent microbial cells allow PDT to treat infections in animal models. Photoantimicrobials have a promising future in the face of the unrelenting increase in antibiotic resistance.

Figure 1. Jablonski diagram showing photochemical pathways in aPDI

The ground state ¹PS absorbs a photon to form excited singlet state ¹PS* that can undergo intersystem crossing (IC) to form the triplet state ³PS*. This long-lived species can undergo energy transfer (Type II) to form singlet oxygen ¹O₂* or elkectron transfer (Type I) to form hydroxyl radicals HO•. Both these ROS are capable of killing a broad spectrum of pathogens.

Figure 2. aPDI with PEI-ce6 and free ce6

(A) Chemical structure of PEI-ce6. (B) Killing of Gram-positive Staphylococcus aureus incubated for 10 min with 10 μM PEI-ce6 or free ce6 and illuminated with increasing fluences of 660 nm light. (C) Same as B but with Gram-negative Pseuodomonas aeruginosa.

Figure 3. aPDI with cationic fullerenes (bucky-balls)

(A) Chemical structures of BB6 (3 cationic charges) and LC16 (10 cationic charges). (B) Killing of S. aureus, Gram-negative Escherichia coli, and fungal yeast Candida albicans incubated for 10 min with 10μM BB6 and illuminated with increasing fluences of broad-band white light (400-700 nm). (C) Killing of S. aureus, and E. coli incubated for 10 min with 10μM LC16 and illuminated with increasing fluences of UVA light (360±20 nm).

Figure 4. aPDI with cationic bacteriochlorins

(A) Chemical structures of asymmetrical dicationic BC38 and symmetrical tetracationic BC31. (B) Killing of Enterococcus faecalis, Gram-negative Acinetobacter baumannii, and fungal yeast Cryptococcus neoformans incubated for 10 min with increasing concentrations of BC38, and illuminated with 10 J/cm² of 732 nm laser. (C) Killing of S. aureus, E. coli, and C. albicans incubated for 10 min with increasing concentrations of BC31, and illuminated with 10 J/cm² of 732 nm laser.

Figure 5. Potentiation of aPDI by addition of inorganic salts

(A) E. coli, methylene blue and red light is potentiated by sodium azide. (B) S. aureus, cationic fullerene LC16 and UVA light is potentiated by potassium iodide. (C) C. albicans, titanium dioxide nanoparticles and UVA light is potentiated by sodium bromide.

Figure 6. aPDI for wound infections in vivo

Mice with excisional wounds received 5X10(6) CFU bioluminescent P. aeruginosa, followed after 30 min by pL-ce6 conjugate (50 μL of 200 μM ce6 equivalent) and after 30 min by successive exposures to aliquots of 660 nm laser. (A1-A8) Representative bioluminescence images of PDT treated mice. (B1-B5) Representative bioluminescence images of dark control mice (conjugate no light). (C) Quantification of bioluminescence signals from the four groups of mice. (D) Kaplan-Meier survival curves of the four groups of mice.