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37POTENTIATION OF ANTIMICROBIAL PHOTODYNAMIC INACTIVATION BY INORGANIC SALTS
MICHAEL HAMBLIN
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, South Africa
ABSTRACT
One of the biggest health problems facing the world today is the inexorable rise of multi-antibiotic resistance amongst a wide range of pathogens, including Gram-positive or Gram-negative bacteria and fungi. Antimicrobial photodynamic inactivation (aPDI) uses visible/NIR excitation of a photosensitizer to produce the reactive oxygen species (ROS) singlet oxygen (Type 2) and hydroxyl radicals (Type1) that are both highly toxic to microbial cells. If the photosensitizer and the light are introduced into the infected area the selectivity is excellent [1]. We have discovered that antimicrobial photodynamic inactivation (aPDI) can be strongly potentiated by addition of the non-toxic salt potassium iodide [2]. This approach works with a wide variety of different photosensitizers including those possessing cationic charges that bind to microbial cells, and those neutral or anionic compounds that are completely ineffective in photoinactivating Gramnegative cells, but can kill > 6 logs in presence of 100 mM KI. The approach is broad spectrum in nature and works with methicillin-resistant Staphylococcus aureus (MRSA), a range of Gram-negative bacteria and Candida albicans. The major mechanism is likely to involve the addition of singlet oxygen to iodide to form peroxyiodide, which then decomposes via two possible routes: (a) formation of the stable species, free iodine and hydrogen peroxide; (b) formation of short-lived radicals I2^- + HOOv When the PS binds to the microbial cells, killing by the short-lived radicals becomes significant, while for Gram-negative cells with Photofrin [3] or Rose Bengal [4], killing by I3- and H2O2 are dominant [5]. This can be studied by comparing "in" (all ingredients together, "after" cells added after light, and "spin" KI and light added after cells were incubated with PS and centrifuged. KI could potentiate RB-PDT in a mouse model of skin abrasions infected with bioluminescent P. aeruginosa demonstrating possible in vivo applications [4]. We also studied two porphyrins TMPyP4 (tetracationic) and TPPS4 (tetraanionic). Surprisingly TPPS4 was an excellent PS for MRSA and Candida, and could eradicate Gram-negative species when KI and light were added after a spin, showing it was bound to the surface [6]. Another tetraanionic phthalocyanine (ClAlPCS4) did not show this behavior. We conclude that TPPS4 behaves as if it has some cationic character in the presence of bacteria.
One of the applications of KI potentiation of aPDI that may have real clinical relevance, is the treatment of oral candidiasis. Patients with immunosuppression are at risk of developing an infection in the mouth caused by the yeast C. albicans, which is painful and difficult to treat. We showed that methylene blue (MB) plus KI excited by red light could successfully treat oral candidiasisin a mouse model [7]. We went on to carry out a clinical trial conducted on 21 adult AIDS patients with C. albicans oral candidiasis who received two treatments using MB + KI excited by red light, and showed that C. albicans CFUs were significantly reduced [8]. Additional applications of aPDI using MB + KI and red light that showed some success, were endodontic disinfection in root canals in teeth [9], and Gram-negative bacterial cystitis (bladder infection) in female rats [10].
Other inorganic salts such as sodium azide, potassium thiocyanate, potassium selenocyanate, potassium bromide and sodium nitrite also produce increased killing of a broad range of pathogens by up to one million times [11]. The mechanisms of potentiation are different for different salts. At one extreme of the salts is sodium azide, that quenches singlet oxygen but can produce azide radicals (presumed to be highly reactive) via electron transfer from photoexcited phenothiazinium dyes, in a so-called paradoxical potentiation process [12]. As discussed above KI is oxidized to molecular iodine by both Type I and Type II PSs, but may also form reactive iodine species. Potassium bromide is oxidized to hypobromite, but only by titanium dioxide photocatalysis (Type I) [13]. Potassium thiocyanate appears to require a mixture of Type I and Type II photochemistry to first produce sulfite, that can then form the sulfur trioxide radical anion [14]. Potassium selenocyanate can react with either Type I or Type II ROS (or indeed with other oxidizing agents) to produce the semi-stable selenocyanogen (SCN)2 that can attack bacteria [15]. Despite the similar chemical structures of thiocyanate and selenocyanate anions, the mechanisms of potentiation of aPDI turned out to be suprisingly different [16]. Finally, sodium nitrite may react with either Type I or Type II PSs to produce peroxynitrate (again, semi-stable) that can kill bacteria and produce nitrotyrosine by tyrosine nitration [17]. Many of these salts (except azide) are non-toxic, and may be clinically applicable.
REFERENCES
[1] M. R. Hamblin, "Antimicrobial photodynamic inactivation: a bright new technique to kill resistant microbes," Curr Opin Microbiol, 33, 67-73 (2016).
[2] D. Vecchio, A. Gupta, L. Huang, G. Landi, P. Avci, A. Rodas, and M. R. Hamblin, "Bacterial photodynamic inactivation mediated by methylene blue and red light is enhanced by synergistic effect of potassium iodide," Antimicrob Agents Chemother, 59(9), 5203-12 (2015).
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[16] L. Huang, W. Xuan, T. Sarna, and M. R. Hamblin, "Comparison of thiocyanate and selenocyanate for potentiation of antimicrobial photodynamic therapy," J. Biophotonics, 12(1), e201800092 (2019).
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