Cationic Porphycenes as Potential Photosensitizers for Antimicrobial Photodynamic Therapy

Structures of typical photosensitizers used in antimicrobial photodynamic therapy are based on porphyrins, phthalocyanines, and phenothiazinium salts, with cationic charges at physiological pH values. However, derivatives of the porphycene macrocycle (a structural isomer of porphyrin) have barely been investigated as antimicrobial agents. Therefore, we report the synthesis of the first tricationic water-soluble porphycene and its basic photochemical properties. We successfully tested it for in vitro photoinactivation of different Gram-positive and Gram-negative bacteria, as well as a fungal species (Candida) in a drug-dose and light-dose dependent manner. We also used the cationic porphycene in vivo to treat an infection model comprising mouse third degree burns infected with a bioluminescent methicillin-resistant Staphylococcus aureus strain. There was a 2.6-log10 reduction (p −2 of red light

Data_Sheet_1_Antimicrobial Blue Light for Prevention and Treatment of Highly Invasive Vibrio vulnificus Burn Infection in Mice.pdf

Vibrio vulnificus is an invasive marine bacterium that causes a variety of serious infectious diseases. With the increasing multidrug-resistant variants, treatment of V. vulnificus infections is becoming more difficult. In this study, we explored antimicrobial blue light (aBL; 405 nm wavelength) for the treatment of V. vulnificus infections. We first assessed the efficacy of aBL against five strains of V. vulnificus in vitro. Next, we identified and quantified intracellular porphyrins in V. vulnificus to provide mechanistic insights. Additionally, we measured intracellular reactive oxygen species (ROS) production and bacterial membrane permeabilization following aBL exposures. Lastly, we conducted a preclinical study to investigate the efficacy and safety of aBL for the prevention and treatment of burn infections caused by V. vulnificus in mice. We found that aBL effectively killed V. vulnificus in vitro in both planktonic and biofilm states, with up to a 5.17- and 4.57-log10 CFU reduction being achieved, respectively, following an aBL exposure of 216 J/cm2. Protoporphyrin IX and coproporphyrins were predominant in all the strains. Additionally, intracellular ROS was significantly increased following aBL exposures (P < 0.01), and there was evidence of aBL-induced permeabilization of the bacterial membrane (P < 0.0001). In the preclinical studies, we found that female mice treated with aBL 30 min after bacterial inoculation showed a survival rate of 81% following 7 days of observation, while only 28% survival was observed in untreated female mice (P < 0.001). At 6 h post-inoculation, an 86% survival was achieved in aBL-treated female mice (P = 0.0002). For male mice, 86 and 63% survival rates were achieved when aBL treatment was given 30 min and 6 h after bacterial inoculation, respectively, compared to 32% survival in the untreated mice (P = 0.0004 and P = 0.04). aBL did not reduce cellular proliferation or induce apoptosis. We found five cytokines were significantly upregulated in the males after aBL treatment, including MCSF (P < 0.001), MCP-5 (P < 0.01), TNF RII (P < 0.01), CXCL1 (P < 0.01), and TIMP-1 (P < 0.05), and one in the females (TIMP-1; P < 0.05), suggesting that aBL may induce certain inflammatory processes. In conclusion, aBL may potentially be applied to prevent and treat V. vulnificus infections.</p

Brain lesion size in the mice.

<p>Example whole brain cross-sections taken from mice sacrificed at day 4 (A–D), day 7 (E–H), day 14 (I–L) and day 28 (M–P). Mice were taken from TBI sham Tx group (A, E, I, M); TBI 1 laser Tx group (B, F, J, N); TBI 3 laser Tx group (C, G, K, O); TBI 14 laser Tx group (D, H, L, P). (Q) Mean (n = 2–3) brain fractional lesion volumes calculated as described at 1, 2, and 4 weeks in brains of mice in 4 groups consisting of sham TBI mice, 810-nm laser treated TBI mice (18 J/cm² delivered at 25 mW/cm² given 1, 3 or 14 laser Tx. ** P<0.01; * p<0.05 vs TBI sham and TBI 14 laser Tx groups. One way ANOVA.</p

NSS scores of the mice.

<p>Mean (n = 8–14) NSS scores measured over 4 weeks of mice in 9 groups consisting of sham TBI mice, sham control mice, 810-nm laser treated TBI mice (18 J/cm² delivered at 25 mW/cm²). (A) Mice and controls given 1 laser Tx or 1 sham Tx at 4 hours post TBI; (B) Mice and controls given 3 daily laser Tx or 3 sham Tx; (C) Mice and controls given 14 daily laser Tx or 14 sham Tx. * p<0.05; ** P<0.01; *** p<0.001. One way ANOVA. (D) Mean areas under curve of NSS time courses from mice in different groups. P values given were determined by one way ANOVA.</p

Visualization 1: Optical lens-microneedle array for percutaneous light delivery

Alignment optimization of OMNA Originally published in Biomedical Optics Express on 01 October 2016 (boe-7-10-4220

Wire grip and motion score for TBI mice.

<p>Mice are assessed the score for the best level they reach.</p

WGMT scores of the mice.

<p>Mean (n = 8–14) WGMT scores measured over 4 weeks, of mice in 9 groups consisting of sham TBI mice, sham control mice, 810-nm laser treated TBI mice (18 J/cm² delivered at 25 mW/cm²). (A) Mice and controls given 1 laser Tx or 1 sham Tx at 4 hours post TBI; (B) Mice and controls given 3 daily laser Tx or 3 sham Tx; (C) Mice and controls given 14 daily laser Tx or 14 sham Tx. (D) Mean areas under curve of WGMT time courses from mice in different groups. P values given were determined by one-way ANOVA.</p

Time course of intrarticular leukocytes.

<p>Comparison of time courses of intraarticular leukocyte counts in the Pre-PDT (−1d) group and the PS-IR- group. <i>n</i> = 5 each. *<i>P</i><0.05, **<i>P</i><0.01.</p

Fluoro Jade C staining of lesion areas in brain sections.

<p>All sections were taken from mice sacrificed at 14 days post-TBI. Green is FJC and blue is DAPI )control for equal numbers of cell nuclei. (A) Sham-TBI control mouse; (B) real-TBI sham Tx mouse. (C) TBI mouse treated with 1 laser Tx. (C) TBI mouse treated with 3 laser Tx (E) TBI mouse treated with 14 laser Tx (F) Mean green staining calculated with Image J from mice (n = 2–3) in the groups in panels A–E. # P<0.05 vs TBI sham; * P<0.05 vs 14 laser Tx. One way ANOVA.</p

Neurological Severity Score (NSS) for TBI Mice.

<p>Mice are awarded 1 point for each failure to complete a task.</p