1. Сombination of Nanoparticles and Photosensitizers in Photodynamic Action on Microorganisms Pavel O. Petrov, Ivan A. Orlov, Alexandr V. Kovalev, Elena S. Tuchina and Valery V. Tuchin Laboratory of Bacteriology Res.-Edu. Institute of Optics and Biophotonics Saratov State University S Saratov S State U University

2. Goal: To study the sensitivity of staphylococci to the combined effects of light, dyes and nanoparticles

3. Genera Staphylococcus: Gram-positive cocci arranged in clusters Occurs frequently on the skin and mucous membranes of humans and animals Cause of acne, boils, endocarditis, osteomyelitis, toxic shock syndrome, nosocomial infections, wound infections, etc. Often resistant to a wide variety of antibiotics, including methicillin Contains carotenoids, staphyloxanthin, 4,4'-diaponeurosporene Staphylococcus aureus Staphylococcus epidermidis

4. S. aureus 209 P S. epidermidis two strains – meticillin-susceptible (MSSE) and meticillin-resistance (MRSE) Staphylococci were grown on nutrient agar and incubated at 37°C Bacteria strains

5. Experimental combinations Blue and Red LEDs + Methylene Blue + Titanium Dioxide Blue and Red LEDs + Brilliant Green + Titanium Dioxide Infrared laser + Indocyanine Green + Titanium Dioxide For the treatment of infections in the tissues For the treatment of infections on the surface of skin and mucous

6. Characteristics and parameters Light Exposure 5, 10, 15, 30 min Light Exposure: 5, 10, 15, 30 min Light source Emission maximum, nm Power desity, mW/cm 2 Manufacturer Blue LED 405±1531.5 Original design, Leonid Dolotov, SSU Red LED 625±2033 IR Laser 805±1550 Aculaser, Photothera Inc., USA Dyes Absorption maximum, nm Concentration Manufacturer Methylene Blue (MB) 6600.0025% Sigma-Aldrich Co., USA Brilliant Green (BG) 440; 650 0.00007% Manufacturing pharmaceutical company, Russia Indocyanine green (ICG) 750-8000.001% Sigma-Aldrich Co., USA Nanoparticles Titanium Dioxide (TiO 2 ) 400-420; 800-1000 0.02% Sigma-Aldrich Co., USA

7. Bacterial suspension (10 000 mc/ml) (Fig. 1) Bacterial suspension in PBS (1000 mc/ml) Bacterial suspension in dye water solution (1000 mc/ml) Darkness (control) Darkness (control) Light exposure (Fig. 2) 24-48 h incubation 37 C on dense nutrient medium Counting the number of colony forming units, using the original digital program (Fig. 3 ) º Fig. 1 Fig. 2 Fig. 3

8. The action of Blue+Red LEDs, Methylene Blue and TiO 2 on microbial colony forming ability

9. The action of Blue+Red LEDs, Brilliant Geen and TiO 2 on microbial colony forming ability

10. The action of IR laser, Indocyanine Green and TiO 2 on microbial colony forming ability

11. Summary The most effective combination for inhibition the growth of the three investigated staphilococci is the LED light (405, 625 nm), Brilliant Green and TiO 2 nanoparticles. This bacteria killing technology has several advantages: a low power radiation and low concentration of dyes can be used; the effectiveness is achieved through various mechanisms (photodynamic, photocatalytic, photothermal) that prevents the formation of bacterial resistance

12. Acknowledgments The research described in this paper has been made possible by: Grant 224014 PHOTONICS4LIFE of FP7-ICT-2007-2 Grant of President of RF "Supporting of Leading Scientific Schools" - NSHA-208.2008.2 Projects 2.1.1/4989 and 2.2.1.1/2950 of RF Program on the Development of High School Potential Project 1.4.09 of Federal Agency of Education of RF The Special Program of RF "Scientific and Pedagogical Personnel of Innovative Russia," Governmental contracts: 02.740.11.0484 and 02.740.11.0770 We are grateful to: Docent Dolotov L.E. (SSU) for the development of the LED light device