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S aratov S tate U niversity. 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 - PowerPoint PPT Presentation
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СombinationСombination of Nanoparticlesof Nanoparticles and and PhotosensitizersPhotosensitizers
in Photodynamic Action on Microorganismsin Photodynamic Action on Microorganisms
Pavel O. Petrov, Ivan A. Orlov, Alexandr V. Kovalev,
Elena S. Tuchina and Valery V. Tuchin
Laboratory of BacteriologyRes.-Edu. Institute of Optics and Biophotonics
Saratov State University
SSaratov SState UUniversity
Goal:
To study the sensitivity of staphylococci to the combined effects
of light, dyes and nanoparticles
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 aureus
Staphylococcus Staphylococcus epidermidisepidermidis
•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
Experimental combinationExperimental combinationss
Blue and Red LEDs + Methylene Blue + Titanium Blue and Red LEDs + Methylene Blue + Titanium DioxideDioxide
Blue and Red LEDsBlue and Red LEDs + Brilliant Green + Titanium + Brilliant Green + Titanium DioxideDioxide
Infrared laser + Indocyanine Green + Titanium Infrared laser + Indocyanine Green + Titanium DioxideDioxide
For the treatment of infections in the tissues
For the treatment of infections on the surface of skin and mucous
Characteristics and parametersCharacteristics and parameters
Light ExposureLight Exposure: 5, 10, 15, 30 5, 10, 15, 30 minmin
Light sourceLight source
Emission maximum, Emission maximum, nmnm
Power desity, Power desity, mW/cmmW/cm22
MManufactureranufacturer
Blue LEDBlue LED 405±15405±15 31.531.5 Original design, Leonid Dolotov, SSURed LEDRed LED 625±20625±20 3333
IR LaserIR Laser 805±15805±15 5050Aculaser,
Photothera Inc., USA
DyesDyes
Absorption maximum, Absorption maximum, nmnm ConcentrationConcentration MManufactureranufacturer
Methylene Blue (MB)Methylene Blue (MB) 660660 0.0025%0.0025% Sigma-Aldrich Co., USA
Brilliant Green (BG)Brilliant Green (BG) 440; 650440; 650 0.00007%0.00007%Manufacturing pharmaceutical
company, Russia
Indocyanine green (ICG)Indocyanine green (ICG) 750-800750-800 0.001%0.001% Sigma-Aldrich Co., USA
NanoparticlesNanoparticles
Titanium Dioxide (TiOTitanium Dioxide (TiO22)) 400-420; 800-1000400-420; 800-1000 0.02%0.02% Sigma-Aldrich Co., USA
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 exposur
e
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
The action of Blue+Red LEDs, Methylene Blue and TiO2 on microbial colony forming ability
S. epidermidis MS100
82
73
56 55
93
85
5551
12
0
20
40
60
80
100
120
0 5 10 15 30Time, min
CF
U, %
Blue+Red LEDs Blue+Red LEDs & MB+TiO2
S.aureus100
75
61
44
11
85
39
148 5
0
20
40
60
80
100
120
0 5 10 15 30Time,min
CF
U, %
Blue+Red LEDs Blue+Red LEDs & MB+TiO2
S. epidermidis MR100
79
6660
26
94
75
46
24
7
0
20
40
60
80
100
120
0 5 10 15 30Time, min
CF
U,
%
Blue+Red LEDs Blue+Red LEDs & MB+TiO2
The action of Blue+Red LEDs, Brilliant Geen and TiO2 on microbial colony forming ability
S. aureus100
78
63
48
15
89
24 24
94
0
20
40
60
80
100
120
0 5 10 15 30Time, min
CF
U, %
Blue+Red LEDs Blue+Red LEDs & MB+TiO2
S. epidermidis MS100
80
71
50 49
88
44
37
20
8
0
20
40
60
80
100
120
0 5 10 15 30Time, min
CFU
, %
Blue+Red LEDs Blue+Red LEDs & MB+TiO2
S. epidermidis MR100
77
65
55
23
87
33
22
115
0
20
40
60
80
100
120
0 5 10 15 30
Time, min
CF
U, %
Blue+Red LEDs Blue+Red LEDs & MB+TiO2
The action of IR laser, Indocyanine Green and TiO2
on microbial colony forming ability
S.aureus100
95
84
6863
84
4841
3326
0
20
40
60
80
100
120
0 5 10 15 30
Time, min
CF
U, %
IR laser IR laser & ICG+TiO2
S.epidermidis MS100
7568
73
30
80
26 25 2217
0
20
40
60
80
100
120
0 5 10 15 30Time, min
CF
U,
%
IR laser IR laser & ICG+TiO2
S.epidermidis MR100
59
39 38
48
73
2124 22
14
0
20
40
60
80
100
120
0 5 10 15 30Time, min
CF
U, %
IR laser IR laser & ICG+TiO2
SummarySummary
• The most effective combination for inhibition the growth of the three investigated staphilococci is the LED light (405, 625 nm), Brilliant Green and TiO2 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
AcknowledgmentsAcknowledgments
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
