Method of increasing the sensitivity of bacteria to biodestruction the action of laser radiation in combination with a photosensitizer

 

(57) Abstract:

The invention relates to medicine and is intended to increase the sensitivity of bacteria to laser radiation in combination with a photosensitizer. Effect of low-intensity laser radiation in combination with a photosensitizer. As photosensitizer using chitosan with methylene blue. The method can effectively increase the sensitivity of bacteria to low-intensity laser radiation. 5 table.

The invention relates to medicine, namely to Microbiology, laser medicine. Low-intensity laser irradiation (LLLT) in practical medicine used primarily for stimulation of disturbed metabolism and tissue trophism (1), in particular, in surgical practice for impact on the wound process in order to accelerate healing of wounds and prevent suppuration. However, NEELY has little bacteriostatic in the complete absence of bactericidal action on biological properties of Staphylococcus aureus (2) and other species of bacteria (3, 4).

To improve the efficiency of laser radiation applied photosensitizers (PS) mainly in Oncology PR is the congestion, increasing the sensitivity of microorganisms to laser study, in the literature there are few messages. For example, a variety of dyes (6): crystal violet thionin, methylene blue, protoporphyrin IX derivatives of hematoporphyrin, toluidine blue Oh, disulfonated aluminum phthalocyanine, which showed sellingbuy activity under the action of laser radiation density from 16 to 320 J/cm2maximum destruction of bacteria was not above 99%.

The disadvantages of these methods include: 1) the need to maintain a constant concentration of solutions of photosensitizers, 2) the inability to prevent the diffusion and spreading of the dye in the tissues, 3) the lack of strong ties dyes with surface structures of microorganisms, 4) lack of effect of concentration of dye on the surface of microorganisms, 5) duration of staining associated with the necessity of using low concentrations of dyes in order to avoid damage to somatic cells, 6) lack of antibacterial activity, 7) absence of immunomodulatory effects, 8) use with high power lasers and high flux density of laser research and / microorganisms to biodestruction the action of laser radiation (7), based on the processing of microorganisms 1% (weight/volume) aqueous solution of phosphorus-tungsten acid (FVC) or ammonium molybdate (MA) for 5-10 sec with subsequent exposure to an equal volume of 0.25% (weight/volume) solution of methylene blue (MC). It was assumed that on the surface of bacteria is formed biofilm with methylene blue, which is a photosensitizer.

The disadvantages of the prototype include: 1) application FVC and MA as compounds containing ions of tungsten and molybdenum, which in varying degrees, can have a toxic effect on cells of the human body, 2) such complexes can serve allergenic factor 3) does not have immunostimulating and wound-healing action.

The authors propose an original way to justify the use of chitosan with methylene blue as a means (photosensitizer) that increases the sensitivity of bacteria to low-intensity laser irradiation. As a drug that can interact with a variety of negatively charged molecules of dyes, proteins, glycoproteins surface of microorganisms we have chosen chitosan.

Chitosan, representing composed of N-acetylglucosamine, causes prolonging the action of drugs, increasing their therapeutic effect. Chitosan allowed the Russian Ministry of health as a food additive - amendment. In addition, chitosan has sorption, deadening and immunomodulatory activity (8, 9). These positive properties, and was the basis for the use of chitosan as an adsorbent capable to enter into the complexation at the same time with different classes of negatively charged molecules (methylene blue - membrane proteins of microorganisms). In this case, the surface of microbial cells using chitosan firmly attached NNN'N'-tetramethylethylene (methylene blue), which is a photosensitizing label for laser studies.

The method consists in the treatment of the wound or inflammatory lesion 1% solution of chitosan 0.25% solution of aqueous hydrochloric acid with 0.25% aqueous solution of methylene blue and influence NEELY in therapeutic radiation mode.

In this regard, the aim of our work was to study the comparative effect of a fundamentally new semiconductor laser ILD 650 "Mustang", (company "Engineering", Moscow) wavelength in the red range ( = 0.63 Ám), operating in pulsed mode the St. aureus, Candida parapsilosis, Candida crusea, Acnietobacter anitratus, and others) and gram-negative (Pseudomonas maltophila, Rs.putida, Hafnia alvei, Rhododorula and others) microorganisms "in vitro" and "in vivo" in its pure form and with the use of FS. The parameters of laser radiation as "in vitro" and "in vivo" were identical, namely, the time of irradiation of the infrared laser was 4 min, Pmax= 10 W with a single dose of 0.02 j/cm2; the exposure time of red pulsed laser also was 4 min, Pmax= 5 watts and a single dose of radiation (f = 80 Hz) of 0.01 j/cm2and when f = 3000 Hz to 0.4 j/cm2(calculation of doses in tables Kozlov Century. And. and Buyline C. A. (10).

To study morphological changes in the course of reparative regeneration of skin wounds using standard histological techniques.

The experiment was carried out in two stages. First investigated the effect of LLLT wavelength range on the viability of microbial cells and the activity of their enzyme systems under the action of laser in pure form and in the pre-treatment of cultures FS.

It was found that the semiconductor lasers of the red and infrared ranges, did not affect the change of life and pathogenicity test organism. While pre-treatment of bacteria of the Federal Assembly of pgcap contributed to a pronounced bactericidal effect. The results of irradiation were mixed. So the number of gram-negative bacteria treated with photosensitizer in sub-bacteriostatic concentration, decreased from 5109CFU/g to 1102CFU/g as the use of IR-radiation, and red pulsed laser in the same degree at a frequency of 80 Hz and 3000 Hz. However, 100% mortality of Staphylococcus aureus, loaded FS, was the only infrared radiation. While red pulse laser when exposed to this strain, regardless of the frequency of radiation, had a reduction in their number.

In the experiment on the animals (42 rat line "Wistar" average weight of 120 g) was taking St. aureus strain 236 having high resilience and expressed pathogenicity (try using other microorganisms was unsuccessful due to their rapid destruction and ultimately pollution wound Staphylococcus aureus). On the back of rats in the interscapular area did scalped, an open wound 1x1 see it was administered daily St suspension. aureus, corresponding to the turbidity standard No. 6 in MC Farland. After 3 days formed the inflammatory process. All the examined animals were divided into 5 groups depending on Primaria group (wound was treated FS) 9 rats were subjected, accordingly, the impulse NEELY red and infrared ranges of the above parameters. In the third and fourth groups (the wound was not processed FS) also in 9 rats and also used identical parameters of laser radiation. NEELY was carried out for 5 days. The control group, the wound which is processed only by the photosensitizer without laser application, amounted to 6 rats. Efficacy was assessed by the change in the culture of bacteria taken from the wound before and after laser treatment.

As a result of the research showed that in rats, the wound which has not been previously processed photoactive substance, the amount allocated from the biopsy strains St. aureus, after applying pulsed LLLT red and infrared ranges were the same as in the control group, regardless of the multiplicity of exposure. In rats, the wound was treated with a photosensitizer, red pulsed laser, did not reduce the number of viable bacteria compared with the control. While the IR laser in rats, the wound was treated with a photosensitizer, significantly reduced the number of St. aureus in biopsies 2-4 times (p<0,05).

The results of microbiological analysis of the content of the RAS experimental animals correlate with optical studies.

At macroscopic examination of the newly formed tissue in the healing of experimental wounds noted that the data are consistent with microscopic changes what is happening in the damaged area (PL. 4, 5).

Microscopic changes in the damaged area, confirm visual observations.

Thus, the use of IR laser in combination with FS changes the speed and nature of implementation of individual stages of post-traumatic regeneration of infected skin wounds, which leads to reduction of total healing time for 3 days.

The results of our study showed that both in vitro and in vivo efficacy of low level laser radiation increases greatly with the use of photosensitizing agents to a greater extent when the IR radiation, which is confirmed by the data of histological research. It has also been found ambiguous enzymatic reaction St. aureus to laser irradiation, indicating their wide adaptation possibilities that require further investigation and explanation. While we have not identified the advantages of a stand-alone application red pulsed radiation in the experiment, compared with infrared, which probably indicates the need for greater understanding and explore options combined use of wavelengths rigestration the action of laser radiation is as photosensitizer is applied 1% solution of chitosan in 0.25% aqueous hydrochloric acid in combination with 0.25% aqueous solution of methylene blue. High molecular weight chitosan is able to partially contact with methylene blue and simultaneously with negatively charged molecules to the surface of bacterial cells. The complex bacterial cell-chitosan-methylene blue becomes susceptible to NEELY red and especially infrared ranges. In addition, chitosan prolong the antiseptic action of methylene blue, has a pronounced antimicrobial, sorption, deathtiny action against bacteria and damaged tissue in the wound has anti-inflammatory, immunomodulatory effect.

Literature

1. Lasers in clinical medicine. Under the arms. Professor C. D. Pletnev // M, Med., 1996, - 430 S.

2. Agarkova, N. The action of laser radiation on some properties of staphylococci. // Ukr. microbiol.Epidemiol. and immunol. - 1979, - N 5 - n-101-103.

3. Bazhenov And R. the Influence of laser radiation on microorganisms. //Honey. Journe. Uzbekist. - 1991, N 7, - S. 26-28.

4. Baibekov I. M., F. Nazyrov,, Ilkhamov F. and others. Morphological aspects of laser effects // Tashkent, is vnesti new domestic photosensitizers in vitro. // In collection: Laser and photodynamic therapy. Obninsk, June 16-18, 1999, - S. 18-19.

6. "The Killing of Helicobacter pylori by low-power light in the presens of the photo sensitiser" Wilson C. E., M. Wilson, A. J. Macrobert, Sedwell J., Bown S. S. // J. Med.Environ., 1996, Apr., 44(4), p. 245-252.

7. Sagittarius E. C., Bogatov centuries, Chervinets C. M., Chistov Century B., Volkov Y. A. - Method of increasing the sensitivity of microorganisms to biodestruction the action of laser radiation. Notification of the positive result of the formal examination (FIPS 10 APR. 1998, from cases N 20), N 97121255/13 (023049).

8. Zonagen Inc. "Chitosan - induced immunopotency" US Patent N 94311532, 23.09.94.

9. Mosquitoes B. A., Treskunov, K. A., Albulov A. I., Fomenko A. C. Application of chitodes and phitochitodes in clinical practice. //In collection: New perspectives in the study of chitin and chitosan", Moscow-Shchyolkovo, 1999, S. 148-150.

10. Kozlov Century, Bolin Century A. Laser therapy with the use of the "Mustang" // M, Aspect Press, 1995, - 143 C.

Method of increasing the sensitivity of microorganisms to biodestruction the action of laser radiation in combination with a photosensitizer, including the effects of low-intensity laser radiation, a photosensitizer, characterized in that the photosensitizer is applied chitosan with methylene blue.

 

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