TEST OF PHTHALOCYANINES ANTIMICROBIAL ACTIVITY

Transcription

1 TEST OF PHTHALOCYANINES ANTIMICROBIAL ACTIVITY Libor KALHOTKA 1, Zuzana HRDINOVÁ 1, Radka KOŘÍNKOVÁ 2, Jitka PŘICHASTALOVÁ 1, Marie KONEČNÁ 1, Lubomír KUBÁČ 2, Jaroslav Lev 3 1 Mendel University in Brno, Brno, Czech Republic, xkalhotk@node.mendelu.cz, xhrdino5@node.mendelu.cz, marie.konecna@mendelu.cz, jitka.prichystalova@mendelu.cz 2 Centre of Organic Chemistry ltd., Rybitvi, Czech Republic, EU, radka.korinkova@cocltd.cz, lubomir.kubac@cocltd.cz 3 ASIO, spol. s r.o., Brno, Czech Republic, EU, lev@asio.cz Abstract The aim of this study is to demonstrate antimicrobial activity of selected positively charged water-soluble phthalocyanines. 12 phthalocyanine derivatives were used for this study. All the tested compounds were synthesized in Centre of Organic Chemistry (COC, Rybitví, Czech republic). Water soluble phthalocyanine derivatives were prepared by peripherally substitution of the phthalocyanine skeleton with the aim to prepare cationic phthalocyanines with an ability to have a wide-spectrum antimicrobial effect. To determine the antimicrobial effects of phthalocyanines, bacterial strains Escherichia coli CCM 3988, Enterococcus faecalis CCM 4224, and Pseudomonas aeruginosa CCM 1960 were used. Phthalocyanine samples were tested in two concentrations and 0.5 g/l. In a sterile vial, phthalocyanine solution with the bacteria was shaken under artificial light at room temperature. The suspension was sampled at 1 st, 2 nd and 3 rd hour. Samples were subsequently inoculated on Petri dishes with nutrient medium. The control sample was prepared by the same procedure. After the cultivation of Petri dishes, accrued colonies were counted and the results were expressed in CFU/ml. Antimicrobial action of phthalocyanines has been demonstrated. Keywords: Phtalocyanines, microorganisms, antimicrobial activity 1. INTRODUCTION Phthalocyanines (Pc) are tetrabenzo-fused 5,10,15,20-tetraazaporphyrins. Phthalocyanines are particularly attractive molecular building blocks for their incorporation into molecular materials.[1] Phthalocyanines belong to the group of photosensitizers together with porphyrins or methylene blue.[2] These biodegradable compounds are capable of generating singlet oxygen and other reactive oxygen species upon interaction with light of suitable wavelength in the presence of dioxygen. Singlet oxygen and other reactive oxygen species have strong biocidal effect on algae, bacteria, fungi and yeasts.[3, 4, 5, 6, 7, 8] The chemical structure of phthalocyanines can be modified. These changes can strongly modulate the photophysical properties of phthalocyanines and affect their interaction with cells and tissues, thereby leading to different photobioical effects.[9] Applications in industry, medicine, and bioy have shown the great importance of phthalocyanine molecules. A newly discovered area of application has been recently published suggesting possible usage of Pc against green algae and cyanobacteria development in the aquatic environment. 8]

2 The aim of this study is to demonstrate antimicrobial activity of selected positively charged water-soluble phthalocyanines. 2. MATERIALS AND METHODS 2.1 Phthalocyanines (Pc) Twelve phthalocyanine derivatives were synthesized in Centre of Organic Chemistry (COC, Rybitví, Czech Republic). Water soluble phthalocyanine derivatives were prepared by peripherally substitution of the phthalocyanine skeleton with the aim to prepare cationic Pc with an ability to have a wide-spectrum antimicrobial effect. Tab. 1: Description of phthalocyanine samples (Pc phthalocyanine) Sample number Sample description (central metal and substituent) 1040/167 Zn with quaternary amine 1040/193 Zn with quaternary amine 1131/6 Al sulpho groups 1131/53 Al with quaternary heterocycle 1131/98 Zn suplhamidic 1131/116 Zn suplhamidic 1131/188 Zn suplhamidic 1131/189 Al with urea derivative 1131/191 Zn suplhamidic 1131/217 Al with quaternary amine 1132/118 Zn hydroxyl 1132/131 Zn suplhamidic 2.1 Microbioical analysis Phthalocyanine samples were mixed with the solution of chosen microorganisms (Pseudomonas aeruginosa CCM 1960, Escherichia coli CCM 3988 and Enterococcus faecalis CCM 4224). 10 mg or 50 mg of phthalocyanine samples was mixed with 100 ml of solution containing microorganisms. Samples were vigorously shaked for 3 hours under room temperature and artificial light source (Aquarium colour 11 W, G23, 211 mm, NBB Bohemia, Czech Republic) Bacterial culture for sample inoculation was cultivated on the solid culture medium (PCA, Biokar Diagnostics, France) and re-suspended in the sterile distilled water. 0.5 ml of sample was taken every 60 minutes for decimal dilutions. 1 ml of diluted sample was transferred on sterile Petri dishes and overlapped by relevant culture medium. Pseudomonas aeruginosa was detected and enumerated on PCA (Biokar Diagnostics, France) at 30 C for 72 hours. Escherichia coli was detected and enumerated on VRBL agar (Biokar Diagnostics, France) at 37 C for 24 hours. Enterococcus faecalis was detected and enumerated on COMPASS Enterococcus agar (Biokar Diagnostics, France) at 45 C for 24 hours. Control blank sample was processed in the same way, but bacterial suspension was free of Pc. After the cultivation of particular Petri dishes accrued colonies were counted and the results were expressed in CFU/ml. 3. RESULTS AND DISCUSION Bacteria are ranked into two main classes depending upon their response to the Gram stain which reflects differences in their morphoy. Gram-negative and positive bacteria differ in the composition of their outer surface, and respond differently to antimicrobial agents. Gram-positive bacteria can easily take up molecules such as photosensitisers and can therefore be readily photoinactivated by most photosensitisers used for conventional PDT. This is not the case for Gram negative bacteria which are however relatively impermeable to neutral or anionic drugs due to their highly negatively charged surface.[6] We carefully chose

3 microorganisms that will represent different bacterial groups with specific properties for antimicrobial effect of phthalocyanines (Pc) testing. P. aeruginosa CCM 1960 and E. coli CCM 3988 are Gram-negative bacteria and E. faecalis CCM4 224 is Gram-positive. Reducion of counts of P. aeruginosa by 2-8 (Tab. 2), E. coli by 2-7 (Tab. 3) and E. faecalis by 1-6 (Tab. 4) was seen during three hours of the test. Tab. 2: Antimicrobial effect to Pseudomonas aeruginosa Pc mg/100ml CFU/ml 0h 1h 2h 3h CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml blanck no Pc , , , , / , , , / , , , / , , , / , , , / , , , / ,00 0 0,00 1 0, / , , , / , , , / , ,51 1 0, / ,00 0 0,00 0 0, / ,32 0 0, , / ,00 9 0,95 1 0,00 blanck no Pc , , , , / , , , / , , , / , , , / , , , / , , , / , , , / , , , / ,00 0 0,00 0 0, / , , , / , , , / , , , / , , ,50 We observed difference in antimicrobial activity on Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and Gram-positive (Enterococcus faecalis). According to the allocation of Pc by charge, negatively charged and neutral Pc are effective against Gram-positive bacteria, but only cationic Pc can inactivate both Gram-positive and Gram-negative bacteria. Strong antimicrobial activity was observed in the Gram positive bacteria for the most Pc. Gram positive bacteria showed small antimicrobial activity for 1131/116 (10 mg/l), 1131/6 (10 mg/l) and 1131/98. Lower antimicrobial activity of higher amount of Pc 1132/118, compared with lower amount of this Pc, was probably caused by lower permeability of light through the solution. Gram-negative bacteria showed a higher resistance to Pc. Strong antimicrobial activity was observed at 1040/167, 1040/193, 1132/131 and 1131/189 for Gram-negative and

4 Gram-positive bacteria compared to weak effect of 1137/7. Inactivation of E. coli and P. aeruginosa by selected Pc was also demonstrated by Minnock et al. [5] Tab. 3: Antimicrobial effect to E. coli 0h 1h 2h 3h Pc mg/100ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml blanck no Pc , , , , / , , , / , , , / , , , / , , , / , , , / , , , / ,60 2 0,30 3 0, / ,00 0 0,00 0 0, / , , , / , , , / , , , / , , ,96 blanck no Pc , , , , / , , , / , , , / , ,65 0 0, / , , , / ,00 0 0,00 0 0, / ,00 0 0,00 0 0, / , , , / , , , / ,00 0 0,00 0 0, / ,00 0 0,00 0 0, / ,00 0 0,00 0 0, / ,00 0 0,00 0 0,00

5 Tab. 4: Antimicrobial effect to Enterococcus faecalis 0h 1h 2h 3h Pc mg/100ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml CFU/ml blanck no Pc , , , , / , ,00 0 0, / ,00 0 0,00 0 0, / ,60 0 0,00 0 0, / ,68 0 0,00 0 0, / ,00 0 0,00 0 0, / ,00 0 0,00 0 0, / , , , / , , , / ,00 0 0,00 0 0, / ,00 0 0,00 0 0,00 blanck no Pc , , , , / , ,14 0 0, / , , , / ,00 0 0,00 0 0, / ,00 0 0,00 0 0, / , , , / , , , / ,33 0 0,00 0 0, / , ,10 3 0,48 blanck no Pc , , , , / , , , / , ,55 3 0, / , , , / , ,51 0 0, / ,00 0 0,00 0 0, / ,00 0 0,00 0 0, / ,45 0 0,00 0 0, / ,49 0 0,00 0 0,00 4. CONCLUSION Applications of Pc showed significant effect for Gram-negative and Gram-positive bacteria. Strong antimicrobial activity was observed at Pc 1040/167, 1040/193, 1132/131 and 1131/189. ACKNOWLEDGEMENT The authors would like to thank the Technoy Agency of Czech Republic for their financial support (project TA ).

6 LITERATURE [1] DE LA TORRE, G., CLAESSENS, CH. G., TORRES, T. Phthalocyanines: old dyes, new materials. Putting color in nanotechnoy. Chem. Commun. 2007, p [2] CLAESSENS, C.G., HAHN, U., TORRES, T. Phthalocyanines: from outstanding electronic properties to emerging applications. Chemical Record (8) p [3] BERTOLONI, G., ROSSI, F., VALDUGA, G., JORI, G., ALI, H., VANLIER, J.E. Photosensitizing activity of watersoluble and lipid-soluble phthalocyanines on prokaryotic and eukaryotic microbial-cells. Microbios.1992 (71) p [4] VALDUGA, G., BERTOLONI G., REDDI, E., JORI, G. Effect of extracellularly generated singlet oxygen on Grampositive and Gram-negative bacteria, J. Photochem. Photobiol. B: Biol (21) p [5] MINNOCK, A., VERNON D. I., SCHOFIELD, J., GRIFFITHS, J., PARISH, J. H., BROWN, S. B. Photoinactivation of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both Gram-negative and Grampositive bacteria. J. Photochem. Photobiol. B: Biol (32) p [6] JORI, G., BROWN, S.B., Photosensitized inactivation of microorganisms. Photochem. Photobiol. Sci (3) p [7] JESENSKÁ, S., PLÍŠTIL, L., PAVEL KUBÁT, P., LANG, K., BROŽOVÁ, L., POPELKA, Š., SZATMÁRY, L., MOSINGER, J. Antibacterial nanofiber materials activated by light. J. BIOMED. MATERIALS RES (4), p [8] JANČULA, D., MARŠÁLEK, B. The toxicity of phthalocyanines to the aquatic plant Lemna minor (duckweed) Testing of 31 compounds. Chemosphere (88) p [9] SEGALLA, A., BORSARELLI,C.D., BRASLAVSKY, S.E., SPIKES, J.D., RONCUCCI,G., DEI,D.,CHITI,G., JORI, G., REDDI, E. Photophysical, photochemical and antibacterial photosensitizing properties of a novel octacationic Zn(II)-phthalocyanine. Photochem. Photobiol. Sci., 2002 (1)