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Antimicrobial PDT with chlorophyll-derived photosensitizer and semiconductor laser
ARTICLE IN PRESS
Medical Laser Application 21 (2006) 177–183 www.elsevier.de/mla
Antimicrobial PDT with chlorophyll-derived photosensitizer and semiconductor laser A. Ulatowska-Jarz˙ aa, J. Zychowiczb, I. Ho"owacza, J. Bauera, J. Razikc, A. Wieliczkoc, H. Podbielskaa,, G. Mu¨llerd, W. Str˛eke, U. Bindigf a
Bio-Optics Group, Institute of Physics, Wroc!aw University of Technology, Wybrzez˙e Wyspian´skiego 27, 50-370 Wroc!aw, Poland Haemato-Poland, 50-376 Wroc!aw, Hoene-Wron´skiego 14, Poland c Department of Epizootiology and Veterinary Administration with Clinic, Faculty of Veterinary Medicine, The Agricultural University of Wroc!aw, pl. Grunwaldzki 45, 50-366 Wroc!aw, Poland d Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Institute of Medical Physics and Laser Medicine, Fabeckstr. 60-62, 14195 Berlin, Germany e Institute of Low Temperatures and Structure Research, Polish Academy of Science, Oko´lna 2, PL-50-119 Wroc!aw, Poland f Laser- und Medizin-Technologie Berlin, Fabeckstr. 60-62, 14195 Berlin, Germany b
Received 12 May 2006; accepted 24 May 2006
Abstract The study was carried out on Escherichia coli strains isolated from poultry and cows. The bacteria were incubated and treated by Photolon, a plant-based photosensibilizator, and exposed after that to laser light at 662 nm. Additionally, the photosensitizer was immobilized in fiberoptic sol–gel coating, constructed as an 8-layer applicator. It was demonstrated that Photolon inhibits bacteria growth. Photolon entrapped into the sol–gel matrix also exhibits photodynamic activity and no bacteria were seen near the doped sol–gel applicator. r 2006 Elsevier GmbH. All rights reserved. Keywords: Antimicrobial photodynamic therapy; E. coli; Sol–gel applicator; Photolon
Introduction Escherichia coli is one of the most widely spread microorganisms. Many strains of E. coli are pathogenic and may cause various diseases, e.g. urinary tract infections, sepsis, meningitis and infectious diarrhea. Pathogenic strains are also capable of contaminating the blood pools or food products. Many strains are resistant to commonly applied disinfection methods and antiCorresponding author. Tel.: +48 71 320 2825; fax: +48 71 328 3696. E-mail address: [email protected] (H. Podbielska).
1615-1615/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.mla.2006.05.003
biotics. Therefore, there is continuous need to search for alternative methods in order to combat the bacteria. One of them is photodynamic therapy. Antimicrobial photodynamic therapy (APDT) combines a nontoxic photoactive dye, photosensitizer (PS), with harmless visible light to generate singlet oxygen and free radicals that kill microbial cells. Currently, the major use of APDT is in disinfection of blood products for bacterial, fungal and viral inactivation and it can also be an alternative method for the decontamination of foods. The studies show that Gram-positive bacteria are susceptible to the photosensitizing action of a variety of
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photosensitizers [1–4]. Lazzeri and colleagues [5] from National University of Argentina have investigated the photodynamic activity of amphiphilic porphine in the different media and in vitro on the Hep-2 human larynx carcinoma cell line. Photosensitized inactivation of E. coli was analyzed using meso-substituted cationic porphine derivatives such as 5,10-di(4-methylphenyl)15,20-di(4-trimethylammoniumphenyl)porphine iodide 1 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphine iodide 2 and its metal complex with Pd(II). The results showed that the higher photoinactivation of cells was found for tricationic porphine, causing an 5.5 log (99.999%) decrease of cell survival. Lazzeri has reported that the high amphiphilic character of investigated photosensitizers may offer a promising model for photosensitizing agents with potential applications in bacteria inactivation by APDT. Additionally, recent work by Scalise et al. [6] has shown the photodynamic efficiencies of cationic Zn(II) N-methylpyridyloxy phthalocyanine (ZnPc 2) and noncharged Zn(II) pyridyloxyphthalocyanine (ZnPc 1) in homogeneous medium bearing a photooxidizable substrate and in vitro using bacterium E. coli. Promising results were obtained for cationic ZnPc 2, which can be an efficient phototherapeutic agent with potential photodynamic inactivation of bacteria cells. Wainwright et al. [7] have investigated the photobactericidal activity of phenothiazinium photosensitizers. Phenothiazinum dyes were tested against Grampositive (S. aureus) and Gram-negative bacteria (E. coli). The material was illuminated using a non-laser light source at a fluence of 1.75 mW/cm2 to enhance antibacterial activity in liquid culture [8]. The results showed a generally increased efficacy of PDT for inactivation of E. coli and S. aureus. Szocs et al. [9] have described the possibility and conditions for the induction of porphyrin synthesis by exogenous d-aminolevulinic acid (ALA) and its applicability for the inactivation of E. coli by PDT. After incubation of bacterial cells with ALA (2 107 cells ml1 with (5–9) 103 mol/l ALA), ALA-treated cells were irradiated by white light of 80 mW/cm2. Under these conditions, an experiment showed significant cell killing (over 99%) at a dose of 4.3 103 kJ/cm2. Obtained results proved that the porphyrin synthesis against Gram-negative E. coli B. can be stimulated by the addition of ALA. APDT can be a new approach for safe food. M. Kreitner et al. from the Institute of Nutritional Sciences (University of Vienna), Institute of Radiodiagnostics and Institute of Dairy Sciences and Bacteriology (University of Agricultural Sciences, Vienna) established an experimental model to examine the photosensitivity of immobilized microorganisms to develop a system where microorganisms with reduced mobility may be targets of a photodynamic effect [10]. They tested the
photosensitivity of several Gram-positive bacteria and yeasts using visible light and photosensitizers. The photosensitizers used for the reduction of microorganisms were hematoporphyrin (HP) and sodium chlorophyllin (CHL), because they are natural constituents of food [11]. Microorganisms such as S. aureus, B. subtilis, and B. cereus, as well as yeasts such as S. cerevisiae, Kloeckera javanica, and Rhodotorula mucilaginosa were under investigation. The cells were incubated with 105 M PS for 1 h. Further, suspended cells were lawned on nutrient agar (bacteria) or wort agar (yeasts) and illuminated immediately with a 1000 W photooptic lamp for 1 h. The results obtained by Kreitner demonstrated that the survival rate of bacteria fixed on solid media ranged from 0.0006% to 0.08% (3–5 log 10 inactivations), and the level of yeasts ranged from 0.1% to 50% (0.3–3 log 10 inactivations).
Materials Photolon Photolon, chlorin e6 ((18-carboxy-20-(carboxymethyl))-8-ethenyl-13-ethyl-2,3-dihydro-3,7,12,17-tetramethyl-21H,23H-porphin-2-propionic acid) (powder from Belmedpreparaty in cooperation with Haemato Ltd. Germany) was used in this study. Aqueous stock solutions (8.2 104 M) were prepared by dissolving the photosensitizer in water (Millipore grade). In organic chemistry, a chlorin is a large heterocyclic aromatic molecule consisting, at the core, of three pyrroles and one reduced pyrrole, all coupled by methine linkages. Compared to the porphyrins, a chlorin is largely aromatic but not aromatic through the entire circumference of the ring. Magnesiumcontaining chlorins are called chlorophylls and are the main photosensitive pigments in chloroplasts. A related compound, with two reduced pyrroles, is called a bacteriochlorin. Because of their strong photosensitivity, chlorins are in use as photosensitizing agents in experimental PDT therapies. Water-soluble derivatives of chlorophyll were first introduced as potential drugs by Snyder (USA) in 1942 [12]. The next important step was done by Allen [13]. He revealed that the major chemical compound of native chlorin mixtures is chlorin e6, which under oral and intravenous administration possesses low toxicity and demonstrates hypotensive, antisclerotic, spasmolytic, anesthetic and antirheumatoid activity. The first PDT usage of a chlorin relates to a pheophorbide a derivative. Some of them were patented as prospective photosensitizers for PDT in Japan in 1984 by Sakata and Nakajima [14]. In 1986, an American group reported on a photosensitizer meeting crucial PDT requirements:
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good tumor affinity and intensive absorption in the middle red part of the visible spectrum [15]. Their choice was mono-L-aspartyl-chlorin e6 (MACE). At present, this compound is at stage III of clinical trial studies in Japan. This group has even patented other functionally advanced chlorin and bacteriopheophorbide a derivatives as photosensitizers for PDT [16]. In the 1990s a Byelorussian group at first headed by G. Gurinovich reported about their research on water-soluble chlorintype photosensitizers derived from nettle [17]. Recently, their work was focused on the spectral-luminescent characteristics of chlorine e6 and – the newly developed photosensitizer – Photolon in whole blood [18]. Photolon – a chlorin derivative – belongs to the onephoton-based photosensitizers [19]. The dye is approved for cancer treatment in some countries [20]. Photolon is referred to a group of new sensitizers for treating cancer by means of PDT [18]. It can be administered intravenously or topically. Recently, some non-oncologic applications have been reported as well [21]. The chemical structure of Photolon has a partial reduced porphyrin moiety. The molecular structure is comparable to chlorine e6, which can be isolated after hydrolysis of the five-membered exocyclic b-ketoester moiety of pheophorbide a. It is well known that absorption and fluorescence characteristics of tetrapyrrolic macrocycles are sensitive to changes in their (molecular) environment. The chemical structure of Photolon is depicted by Fig. 1.
Photolon doped sol–gel fiberoptic applicators Sol–gel materials are porous, glass-like solid bodies that find many interesting applications, among others, in the biomedical field [22,23]. All chemical compounds for the sol–gel production of coated fibers examined here were obtained from commercial sources and used without further purification: deionized water, ethyl
Fig. 1. Chemical structure of Photolon (R1 ¼ CH2CH2 COOH, R2 ¼ CH2COOCH3, R3 ¼ COOH) according to [19].
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alcohol C2H5OH 96% v/v (Polish Chemicals), Triton X-100 (Aldrich), and tetraethylorthosilane Si(C2H5O)4, TEOS (Aldrich), hydrochloric acid HCl 35% w/w (Polish Chemicals). Recently, we demonstrated that Photolon entrapped in a sol–gel matrix preserves its photochemical activity [24]. The sol–gel layers were produced from sols prepared from the silicate precursor TEOS mixed with ethyl alcohol. The sol–gel material was prepared with factor R ¼ 20, where R denotes the solvent to precursor molar ratio. Hydrochloric acid was added as catalyst in proportion to ensure acid hydrolysis (pHE2). The mixture was stirred at room temperature for 4 h by means of a magnetic stirrer (speed 400 rpm). Hard clad silica (HCS) fibers of low OH (CFO 149312) origin laser components were used (fused silica core f1 ¼ 400 mm, with cladding f2 ¼ 430 mm; with ETFE buffer diameter 730 mm). They feature high transmission values from UV to IR (200–2400 nm). The external jacket was mechanically removed from a distance of 3.5 cm. Then the cladding was treated by a hot torch. The residuals were removed with linen cloth and cleaned with ethanol. An aqueous stock solution of Photolon was prepared by dissolving 0.5 mg of dye in 1 ml deionized water. Next 1 ml of ethyl alcohol was added. Aliquots of 50 ml stock solution and 20 ml of Triton X-100 were added to 1 ml of the freshly prepared sols. The freshly prepared sol was used to produce doped fiberoptic coatings according to the procedure described. The stock solutions and the sol–gel modified fibers were stored in darkness at room temperature. A modified dip-coating method was applied to cover the bare fibers with sol–gels. The fibers were placed vertically in a plastic container with a specially designed bottom possessing an opening with a controlled diameter, to ensure the required speed (1 cm/h) of sol outflow. Just before the deposition procedure, the surfactant Triton X-100 was added to the liquid sol. Prior to the applicator construction, some spectrophotometer studies of Photolon in the sol–gel matrix were performed. Fluorescence spectra were obtained by means of the fluorescence spectrometer LS-50B (Perkin Elmer/UK). Spectra were recorded in the wavelength range 350–750 nm (100 nm/min, excitation slit 3 nm, emission slit 5 nm) by the use of quartz micro-cuvettes (optical length 0.5 cm, Hellma/Germany). The fluorescence intensity of Photolon entrapped in sol–gel (1.6 104 M Photolon) is depicted in Fig. 2. The visible maxima are observed for the blue (411, 403 nm) and red regions (648, 670 nm) of the spectrum. These spectra were measured for an eight -layer coating (fresh preparation). Recently, we demonstrated that the number of layers building the sol–gel applicators for interstitial laser therapy influences significantly the light
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distribution from the applicator and overall applicator performance in interstitial coagulation [25,26]. The optimal number of layers according to the results is 8.
Bacteria preparation The studies were carried out on E. coli strains isolated from poultry and cows (from the collection of the Department of Epizootiology and Veterinary Administration with Clinic). All strains were cultured on MacConkey agar (bioMerieux, no cat. D6612) and tryptose-soya broth (bioMerieux, no cat. 51019). E. coli were cultured according to standard bacteriological procedures. Strains were determined by routine laboratory methods. The bacteria were identified on the basis of their metabolic properties using API 20E tests (bioMerieux, no cat. 20100). Bacterial strains were inoculated into tryptose-soya broth and incubated at 37 1C overnight. The bacteria suspension density was set with the use of a spectrophotometer at l ¼ 560 nm to an equivalent of 1. The appropriate dilutions from 101 to 106 in 0.85% NaCl were prepared and then from each dilution 0.1 ml was cultured on MacConkey agar.
fluorescence intensity / a.u.
40 403
i) ii)
i)
a)
b)
ii) ii) 50 501 53 531
61 611
350
400
500 wavelength/nm
600
a) ex 400 nm b ) ex 410 nm m
700
750
Fig. 2. Fluorescence intensity of Photolon entrapped in sol–gel coating on optical fiber core, eight layers.
Table 1.
CFU ¼
C , ðN1 þ 0:1N2Þ d a
where C is the number of all counted colonies, N1 the number of plates for lower bacteria dilution, N2 the number of plates for higher bacteria dilution, d the dilution factor and a the volume of bacteria deposited on the plate. The experimental groups examined in this study are shown in Table 1. For each group five plates were seeded by bacteria.
Results
64 648
530
The colonies were illuminated by laser light. To some of colonies the Photolon was added. Photolon-doped sol–gel interstitial applicators were also examined. For illumination the semiconductor pig-tailed laser (Laser Secura, Poland) lasing at 662 nm was used. The output power chosen was 200 mm and the illumination time for each plate was 10 min. After the illumination, the plates were incubated at 37 1C for 24 h. The next day the colonies on the plates were counted (we chose the plates with 10–300 CFU/plate). Colony forming units (CFU) were calculated according to the formula
67 670
em 670 nm em 648 nm
41 411
Methods
The CFU for each group of samples was counted after the post-illumination incubation period. Some of the exemplary results are presented in Figs. 3 and 4. Fig. 3 demonstrates that the photodynamic activity of Photolon leads to inhibition of bacteria growth. This activity can also be seen for Photolon immobilized in a sol–gel applicator (Fig. 4a). For better visualization of the colonies the Sobel filter was applied. Analysis was performed with the OPTIMAS image processing program. The diagram (Fig. 5) demonstrates the results (CFU) as a percentage of mean CFU values in the control group.
Description of examined samples.
No (a—stays for higher bacteria dilution)
E. coli (low dilution)
E. coli (high dilution)
1–1a 2–2a 3–3a 4–4a
Control (not illuminated, no Photolon) Not illuminated, Photolon added Illuminated, no Photolon Illuminated, Photolon doped sol–gel applicator Not illuminated, Photolon doped sol–gel applicator Illuminated, with Photolon
Control (not illuminated, no Photolon) Not illuminated, Photolon added Illuminated, no Photolon Illuminated, Photolon doped sol–gel applicator Not illuminated, Photolon doped sol–gel applicator Illuminated, with Photolon
5–5a 6–6a
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Sample No
E. coli (low dilution)
181
E. coli (high dilution)
1 – 1a
6 – 6a
Fig. 3. E. coli colonies after incubation, (1), control without photosensitizer, not illuminated, (6), illuminated samples treated with Photolon.
Fig. 5. Mean values of CFU for each experimental group calculated in [%] of mean CFU values for control group. The visible colonies inhibition is observed for Photolon treated bacteria illuminated with laser light. There is also inhibition seen for samples with sol–gel applicator.
bacteria in various environments (e.g. milk, various liquids, food, etc.).
Acknowledgement
A
B
Fig. 4. The Photolon doped sol–gel applicator was placed in bacteria environment. After illumination and incubation, no bacteria are seen near the applicator (A). The image processing (Sobel filter) applied to image (A) visualized the colonies. They are not observed near the applicator.
The support of the Polish Ministry of Science, Grant KBN No. 4T11E01124 is gratefully acknowledged. This work was also supported by the joint program of DAAD (German Academic Exchange Service) and the Polish Ministry of Science for endorsement of cooperative research projects.
Zusammenfassung
Discussions The E. coli was the subject of this in vitro study. The photodynamic activity of a plant-based sensitizer was examined. The highest mean value of CFU was observed in the control group, which remained untreated in an incubator. Some bacteriotoxic effect was observed in the remaining groups that for the experiment were taken out from the incubator. The visible effect of photodynamic activity was observed for illuminated samples, treated by Photolon. The visible colonies inhibition was observed in this case. There is also significant inhibition seen for samples with the sol–gel applicator. The result obtained for the applicator may be exploited for some local PDT applications, e.g. interstitial therapy. The other modality may be the application of such an applicator to combat
Antimikrobielle PDT mit einem dem Chlorophyll verwandten Photosensibilisator und Halbleiterlaser Die vorliegende Studie wurde an E. coli-Sta¨mmen durchgefu¨hrt, die aus Geflu¨gel und Rind isoliert wurden. Die Bakterien wurden inkubiert, mit Photolon, einem pflanzlichen Photosensibilisator, behandelt und mit Laserlicht der Wellenla¨nge 662 nm bestrahlt. Zusa¨tzlich wurde der Photosensibilisator in die Sol–Gel–Beschichtung eines aus acht Schichten bestehenden Applikators immobilisiert. Es wurde nachgewiesen, dass Photolon das bakterielle Wachstum inhibiert. Im Bereich des dotierten Sol–Gel–Applikators wurde kein bakterielles Wachstum beobachtet. Das in die Sol–Gel–Matrix implementierte Photolon weist somit eine photodynamische Aktivita¨t auf. Schlu¨sselwo¨rter: Antimikrobielle photodynamische Therapie; E. coli; Sol–Gel–Applikator; Photolon
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Resumen Terapia Fotodina´mica (PDT) antimicrobiana mediante un fotosensibilizador derivado de la clorofila y la´ser semiconductor Este estudio fue realizado utilizando diferentes cepas de E. coli aisladas de vacas y aves de corral. Las bacterias fueron incubadas y tratadas con Photolon, un compuesto fotosensibilizador a base de plantas, y posteriormente expuestas a un la´ser de 662 nm. Adicionalmente, el fotosensibilizador fue inmobilizado en una cubierta fibroo´ptica sol–gel, construı´ da como un aplicador de 8 capas. Se demostro´ que el Photolon inhibe el crecimiento bacteriano. El Photolon atrapado en la matriz sol–gel tambie´n posee esta actividad fotodinamica ya que no se observo´ crecimiento bacteriano alguno en los alrededores del aplicador sol–gel. Palabras clave: Terapia fotodina´mica antimicrobiana; E. coli; aplicador sol–gel; Photolon
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[9] Szocs K, Gabor F, Csik G, Fidy J. d-Aminolevulinic acidinduced porphyrin synthesis and photodynamic inactivation of Escherichia coli B. J Photochem Photobiol B: Biol 1999;50:8–17. [10] Kreitner M, Wagner K-H, Alth G, Ebermann R, Foißy H, Elmadfa I. Haematoporphyrin- and sodium chlorophyllin-induced phototoxicity towards bacteria and yeasts – a new approach for safe foods. Food Control 2001;12:529–33. [11] Wilson M, Dobson J, Harley W. Sensitization of oral bacteria to killing by low-power laser radiation. Current Microbiol 1992;25:77–81. [12] Snyder EG. Preparation of chlorine, Patent 2,274,101 USA, (02/1942). [13] Allen EA. New porphyrinic and chlorophyllic compositions and process therefore, Patent 3,102,891 USA; 1963. [14] Sakata I, Nakajima S. Pheophorbide derivatives and alkaline salts thereof, Patent 4,709,022 USA (11/1987), C.A., 103 (15), 123271s, 1985. [15] Bommer JC, Sveida ZJ, Burnham B.F., 1986. Further studies on the relationship between tetrapyrrole structure and usefulness as photosensitizers. In: Proceedings of 1st International Conference of Clinical Applications of Photosensitization for Diagnosis and Treatment, Tokyo, p. 129. [16] Bommer JC, Burnham BF. Tetrapyrrol therapeutic agents, Patent 4,656,186 USA (04/1987), C.A. 106, 85048b, 1987. [17] Kostenich GA, Zhuravkin IN, Zhavrid EA. Experimental grounds for using chlorin e6 in the photodynamic therapy of malignant tumors. J Photochem Photobiol 1994;22:211. [18] Parkhots MV, Knyukshto VN, Isakov GA, Petrov PT, Lepeshkevich SV, Khairullina AYA, et al. Spectralluminescent studies of the Photolon photosensitizer in model media and in blood of oncological patients. J Appl Spectr 2003;70:921–6. [19] Karotki A. Simultaneous two-photon absorption of tetrapyrrolic molecules: from femtosecond coherence experiments to photodynamic therapy, Ph.D. Dissertation. Bozeman, Montana: Montana State University; 2003. [20] Website: www.belmedpreparaty.com [21] Podbielska H, Str˛ek W, Deren´ P, Bednarkiewicz A. New approach to PDT with chlorophyll based photosensitizer and semiconductor laser. Physica Medica 2004;20(Supplement 1):61–2. [22] Podbielska H, Ulatowska-Jarz˙ a A. Sol–gel technology for biomedical engineering. Bulletin of the Polish Academy of Sciences 2005;53(3):261–71. [23] Podbielska H, Ulatowska-Jarz˙ a A, Mu¨ller G, Eichler HJ. Sol gels for optical sensors. In: Baldini F, Chester AN, Homola J, Martellucci S, editors. Optical Chemical Sensors, vol. 224. Berlin: Springer; 2006. p. 353–85 Chapter. 17. [24] Ulatowska-Jarz˙ a A, Bindig U, Podbielska H, Ho"owacz I, Str˛ek W, Mu¨ller G, et al. Spectroscopic properties of chlorophyll based photosensitive dye entrapped in sol–gel fiberoptic applicators. Mater Sci 2005;23:111–22.
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Medical Laser Application 21 (2006) 177–183 www.elsevier.de/mla
Antimicrobial PDT with chlorophyll-derived photosensitizer and semiconductor laser A. Ulatowska-Jarz˙ aa, J. Zychowiczb, I. Ho"owacza, J. Bauera, J. Razikc, A. Wieliczkoc, H. Podbielskaa,, G. Mu¨llerd, W. Str˛eke, U. Bindigf a
Bio-Optics Group, Institute of Physics, Wroc!aw University of Technology, Wybrzez˙e Wyspian´skiego 27, 50-370 Wroc!aw, Poland Haemato-Poland, 50-376 Wroc!aw, Hoene-Wron´skiego 14, Poland c Department of Epizootiology and Veterinary Administration with Clinic, Faculty of Veterinary Medicine, The Agricultural University of Wroc!aw, pl. Grunwaldzki 45, 50-366 Wroc!aw, Poland d Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Institute of Medical Physics and Laser Medicine, Fabeckstr. 60-62, 14195 Berlin, Germany e Institute of Low Temperatures and Structure Research, Polish Academy of Science, Oko´lna 2, PL-50-119 Wroc!aw, Poland f Laser- und Medizin-Technologie Berlin, Fabeckstr. 60-62, 14195 Berlin, Germany b
Received 12 May 2006; accepted 24 May 2006
Abstract The study was carried out on Escherichia coli strains isolated from poultry and cows. The bacteria were incubated and treated by Photolon, a plant-based photosensibilizator, and exposed after that to laser light at 662 nm. Additionally, the photosensitizer was immobilized in fiberoptic sol–gel coating, constructed as an 8-layer applicator. It was demonstrated that Photolon inhibits bacteria growth. Photolon entrapped into the sol–gel matrix also exhibits photodynamic activity and no bacteria were seen near the doped sol–gel applicator. r 2006 Elsevier GmbH. All rights reserved. Keywords: Antimicrobial photodynamic therapy; E. coli; Sol–gel applicator; Photolon
Introduction Escherichia coli is one of the most widely spread microorganisms. Many strains of E. coli are pathogenic and may cause various diseases, e.g. urinary tract infections, sepsis, meningitis and infectious diarrhea. Pathogenic strains are also capable of contaminating the blood pools or food products. Many strains are resistant to commonly applied disinfection methods and antiCorresponding author. Tel.: +48 71 320 2825; fax: +48 71 328 3696. E-mail address: [email protected] (H. Podbielska).
1615-1615/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.mla.2006.05.003
biotics. Therefore, there is continuous need to search for alternative methods in order to combat the bacteria. One of them is photodynamic therapy. Antimicrobial photodynamic therapy (APDT) combines a nontoxic photoactive dye, photosensitizer (PS), with harmless visible light to generate singlet oxygen and free radicals that kill microbial cells. Currently, the major use of APDT is in disinfection of blood products for bacterial, fungal and viral inactivation and it can also be an alternative method for the decontamination of foods. The studies show that Gram-positive bacteria are susceptible to the photosensitizing action of a variety of
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photosensitizers [1–4]. Lazzeri and colleagues [5] from National University of Argentina have investigated the photodynamic activity of amphiphilic porphine in the different media and in vitro on the Hep-2 human larynx carcinoma cell line. Photosensitized inactivation of E. coli was analyzed using meso-substituted cationic porphine derivatives such as 5,10-di(4-methylphenyl)15,20-di(4-trimethylammoniumphenyl)porphine iodide 1 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphine iodide 2 and its metal complex with Pd(II). The results showed that the higher photoinactivation of cells was found for tricationic porphine, causing an 5.5 log (99.999%) decrease of cell survival. Lazzeri has reported that the high amphiphilic character of investigated photosensitizers may offer a promising model for photosensitizing agents with potential applications in bacteria inactivation by APDT. Additionally, recent work by Scalise et al. [6] has shown the photodynamic efficiencies of cationic Zn(II) N-methylpyridyloxy phthalocyanine (ZnPc 2) and noncharged Zn(II) pyridyloxyphthalocyanine (ZnPc 1) in homogeneous medium bearing a photooxidizable substrate and in vitro using bacterium E. coli. Promising results were obtained for cationic ZnPc 2, which can be an efficient phototherapeutic agent with potential photodynamic inactivation of bacteria cells. Wainwright et al. [7] have investigated the photobactericidal activity of phenothiazinium photosensitizers. Phenothiazinum dyes were tested against Grampositive (S. aureus) and Gram-negative bacteria (E. coli). The material was illuminated using a non-laser light source at a fluence of 1.75 mW/cm2 to enhance antibacterial activity in liquid culture [8]. The results showed a generally increased efficacy of PDT for inactivation of E. coli and S. aureus. Szocs et al. [9] have described the possibility and conditions for the induction of porphyrin synthesis by exogenous d-aminolevulinic acid (ALA) and its applicability for the inactivation of E. coli by PDT. After incubation of bacterial cells with ALA (2 107 cells ml1 with (5–9) 103 mol/l ALA), ALA-treated cells were irradiated by white light of 80 mW/cm2. Under these conditions, an experiment showed significant cell killing (over 99%) at a dose of 4.3 103 kJ/cm2. Obtained results proved that the porphyrin synthesis against Gram-negative E. coli B. can be stimulated by the addition of ALA. APDT can be a new approach for safe food. M. Kreitner et al. from the Institute of Nutritional Sciences (University of Vienna), Institute of Radiodiagnostics and Institute of Dairy Sciences and Bacteriology (University of Agricultural Sciences, Vienna) established an experimental model to examine the photosensitivity of immobilized microorganisms to develop a system where microorganisms with reduced mobility may be targets of a photodynamic effect [10]. They tested the
photosensitivity of several Gram-positive bacteria and yeasts using visible light and photosensitizers. The photosensitizers used for the reduction of microorganisms were hematoporphyrin (HP) and sodium chlorophyllin (CHL), because they are natural constituents of food [11]. Microorganisms such as S. aureus, B. subtilis, and B. cereus, as well as yeasts such as S. cerevisiae, Kloeckera javanica, and Rhodotorula mucilaginosa were under investigation. The cells were incubated with 105 M PS for 1 h. Further, suspended cells were lawned on nutrient agar (bacteria) or wort agar (yeasts) and illuminated immediately with a 1000 W photooptic lamp for 1 h. The results obtained by Kreitner demonstrated that the survival rate of bacteria fixed on solid media ranged from 0.0006% to 0.08% (3–5 log 10 inactivations), and the level of yeasts ranged from 0.1% to 50% (0.3–3 log 10 inactivations).
Materials Photolon Photolon, chlorin e6 ((18-carboxy-20-(carboxymethyl))-8-ethenyl-13-ethyl-2,3-dihydro-3,7,12,17-tetramethyl-21H,23H-porphin-2-propionic acid) (powder from Belmedpreparaty in cooperation with Haemato Ltd. Germany) was used in this study. Aqueous stock solutions (8.2 104 M) were prepared by dissolving the photosensitizer in water (Millipore grade). In organic chemistry, a chlorin is a large heterocyclic aromatic molecule consisting, at the core, of three pyrroles and one reduced pyrrole, all coupled by methine linkages. Compared to the porphyrins, a chlorin is largely aromatic but not aromatic through the entire circumference of the ring. Magnesiumcontaining chlorins are called chlorophylls and are the main photosensitive pigments in chloroplasts. A related compound, with two reduced pyrroles, is called a bacteriochlorin. Because of their strong photosensitivity, chlorins are in use as photosensitizing agents in experimental PDT therapies. Water-soluble derivatives of chlorophyll were first introduced as potential drugs by Snyder (USA) in 1942 [12]. The next important step was done by Allen [13]. He revealed that the major chemical compound of native chlorin mixtures is chlorin e6, which under oral and intravenous administration possesses low toxicity and demonstrates hypotensive, antisclerotic, spasmolytic, anesthetic and antirheumatoid activity. The first PDT usage of a chlorin relates to a pheophorbide a derivative. Some of them were patented as prospective photosensitizers for PDT in Japan in 1984 by Sakata and Nakajima [14]. In 1986, an American group reported on a photosensitizer meeting crucial PDT requirements:
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good tumor affinity and intensive absorption in the middle red part of the visible spectrum [15]. Their choice was mono-L-aspartyl-chlorin e6 (MACE). At present, this compound is at stage III of clinical trial studies in Japan. This group has even patented other functionally advanced chlorin and bacteriopheophorbide a derivatives as photosensitizers for PDT [16]. In the 1990s a Byelorussian group at first headed by G. Gurinovich reported about their research on water-soluble chlorintype photosensitizers derived from nettle [17]. Recently, their work was focused on the spectral-luminescent characteristics of chlorine e6 and – the newly developed photosensitizer – Photolon in whole blood [18]. Photolon – a chlorin derivative – belongs to the onephoton-based photosensitizers [19]. The dye is approved for cancer treatment in some countries [20]. Photolon is referred to a group of new sensitizers for treating cancer by means of PDT [18]. It can be administered intravenously or topically. Recently, some non-oncologic applications have been reported as well [21]. The chemical structure of Photolon has a partial reduced porphyrin moiety. The molecular structure is comparable to chlorine e6, which can be isolated after hydrolysis of the five-membered exocyclic b-ketoester moiety of pheophorbide a. It is well known that absorption and fluorescence characteristics of tetrapyrrolic macrocycles are sensitive to changes in their (molecular) environment. The chemical structure of Photolon is depicted by Fig. 1.
Photolon doped sol–gel fiberoptic applicators Sol–gel materials are porous, glass-like solid bodies that find many interesting applications, among others, in the biomedical field [22,23]. All chemical compounds for the sol–gel production of coated fibers examined here were obtained from commercial sources and used without further purification: deionized water, ethyl
Fig. 1. Chemical structure of Photolon (R1 ¼ CH2CH2 COOH, R2 ¼ CH2COOCH3, R3 ¼ COOH) according to [19].
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alcohol C2H5OH 96% v/v (Polish Chemicals), Triton X-100 (Aldrich), and tetraethylorthosilane Si(C2H5O)4, TEOS (Aldrich), hydrochloric acid HCl 35% w/w (Polish Chemicals). Recently, we demonstrated that Photolon entrapped in a sol–gel matrix preserves its photochemical activity [24]. The sol–gel layers were produced from sols prepared from the silicate precursor TEOS mixed with ethyl alcohol. The sol–gel material was prepared with factor R ¼ 20, where R denotes the solvent to precursor molar ratio. Hydrochloric acid was added as catalyst in proportion to ensure acid hydrolysis (pHE2). The mixture was stirred at room temperature for 4 h by means of a magnetic stirrer (speed 400 rpm). Hard clad silica (HCS) fibers of low OH (CFO 149312) origin laser components were used (fused silica core f1 ¼ 400 mm, with cladding f2 ¼ 430 mm; with ETFE buffer diameter 730 mm). They feature high transmission values from UV to IR (200–2400 nm). The external jacket was mechanically removed from a distance of 3.5 cm. Then the cladding was treated by a hot torch. The residuals were removed with linen cloth and cleaned with ethanol. An aqueous stock solution of Photolon was prepared by dissolving 0.5 mg of dye in 1 ml deionized water. Next 1 ml of ethyl alcohol was added. Aliquots of 50 ml stock solution and 20 ml of Triton X-100 were added to 1 ml of the freshly prepared sols. The freshly prepared sol was used to produce doped fiberoptic coatings according to the procedure described. The stock solutions and the sol–gel modified fibers were stored in darkness at room temperature. A modified dip-coating method was applied to cover the bare fibers with sol–gels. The fibers were placed vertically in a plastic container with a specially designed bottom possessing an opening with a controlled diameter, to ensure the required speed (1 cm/h) of sol outflow. Just before the deposition procedure, the surfactant Triton X-100 was added to the liquid sol. Prior to the applicator construction, some spectrophotometer studies of Photolon in the sol–gel matrix were performed. Fluorescence spectra were obtained by means of the fluorescence spectrometer LS-50B (Perkin Elmer/UK). Spectra were recorded in the wavelength range 350–750 nm (100 nm/min, excitation slit 3 nm, emission slit 5 nm) by the use of quartz micro-cuvettes (optical length 0.5 cm, Hellma/Germany). The fluorescence intensity of Photolon entrapped in sol–gel (1.6 104 M Photolon) is depicted in Fig. 2. The visible maxima are observed for the blue (411, 403 nm) and red regions (648, 670 nm) of the spectrum. These spectra were measured for an eight -layer coating (fresh preparation). Recently, we demonstrated that the number of layers building the sol–gel applicators for interstitial laser therapy influences significantly the light
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distribution from the applicator and overall applicator performance in interstitial coagulation [25,26]. The optimal number of layers according to the results is 8.
Bacteria preparation The studies were carried out on E. coli strains isolated from poultry and cows (from the collection of the Department of Epizootiology and Veterinary Administration with Clinic). All strains were cultured on MacConkey agar (bioMerieux, no cat. D6612) and tryptose-soya broth (bioMerieux, no cat. 51019). E. coli were cultured according to standard bacteriological procedures. Strains were determined by routine laboratory methods. The bacteria were identified on the basis of their metabolic properties using API 20E tests (bioMerieux, no cat. 20100). Bacterial strains were inoculated into tryptose-soya broth and incubated at 37 1C overnight. The bacteria suspension density was set with the use of a spectrophotometer at l ¼ 560 nm to an equivalent of 1. The appropriate dilutions from 101 to 106 in 0.85% NaCl were prepared and then from each dilution 0.1 ml was cultured on MacConkey agar.
fluorescence intensity / a.u.
40 403
i) ii)
i)
a)
b)
ii) ii) 50 501 53 531
61 611
350
400
500 wavelength/nm
600
a) ex 400 nm b ) ex 410 nm m
700
750
Fig. 2. Fluorescence intensity of Photolon entrapped in sol–gel coating on optical fiber core, eight layers.
Table 1.
CFU ¼
C , ðN1 þ 0:1N2Þ d a
where C is the number of all counted colonies, N1 the number of plates for lower bacteria dilution, N2 the number of plates for higher bacteria dilution, d the dilution factor and a the volume of bacteria deposited on the plate. The experimental groups examined in this study are shown in Table 1. For each group five plates were seeded by bacteria.
Results
64 648
530
The colonies were illuminated by laser light. To some of colonies the Photolon was added. Photolon-doped sol–gel interstitial applicators were also examined. For illumination the semiconductor pig-tailed laser (Laser Secura, Poland) lasing at 662 nm was used. The output power chosen was 200 mm and the illumination time for each plate was 10 min. After the illumination, the plates were incubated at 37 1C for 24 h. The next day the colonies on the plates were counted (we chose the plates with 10–300 CFU/plate). Colony forming units (CFU) were calculated according to the formula
67 670
em 670 nm em 648 nm
41 411
Methods
The CFU for each group of samples was counted after the post-illumination incubation period. Some of the exemplary results are presented in Figs. 3 and 4. Fig. 3 demonstrates that the photodynamic activity of Photolon leads to inhibition of bacteria growth. This activity can also be seen for Photolon immobilized in a sol–gel applicator (Fig. 4a). For better visualization of the colonies the Sobel filter was applied. Analysis was performed with the OPTIMAS image processing program. The diagram (Fig. 5) demonstrates the results (CFU) as a percentage of mean CFU values in the control group.
Description of examined samples.
No (a—stays for higher bacteria dilution)
E. coli (low dilution)
E. coli (high dilution)
1–1a 2–2a 3–3a 4–4a
Control (not illuminated, no Photolon) Not illuminated, Photolon added Illuminated, no Photolon Illuminated, Photolon doped sol–gel applicator Not illuminated, Photolon doped sol–gel applicator Illuminated, with Photolon
Control (not illuminated, no Photolon) Not illuminated, Photolon added Illuminated, no Photolon Illuminated, Photolon doped sol–gel applicator Not illuminated, Photolon doped sol–gel applicator Illuminated, with Photolon
5–5a 6–6a
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Sample No
E. coli (low dilution)
181
E. coli (high dilution)
1 – 1a
6 – 6a
Fig. 3. E. coli colonies after incubation, (1), control without photosensitizer, not illuminated, (6), illuminated samples treated with Photolon.
Fig. 5. Mean values of CFU for each experimental group calculated in [%] of mean CFU values for control group. The visible colonies inhibition is observed for Photolon treated bacteria illuminated with laser light. There is also inhibition seen for samples with sol–gel applicator.
bacteria in various environments (e.g. milk, various liquids, food, etc.).
Acknowledgement
A
B
Fig. 4. The Photolon doped sol–gel applicator was placed in bacteria environment. After illumination and incubation, no bacteria are seen near the applicator (A). The image processing (Sobel filter) applied to image (A) visualized the colonies. They are not observed near the applicator.
The support of the Polish Ministry of Science, Grant KBN No. 4T11E01124 is gratefully acknowledged. This work was also supported by the joint program of DAAD (German Academic Exchange Service) and the Polish Ministry of Science for endorsement of cooperative research projects.
Zusammenfassung
Discussions The E. coli was the subject of this in vitro study. The photodynamic activity of a plant-based sensitizer was examined. The highest mean value of CFU was observed in the control group, which remained untreated in an incubator. Some bacteriotoxic effect was observed in the remaining groups that for the experiment were taken out from the incubator. The visible effect of photodynamic activity was observed for illuminated samples, treated by Photolon. The visible colonies inhibition was observed in this case. There is also significant inhibition seen for samples with the sol–gel applicator. The result obtained for the applicator may be exploited for some local PDT applications, e.g. interstitial therapy. The other modality may be the application of such an applicator to combat
Antimikrobielle PDT mit einem dem Chlorophyll verwandten Photosensibilisator und Halbleiterlaser Die vorliegende Studie wurde an E. coli-Sta¨mmen durchgefu¨hrt, die aus Geflu¨gel und Rind isoliert wurden. Die Bakterien wurden inkubiert, mit Photolon, einem pflanzlichen Photosensibilisator, behandelt und mit Laserlicht der Wellenla¨nge 662 nm bestrahlt. Zusa¨tzlich wurde der Photosensibilisator in die Sol–Gel–Beschichtung eines aus acht Schichten bestehenden Applikators immobilisiert. Es wurde nachgewiesen, dass Photolon das bakterielle Wachstum inhibiert. Im Bereich des dotierten Sol–Gel–Applikators wurde kein bakterielles Wachstum beobachtet. Das in die Sol–Gel–Matrix implementierte Photolon weist somit eine photodynamische Aktivita¨t auf. Schlu¨sselwo¨rter: Antimikrobielle photodynamische Therapie; E. coli; Sol–Gel–Applikator; Photolon
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Resumen Terapia Fotodina´mica (PDT) antimicrobiana mediante un fotosensibilizador derivado de la clorofila y la´ser semiconductor Este estudio fue realizado utilizando diferentes cepas de E. coli aisladas de vacas y aves de corral. Las bacterias fueron incubadas y tratadas con Photolon, un compuesto fotosensibilizador a base de plantas, y posteriormente expuestas a un la´ser de 662 nm. Adicionalmente, el fotosensibilizador fue inmobilizado en una cubierta fibroo´ptica sol–gel, construı´ da como un aplicador de 8 capas. Se demostro´ que el Photolon inhibe el crecimiento bacteriano. El Photolon atrapado en la matriz sol–gel tambie´n posee esta actividad fotodinamica ya que no se observo´ crecimiento bacteriano alguno en los alrededores del aplicador sol–gel. Palabras clave: Terapia fotodina´mica antimicrobiana; E. coli; aplicador sol–gel; Photolon
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