Photodynamic therapy (PDT) [1] is a treatment that is used for the destruction of certain tumors. PDT involves the combination of visible light and an organic dye - photosensitizer. Each factor is harmless by itself, but when is combined with oxygen, they can produce lethal cytotoxic agents that can inactivate tumor cells. Upon illumination, the photosensitizer is excited from the ground state to the first excited single state, followed by conversion to the triplet state via intersystem crossing. The excited triplet state can react in two ways. A Type I mechanism involves hydrogen-atom abstraction or electron-transfer reactions between the excited state of the photosensitizer and the substrate to yield free radicals and radicals ions. A Type II results from an energy transfer between the excited triplet state of the photosensitizer and the ground state molecular oxygen generating the first excited state of oxygen, singlet oxygen. The absence of light in many regions of the body may preclude the use of the photosensitizers as the therapeutic compounds for PDT. It has been found that the chemiluminescent oxidation of luciferin is able to generate a sufficiently intense and long-lived emission to induce antiviral activity of hypericin [2], a well-known photosensitizer.

Weak chemiluminescence accompanies the catalytic decomposition of hydrogen peroxide. It is possible to enhance this light emission and therefore also the population of the excited states by means of oxidizable substrates with the high quantum yield of excitation (energizers e.g. phthalhydrazide (PH) and luminol (LU) [3,4]. It is possible to transfer their excitation energy to the suitable acceptor (e.g. photosensitizer).

Instrumentation: Luminometer BioOrbit 1250 (Finland), Luminoskan and Fluoroskan Ascent (Labsystems, Finland) and inversion fluorescent microscope Olympus IX 70, Laser LASOTRONIC MED-130 (Germany).

Reagents: phthalocyanines (ClAlPcS₂, ZnPcS₂, CuPcS₄ (CU), NiPcS₄ (NI)), phthalhydrazide (PH), protoporphyrin IX (PP), methylene blue (MB), eosin B (EB), CuSO₄, H₂O₂ (Sigma-Aldrich, Germany), hypericin (HY), hypocrellin A (HA), luminol (LU), calcein AM (Molecular Probes, USA), all media for MIC determination were obtained from HiMedia Laboratories (Bombay, India).

Bacterial strains: Escherichia coli 3954, 3988 and 4225, Pseudomonas aeruginosa 3955, Staphylococcus aureus 3953 and 4223, and Enterococcus faecalis 4224 (Czech Collection of Microorganisms, Brno).

Cell cultures: NIH3T3 (mouse fibroblast), MCF7 (human breast adenocarcinoma), HOS (osteosarcom) (Masaryk Memorial Cancer Institute, Brno-Czech Republic)

The migration activity of splenocytes [5] derived from rabbit spleen fragments was determined as follows. Aseptically excised rabbit (Chinchilla) spleen was cut on a sterile paraffin plate into pieces 1 mm in diameter. These fragments were washed in minimal essential medium (MEM). Increasing concentrations of the additives (photosensitizers, phthalhydrazide, CuSO₄, H₂O₂) in MEM with 10% fetal calf serum (FCS) were pipetted in volumes of 0,2 mL into wells (plates 8x12, flat bottom). Each combination of the additives was placed into four wells; one fragment was transferred into each well. The fragments in cultivation medium only were used as a control. After 24 hours of cultivation at 37^oC in 5% CO₂, the migration zones of the spleen cells were measured [5]. The results are presented as the migration index (MI) - the ratio between the experimental migration zone (Fig.I.B.) to the control zone (Fig.I.A.). Compounds were tested in two independent experiments in triplicate.

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 Minimal inhibitory concentration (MIC) [6] Antimicrobial activity was tested on the standard strains of Escherichia coli 3954, 3988 and 4225, Pseudomonas aeruginosa 3955, Staphylococcus aureus 3953 and 4223, and Enterococcus faecalis 4224 (Czech Collection of Microorganisms, Brno). Bacteria were first precultured 2 h at 37°C in 5 mL of Mueller-Hinton broth, then diluted with sterile distilled water in a 1:10 ratio, and inoculated into the wells (sterile plates 8x12, flat bottom) containing tested compound in cultivating medium. Gram-positive bacteria were cultivated in a medium containing 37g Brain Heart Infusion in 1 L of distilled water. Medium for Gram-negative bacteria consisted of 5 g Protose-BE, 17,5 g Casein Acid Hydrolysate and 0,6 g Na₂CO₃ in 1 L of distilled water. Minimal inhibitory concentrations (MICs) were determined by broth microdilution method. The stock solutions of tested compounds were prepared in distilled water. The plates were incubated 18 h at 37°C and MIC was determined as the lowest concentration of the tested compounds at which bacterial growth was not observed (content of well stays without turbidity). Compounds were tested in concentrations in geometrical dilution steps, in three independent experiments in triplicate.

Test of viability [7] NIH3T3 (mouse fibroblast), MCF7 (human breast adenocarcinoma) and HOS (osteosarcom) were used as a standard testing system. Twice washed trypsinized cells were divided in the amount of 10⁴ to each well and filled in DMEM with 10% FCS in a total volume of 80 µL. After 24 hours of cultivation at 37°C in 5% CO₂ the photosensitizer with chemiluminescent system (20 µL) was added. The total volume of 100 µL (cells with additives) were cultivated for 24 hours. For identification of live and dead cells the fluorescent probes calcein AM was used. For the elimination of the influence of fluorescence from the photosensitizer the testing wells were aspirated, washed with 100 µL PBS and filled with 100 µL PBS and 100 µL calcein AM. The results were proved by fluorometric measurement with Fluoroskan Ascent. Morphological changes of the cells during cultivation have been observed in an Olympus IX 70 inversion fluorescent microscope. Compounds were tested in three independent experiments in quadruple.

The chemiexcitation of the xanthene dyes and photosensitizers (e.g. hypericin, phthalocyanines) induced by the hydrogen peroxide decomposition and autooxidation by dissolved oxygen in the micellar solutions of the cetyltrimethylammonium cation was studied in our previous work [4]. We subscribe to the CIEEL theory a formation of the micellar complex of electron transfer between hydrogen peroxide and dyes and formation of their excited states in the chemiluminescent system. Observed facts can be phenomenologically explained using the idea of the formation of micellar complexes of dyes according to Poisson Distribution Model (PDM). Both chemiluminescent intensity and chemiexcitation rate depend on the rate of hydrogen peroxide decomposition. The chemiexcitation does not depend on the structural type of dye. The final step is peroxide decomposition, back electron transfer to dyes and formation of their excited states. The energy transfer by energy pooling is only the minority way. The goal of this work was to find the efficiency of the chemiexcitation of the photodynamic effect on the biological material.

As a first we confirmed that the photosensitizers chemiexcited by phthalhydrazide can produce the photodynamic damage on the rabbit’s spleen (test of vitality of the migration activity of splenocytes derived from rabbit's spleen fragments).

This test turned up to be the simple, versatile and reliable for photodynamic effect in connection with sensitized chemiluminescence. For the dependence on the elementary concentrations of the photosensitizer, compounds of the chemiluminescent system and on their combinations, the data display the changes of the migration index. The changes can be neglected for the compounds of the chemiluminescent system but they are significant for the individual photosensitizer and its combination with the chemiluminescent compounds. Dependencies of MI on the photosensitizer concentration, eventually on the photosensitizer concentration at the presence of compounds of the chemiluminescent system, complied with simple Langmuir isotherms. For this reason we were able to assume that photosensitizer is adsorbed in the biological membrane and the presented data indicate that observed lethality is a result of photodynamic effect. Tables I. and II. show review c₅₀ (concentration calculated according to Langmuir isotherms, at which MI decrease in 50%) for the chemiexcited photodynamic effect.

Bacterial resistance against antibiotic treatment is becoming an increasing problem in medicine. Therefore methods to destroy microorganisms by other means are being investigated, one of which is photodynamic therapy. It has already been shown that a variety of Gram-positive and Gram-negative bacteria can be killed in vitro by PDT [8]. Therefore we have studied the chemiexcitation of photosensitizer (phthalhydrazide (2.10^-4 M) CuSO₄ (1.10^-6 M) and H₂O₂ (2,5.10^-5 M)) on Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration has been found for the presence of the photosensitizers and for the photosensitizer in the presence of the chemiluminescent system: Gram-positive bacteria Enterococcus faecalis: 0,545 mg/mL ClAlPcS_2, 0,327 mg/mL ClAlPcS₂ in the presence of PH, and Staphylococcus aureus: 0,545 mg/mL ClAlPcS₂, 0,188 mg/mL ClAlPcS₂ in the presence of PH, 1,114 mg/mL ZnPcS₂, 0,265 mg/mL ZnPcS₂ in the presence of PH, Gram-negative bacteria Pseudomonas aeruginosa: 0,667 mg/mL ClAlPcS₂, 0,333 mg/mL ClAlPcS₂ in the presence of PH more than 1,2 mg/mL ZnPcS₂, 0,133 mg/mL ZnPcS₂ in the presence of PH, Escherichia coli: more than 1,2 mg/mL ClAlPcS₂, 0,375 mg/mL ClAlPcS₂ in the presence of PH, more than 1,2 mg/mL ZnPcS₂, 0,265 mg/mL ZnPcS₂ in the presence of PH.

* - the concentration used was up to 1,5 mg/mL. Number in the brackets shows the concentration of phthalocyanines (mg/mL) in the presence of phthalhydrazide but without CuSO₄ and H₂O₂.

Table V. shows the cytotoxicity (IC₅₀ for NIH3T3) of the photosensitizer (without the chemiexcitation) and of the photosensitizer chemiexcited by phthalhydrazide or by luminol.

Table VI. shows the cytotoxicity (IC₅₀ for MCF7) of the photosensitizer (without the chemiexcitation) and of the photosensitizer chemiexcited by phthalhydrazide or by luminol.

1. Sharman W.S., Allen C.M., van Lier J.E.: Photodynamic therapeutics: basic principles and clinical applications, Drug Discov Today 1999; 4 (11): 507-517.
2. Lasovský J., Rypka M., Slouka J.: Chemiluminescence enhanced by intramicellar energy transfer. J Lumin 1995; 65: 25-32.
3. Lasovský J., Bancířová M., Hrbáč J., Otyepka M.: Chemiexcitation of the xanthene dyes by phthalhydrazide and hydrogen peroxide in the micellar environment of cetyltrimethylammonium bromide. In: Roda A., Pazzagli M., Kricka L. J., Stanley P. E. editors. Bioluminescence and Chemiluminescence: Perspectives for the 21^st Century. Chichester: John Wiley & Sons. Ltd. 1998: 555-558.
4. Carpenter S., Fehr M. J., Kraus G. A., Petrich J. W.: Chemiluminescent activation of the antiviral activity of Hypericin: A molecular flashlight. Proc Natl Acad Sci USA 1994; 91: 12273-7.
5. Bancířová M. Ph.D. Thesis: Micellar Catalysis, Palacký University, Faculty of Natural Sciences 1997.
6. Urbášková P. at al.: Vyšetření pro antimikrobiálni terapii. Praha, Avicenum, 1985.
7. Kolářová H., Kubínek R., Lenobel R., Bancířová M., Strnad M., Jírová D., Lasovský J.: In vitro photodynamic therapy with phthalocyanines on the MCF7 cancer cells. Internet Journal of Photochemistry and Photobiology. 1999

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8. Phillips D.: Chemical mechanisms in photodynamic therapy with phthalocyanines. Prog Reaction Kinetics 1997; 22: 175-300.