Grigor V. Gyulkhandanyan

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods.

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800 nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.

Investigation of photodynamic activity of water-soluble porphyrins in vitro and in vivo

Photodynamic therapy (PDT) is the method of photosensitized tumor treatment. It is based on the photosensitizer (PS) selective accumulation in tumors, its subsequent activation under the light influence and oxygen active form formation that results in tumor destruction. Photodynamic action of some new water-soluble porphyrins was investigated in our laboratory. Dose-dependent effect of these porphyrins was shown on PC-12 murine pheochromocytoma cell line. The results revealed that the efficiency of the investigated porphyrins decreased in the following way: TOEPyP (meso-tetra-(4-N-oxyethylpyridyl)porphyrin) > Zn-TOEPyP > Ag-TOEPyP. It was shown that TOEPyP possessed nearly the same photodynamic activity (LD50) as well-known photosensitizer chlorin e6. These porphyrins have also demonstrated quite high photodynamic activity in vivo. The results were obtained in the experiments on white mice with engrafted C-180 (Crokers sarcoma). Antitumor activity of these porphyrins in the dark was 30-40%, whereas photodynamic activity was 45-60%.

Complexation of porphyrins with nanoparticles of zeolite

The binding of nanoparticles zeolite with a number of cationic porphyrins are studied. Previously, it was established that the main mechanism of binding the zeolite nanoparticles with cationic porphyrins is an ionic bond. Since binding of porphyrins as ligands to nanoparticles at the initial stage of interaction is determined by the Brownian motion of porphyrins, it is obvious that the interaction of porphyrins with nanoparticles may depend on the temperature. In the present paper by methods of absorption and fluorescence spectroscopy was studied the complexation of porphyrins with zeolite nanoparticles at different temperature conditions. It was established that there is a clear temperature dependence of the complexation of cationic metalloporphyrins with zeolite naonoparticles, and for correct determination of the percentage of binding must be strict thermostating of the experimental conditions.

Photophysical properties and photodynamic efficiency of cationic porphyrins

Photodynamic inactivation of some microorganisms (St. aureus, E.coli) was investigated and their dependence on photo-physical properties of photosensitizers (PS) (cationic porphyrins and metalloporphyrins) was shown. One of the most important criteria for the effectiveness of the PS`s is the quantum yield of singlet oxygen (γΔ). Our investigations were shown that γΔ of metalloporphyrins, containing Zn, significantly higher than of metal-free porphyrins (85-97% and 77-79%, respectively). Previousl y experimentally we were found that under the action cationic porphyrins and metalloporphyrins on Gram (+) and Gram (-) microorganisms efficiency of metalloporphyrins Zn-TOEt4PyP and Zn-TBut4PyP in 3-5 times was higher than the metal-free porphyrins. In this study under the action of porphyrins and their Zn-derivatives on microorganism St. aureus such an effect was confirmed. Using the LED with a peak emission of 405 nm and a power density of 70 mW/cm2, and irradiation time of microorganisms from 5 to 30 minutes we have found, that at a concentration of 0.1 ug/ml the highest efficiency is observed of metalloporphyrin Zn-TBut3PyP. Upon irradiation of 10 and 15 min his efficiency is 3-5 times higher than the metal-free porphyrin TOEt4PyP, and irradiation for 30 min via Zn-TBut3PyP is practically completely kills microorganisms. These data correlate with the quantum yield of singlet oxygen for photosensitizers. The 30 mindirect sun exposure (power density of 70 mW/cm2) of photosensitizer solutions showed that a significant photobleaching of porphyrins and metalloporphyrins does not occur. Thus, Zn-containing cationic metalloporphyrins are highly efficient photosensitizers for photodynamic inactivation of microorganisms and PDT.

Complexation of porphyrins with silver and zeolite nanoparticles

It is known that nanoparticles of colloidal silver and zeolites due to the porosity have an extremely large specific surface, which is an order of magnitude increases their sorption capacity. Previously we synthesized a set of water-soluble cationic porphyrins and metalloporphyrins and in the laboratory in vitro had shown their high effectiveness against the various cancer cell lines, and against a variety of microorganisms. The aim of this work was to study of processes sorption/desorption of porphyrins on nanoparticles of silver and zeolites. The interaction of cationic porphyrins with silver nanoparticles of 20 nm diameter was studied in the visible spectrum, in the range 350-800 nm. Investigation of sorption dynamics of porphyrins in the silver nanoparticles using two porphyrins: a) meso-tetra (4-N-butyl pyridyl) porphyrin (TBut4PyP), b) Ag-TBut4PyP, as well as of photosensitizer Al-phthalocyanine was carried out. Analysis of the dynamics of change in the absorption spectra for porphyrins TBut4PyP, Ag-TBut4PyP, Zn-TBut4PyP and Zn-TOEt4PyP by adding of nanoparticles of colloidal silver and zeolites leads to the conclusions: 1. nanoparticles of colloidal silver and zeolites are promising adsorbents for cationic porphyrins (sorption of 55-60% and 90-95%, respectively); 2. sorbents stable long (at least 24 hours) keeps the cationic porphyrins; 3. on nanoparticles of colloidal silver and zeolites an anionic and neutral porphyrins not be adsorbed or adsorbed bad.

Study of photodynamic activity of hypericin and synthetic photosensitizers on haemolysis of erythrocytes in vitro

Influence of hypericin and synthetic (see manuscript for formula) on haemolysis of human erythrocytes was investigated. It was shown that both hypericin and synthetic porphyrins (TOEPyP and Zn-TOEPyP) did not cause haemolysis in the dark (24 hrs incubation), whereas Ag-TOEPyP leaded to haemolysis already after 40 min incubation, i.e. was cytotoxic. Hypericin (25 μM -125 μM) possessed haemolytic activity upon light exposure (visible spectrum, 30 mW/cm2, 5 min and more). Total haemolysis of erythrocytes was observed at 15 min exposure to light at all the investigated concentrations of hypericin. Dose-dependent haemolytic effect of TOEPyP and Zn-TOEPyP depending on light exposure time was also investigated. TOEPyP demonstrated considerably higher haemolytic activity compared to Zn-TOEPyP. Ag-TOEPyP demonstrated the weakest photodynamic activity. The effect of ascorbic acid on porphyrin-induced haemolysis was also revealed. Ascorbic acid at the concentration of 0.15 μM and above significantly increased haemolysis induced by hypericin, whereas at concentration of 0.75 μM and less it appeared to possess protective property. TOEPyP and Zn-TOEPyP did not demonstrate photodynamic properties in the presence of ascorbic acid (3.75 μM and more). The ascorbic acid at the concentrations below 3.75 μM did not have any influence on erythrocyte haemolysis induced by TOEPyP, while it increased haemolytic effect of Zn-TOEPyP. Thus, the full inhibition of photohaemolysis induced by (see manuscript for formula) by singlet oxygen quenchers - ascorbic acid and tryptophan - was shown. This testifies to the fact, that the photohaemolysis induced by these porphyrins is caused by Type II reactions.

Anticancer activity of the new photosensitizers: dose and cell type dependence

The necessity of researches of antitumor efficiency of new photosensitizers (PS) is explained by the opportunity of their application in photodynamic therapy of tumors. PS, selectively accumulated in cancer cells and activated by the light, generate the active oxygen species that cause apoptosis. Earlier, it was shown that PS chlorin e6 (0.3-0.5 μg/ml) induces rat embryo fibroblast-like cell apoptosis. In present work antitumor activity of the new photosensitizers, water-soluble cationic porphyrins and their metal complexes, is investigated. The dose-dependent destruction of cancer cells was shown on PC-12 (pheochromocytoma, rat adrenal gland) and Jurkat (human lymphoma) cell lines. Meso-tetra-[4-N-(2 `- oxyethyl) pyridyl] porphyrin (TOEPyP) and chlorin e6 possessed the same toxicity at LD50 dose on PC-12 cell line, whereas phototoxicity of TOEPyP was 3 times less compared to chlorin e6(LD50=0.2 and 0.075 μg/ml accordingly). The results have shown weak photosensitizing effect of Zn-and Ag-derivatives of TOEPyP on PC-12 cell line. TOEPyP and Zn-TOEPyP (0.1 - 50 μg/ml) were non-toxic for Jurkat cell line, whereas Ag-TOEPyP was toxic at 10 μg/ml (LD90). TOEPyP and chlorin e6 have shown phototoxic effect in the same dose range (LD50=0.5 and 0.3 μg/ml accordingly). The investigation of toxic and phototoxic effects of the new porphyrins revealed significantly different sensitivity of various cell lines to PSs.