Photosensitizers (porphyrins) attack cancer cells and during photoactivation contribute to the generation of reactive oxygen species that damage various structures of cancer cells, which leads to their destruction. Porphyrins reach tumors through the blood and modelling of conditions for the transfer of porphyrins to the tumor (binding to blood proteins, the influence of pH and salt composition of the medium) is one of the most important problem of photodynamic therapy (PDT) of tumor. Ceruloplasmin (CP) is an important copper-containing plasma protein that actively binds photosensitizers (PS) and can play a certain role in the transfer of photosensitizers to the tumor. Changes in pH, as well as of salt composition in the tumor tissue and its cells can cause modifications in the complexes [CP + PS] and a possible separation of the PS from the complex can change the photodynamic process of tumor destruction. In this paper, it was shown that: 1) with a change in the pH of the medium from neutral to acidic, as well as with a change in the salt composition of the medium, significant conformational changes of ceruloplasmin and the transition of photosensitizers to the surface (for cationic porphyrins) or to the inside of the protein macromolecule (for chlorin e6) occur, with a partial separation of photosensitizers from protein; 2) CP can form complexes with photosensitizers and be an active carrier of PSs in the blood.
The relevance and importance of the development of new areas of tumor therapy is defined in connection with a strong increase in the level of oncological diseases. The latest global cancer data indicates a high mortality rate: the incidence of cancer increases to 18.1 million new cases and 9.6 million cancer deaths in 2018. Advances in nanostructures and nanotechnologies at the molecular scale in future can revolutionize several aspects of the diagnosis and treatment of body tumors. Search of highly specific and highly effective new therapeutic compounds is one of the most important directions in treatment of oncological diseases by noninvasive way. Currently, in clinics method of photodynamic therapy (PDT) of tumors based on photosensitizers (PSs) and light irradiation is widely used. At its application, the cancer cells are unable to develop resistance to such method of destruction.
Anna Gyulkhandanyan, Marina Parkhots, Valeri Knyukshto, Sergei Lepeshkevich, Boris Dzhagarov, Anna Zakoyana, Aram Gyulkhandanyan, Marina Sheyranyan, Guevork Kevorkian, Grigor Gyulkhandanyana
In photodynamic therapy (PDT) of tumors, targeted therapy is one of the most successful directions. The goal of the present work was to study the formation of new potential photosensitizers, based on transferrin (Tf) and cationic porphyrins, for targeted binding with transferrin receptors of cancer cells. We studied non-covalent binding of three cationic porphyrins 1) meso-tetra [4-N-(2'-oxyethtyl) pyridyl] porphyrin (TOEt4PyP) 2) Zn-TOEt4PyP and 3) Zn-mesotetra [4-N-butyl pyridyl] porphyrin (Zn-TBut4PyP) with human transferrin by absorption and fluorescent spectroscopy as well as by gel filtration methods. It was shown that the investigated porphyrins and metalloporphyrins bind stably enough to the protein molecule. It was found that the porphyrins having Zn ion in porphyrin core as well as the peripheral OH - groups are linked better to the transferrin molecules. It can be apparently explained by Zn coordination with transferrin amino acids and the formation of the hydrogen bonds between OH - groups of the porphyrin and transferrin amino acids. It was shown that, for the transferrin-porphyrin complexes, singlet oxygen luminescence is significantly decreased due to the presence of transferrin amino acids which are efficient quenchers of singlet oxygen.
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.
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.
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.
Antimicrobial photodynamic inactivation has been successfully used against Gram (+) microorganisms, but most of the
photosensitizers (PSs) on Gram (-) bacteria acts weakly. PSs are the natural or synthetic origin dyes, mainly porphyrins.
We have synthesized more than 100 new cationic porphyrins and metalloporphyrins with different functional groups
(hydroxyethyl, butyl, allyl, methallyl) and metals (cobalt, iron, copper, zinc, silver and other); from the nettle have also
been purified pheophytin (a+b) and pheophytin (a) and have synthesized their Ag-and Zn-metalloporphyrins. It was
found that in the dark (cytotoxic) mode, the most highly efficiency against microorganisms showed Agmetalloporphyrins
of both types of porphyrins (synthetic and natural). Metalloporphyrin of natural origin Ag-pheophytin
(a + b) is a strong antibacterial agent and causes 100% death as the Gram (+) microorganisms (St. aureus and MRSA) and
the Gram (-) microorganisms (E.coli and Salmonella). It is established that for the destruction of Gram (+) and Gram (-)
microorganisms in photodynamic mode cationic water-soluble synthetic metalloporphyrins, especially Zn-TBut4PyP,
many times more effective than pheophytins. In vivo conditions on mice established that the best therapeutic activity
against various strains of the microorganism St. aureus has the synthetic metalloporphyrin Ag-TBut4PyP. It is
significantly more efficient than known drug "Chlorophyllipt" (2.5-3 times) and leads the survival rate of animals up to
50-60%.
Porphyrins binding and transport to tumor is the one of the central tasks of photodynamic therapy of tumor (PDT). The
main carriers of porphyrins (photosensitizers) in the blood are lipoproteins, serum albumin and hemoglobin. In studying
the phenomenon of complexation of proteins with ligands must take into considering the real conditions that exist in the
organism and, in particular, take into considering the presence of fatty acids in blood. Up to date the role of fatty acids
(palmitic and stearic) in the binding of porphyrins with proteins not been determined. A key step in solving of these
problems is to determine the binding constants of porphyrin-protein pairs and effect of fatty acids on this process. The
most direct and sufficiently accurate methods of solving such problems are complementary methods of absorption and
fluorescence spectroscopy. The results of spectral studies on the binding of porphyrins to serum albumin and hemoglobin
in the presence of fatty acids demonstrated a significant decrease in the degree of binding pair porphyrin-albumin and
porphyrin-hemoglobin with increasing concentrations of fatty acids in solution. The results lead to the conclusion that for
hemoglobin the presence in a solution of fatty acids on binding to the porphyrins affected more significantly than for
serum albumin. Thus, in natural conditions, when in the blood presented fatty acids the preference between hemoglobin
and serum albumin in the binding and in the transport of porphyrins should be given to serum albumin.
Photodynamic inactivation (PDI) of microorganisms is successfully applied against Gram (+)
microorganisms. However the majority of photosensitizers poorly affect on Gram (-) microorganisms. At
present number of works have shown that cationic photosensitizers are able to induce photoinactivation both
Gram (+) and Gram (-) microorganisms. The purpose of this work was definition of more effective new
cationic pyridylporphyrins with various coordinated metals and functional groups for destruction of Gram (-)
microorganisms. The efficiency of new cationic porphyrins and metalloporphyrins (9 compounds) was tested
against Gram (-) microorganism E. coli (strain Κ-12). The testing results show high efficiency of
metalloporphyrins, especially silver complexes, against E. coli microorganism under dark conditions. 50 %
and 100 % cell growth inhibitory concentrations (IC50 and IC100 values, accordingly) of studied metallocomplexes
are considerably lower in comparison with metal-free porphyrins. At the same time the Zncomplexes
of porphyrins are more phototoxic than their metal-free analogues. Zn-metalloporphyrins with
allyl and butyl functional groups were shown the highest efficiency against E. coli. The photodynamic action
of cationic Zn -TBut4PyP metalloporphyrin against Gram(+) (St. aureus and St. epidermis) and Gram(-)
(E.coli, strain K-12 and Salmonella sp.) microorganisms was investigated. It is revealed, that Gram (+)
microorganisms were 3-5 times more susceptible to the compounds' phototoxic influence than Gram (-)
microorganisms.
Transmembrane potentials appear to be cell state sensitive characteristics and can give information about cell damage initial stage. Photodynamic action of the photosensitizer chlorin e6 on plasmatic and mitochondrial membrane potentials of the rat thymus lymphocytes was studied using voltage-sensitive dye rhodamine 6G. It has been revealed that mitochondrial membrane potential is more sensitive characteristic of membrane disfunction than plasmatic one at the cell photodamage.
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 (Croker's sarcoma). Antitumor activity of these porphyrins in the dark was 30-40%, whereas photodynamic activity was 45-60%.
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.
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.
The interaction of aromatic amino acids, superoxide dismutase (SOD) and bovine serum albumin (BSA) with chlorin e6 (Chl) and formation of complexes were studied. The decrease of the efficiency of photochemical destruction in the absence of oxygen was observed. The changes of SOD absorption spectra in UV region agree with our former results on irreversible decrease of protein's tryptophan (Trp) luminescence under irradiation. At the same time no changes in enzymatic activity of irradiated SOD in the presence of Chl were detected. The irradiation effect on K+ and H+ ions movement across the membrane of erythrocytes loaded with Chl was studied.
The pharmacokinetic behavior of chlorin e6 in different organs and tumor tissues of rats infected with Sarcoma-45 has been investigated with help of the fiber-optic spectrofluorometer at excitation by harmonics of YAG:Nd laser ((lambda) equals 355, 532 and 660 nm). The most intensive chlorine e6 fluorescence in tumor tissue was observed in 18 hours after pigment injection. The optimal time of fluorescence diagnosis has been determined (in 27 hours after pigment injection). The maximum of the tumor-to- normal tissue ratio of fluorescence was equal 10 at 666 nm. The rates of the accumulation and elimination of chlorin e6 in the skin, hypodermic tissues and tumors of rats were determined in vivo.
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