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3.3. The analysis of photodynamic activity tetraariltetratsianoporfirazinov concerning tumoral crates in culture

By us ability Pz (I) PSHCH has been shown to cause photoinduced  destruction of crates (drawing 26). At illumination galogenovoj a valve of crates, predynkubirovannyh with dye, in 10-20 minutes it was observed "puzyrenie" membranes of crates and their  destruction while at inkubatsii in darkness or illumination of crates without dye they maintained the viability.


The chemical structure of explored linkings has allowed to make the guess of their ability to generate singletnyj oxygen at illumination. For acknowledgement of the given guess (table 6) has been measured quantum vyhodgeneriruemogo Pz (I) in various mediums. As standard linkings with known values of a quantum yield used TPPS and TMPyP.
Table 6.

Quantum yield singletnogo the oxygen generated Pz (I) in

Various mediums

The sample Solvent Fa
TPPS D2O 0.70
Pz (I) D2O, polymeric brushes 0.29
Pz (I) D2O, sucrose (67 %) L

0.21

TMPyP D2O 0.74

— Calculated against TMPyP


From table data it is visible, that TPPS and TMPyP essentially surpass Pz (I) in ability to generate singletnyj oxygen. The quantum yield 1O2 for Pz (I) PSHCH at measuring in dejterievoj to water has made 0,29, however it is necessary to score, that in connection with features of photophysical properties

Fluorescent molecular curls this quantity will depend substantially on parametres of medium.

For a quantitative assessment of photodynamic effect we had been developed a light-emitting diode radiator with replaceable light-emitting diode matrixes for reception of the uniform light stream in 96-lunochnyh tablets (drawing 27).

Drawing 27. A light-emitting diode radiator for making of the uniform light stream in 96-lunochnyh tablets.

1 — a replaceable light-emitting diode matrix; 2 — guiding devices; 3 — the guidance block; 4 — switches of clusters of light-emitting diodes; 5 — a power controller; 6 — a calorstat; 7 — 96-lunochnyj a tablet. 8 — the tumbler of insert/lockout of a food, 9 — a display valve


The created radiator allows to spend an irradiation of cultures of crates with intensivnostjami to 90 mvt/sm on a wave length of 625 nanometers and to 30 mvt/sm on 590 nanometers that provides accumulation of doses necessary for photodynamic effect for rather short time intervals (unities

Minutes).

The relative change of intensity measured within 30 minutes for a matrix of 625 nanometers at two operating modes (20 and 40 mvt/sm), has made less than 1 % at a stabilisation time of operating conditions no more than 1 minute from the moment of insert.

The narrow directional diagramme of the used light-emitting diode radiants provides hit of the basic part of intensity of radiation for a bottom "target" lunki a tablet. Nevertheless it is necessary to consider hit of some part of radiation in next lunki. In this connection have been carried out examination of uniformity of allocation of intensity of radiation on the area of a tablet and an estimate of influence of radiation of an individual light-emitting diode cluster on next lunki a tablet.

In drawing 28.а the photographic image of a tablet gained through a leaf of the scatterer, anchored at the bottom of a tablet in front of the cabinet is presented. We had been oozed the central part of a tablet (60 lunok) in which all lunki are in identical requirements, and regional rows lunok (36 lunok). According to effects of a numerical analysis of the image (fig. 28.б), medial intensity in a peripheral part of a tablet on 10-15 % more low, than in the central part. Intensity allocation in individual lunkah a tablet for both bands can be featured the normal law, thus the relative standard diversion characterising straggling of values, in both cases does not exceed 4-5 %.


Drawing 28. Allocation of intensity of radiation on the tablet area: and — the photographic image of a bottom of the tablet, gained through the leaf of the scatterer anchored at the bottom of a tablet in front of the cabinet; — the histogram of allocation of intensity of the radiation averaged on the area lunki of a tablet. Dashed lines correspond to approximation

Normal distribution

The construction of a radiator provides independent insert/lockout of 24 clusters on 4 light-emitting diodes in everyone. It provides possibility of an independent irradiation of corresponding groups lunok. At an invariable power density of radiation the dose of an irradiation for different groups lunok can be changed at the expense of an operating time of light-emitting diodes corresponding to them. It has been shown, that the part of radiation getting to the next number lunok, makes to 25 % from "target" lunok (drawing 29). The part of radiation getting to following, even more remote number, does not exceed 4-5 %.

Drawing 29. Influence of radiation of an individual light-emitting diode cluster on next lunki a tablet: and — the photographic image of a site of the tablet shined with a light-emitting diode cluster; — allocation of intensity of radiation on central (0) and next (1 and 2) to rows lunok. For 100 % intensity of radiation in the central number is accepted


Influence of a flare next lunok at insert of a light-emitting diode cluster has been checked up in experiment on culture of crates А-431, predynkubirovannyh with preparation fotosensj in concentration 10 microns. At tablet illumination one number of diodes (two clusters, located in one line), the dose of radiation transiting in a bottom lunok has been used only,

G ~\

Located immediately under light-emitting diodes, has made 15 Dzh/sm

2

At a power density 20 mvt/see



As a result of an irradiation viability of crates in lunkah under light-emitting diodes has made no more than 10 % from the control (drawing 30). Essential influence was observed and in the next rows lunok (in drawing 30 are designated as «-1» and «1»), at the chosen concentration of a photosensitizer and a dose of impinging radiation viability in these lunkah has made only 50-60 % from not irradiated variant. At the same time any biologically significant influence of the individual included number of light-emitting diodes on lunki, located through 1 number and further, it was not observed. Thus, at an irradiation of cellular culture in 96 lunochnom a tablet (12 rows lunok) are possible allocation of several independent groups lunok and processing by their various doses of light.

In drawing 31 dependence of viability of crates of line Т24, predynkubirovannyh with preparation fotosensj (10 microns), from a radiation dose is shown. Two experiments have been spent, in first of which an irradiation different doses carried out for the crates landed on the different


Tablets, and in second — for the crates landed in parallel rows lunok on the same tablet. In all cases the radiation power density made 20 mvt/sm, and a radiation dose varied at the expense of an operating time of light-emitting diodes. In case of use of one tablet different doses of radiation gained by consecutive insert of light-emitting diode clusters: the the major dose was necessary for typing, the included a corresponding cluster, thus lockout of all installation yielded simultaneously earlier. Good coincidence of the data gained by means of various approaches to statement of experiment, shows, that use of independent insert/lockout of clusters of light-emitting diodes allows to reduce essentially blanket time of experiment without any significant influence on effect of examination.


The created installation has been used at examination of light activity Pz (I) PSHCH.

Examination has been carried out prestress experiment

Toxicity of "polymeric brushes" — novosintezirovannogo a polymetric compound which activity in the relation eukarioticheskih crates is not studied yet. Concerning a line of crates А-431 of significant cytotoxic effect for the given polymetric compound in concentrations to 0,0035 % it is not revealed (drawing 32). It is necessary to score, that at reception nanochastits on the basis of polymeric brushes, dopirovannyh porfirazinami, concentration of a polymetric compound of 0,003 % corresponds to concentration porfirazina 100 microns.

Drawing 32. Dependence of viability of crates A 431 from

Concentrations in the environment of PSHCH


In drawing 33 the curves illustrating are presented

Viability of crates of line А431, predynkubirovannyh in the medium containing Pz (I) PSHCH. From drawing it is well visible, that explored

The photosensitizer possesses the expressed phototoxicity.

2

Ingibirujushchaja concentration ИК50 Pz (I) PSHCH at an irradiation in a dose of 10 Dzh/sm has made 20 microns whereas for lack of illumination it exceeds 60 microns.


Concentrations in the environment of Pz (I) PSHCH. Inkubatsija crates of line А431 in medium with dye in darkness (a black line) or at an irradiation light of 625 nanometers in

To dose of 10 Dzh/sm (a red line)

Unlike bezmetalnogo porfirazina, toxicity YbPz (I) in darkness and at illumination statistically significantly did not differ (drawing 34), thus the dark toxicity of a complex exceeded that for bezmetalnogo Pz (I) a little.


Concentrations in the environment of YbPz (I) PSHCH. Inkubatsija crates of line А431 in medium with dye in darkness (a black line) or at an irradiation light of 625 nanometers in

To dose of 10 Dzh/sm (a red line)

Presumably, decrease in photodynamic effect in the presence of atom of a ytterbium in a chromophore molecule is related to the following chain of events (drawing 35). At uptake of quantum of light a chromophore (fluorescence) or conversion in tripletnoe a state transfers in singletnoe an excited state with the subsequent emissive relaxation. The chromophore in tripletnom a state is capable to phosphoresce, and also to interreact with tripletnym oxygen of air with its formation highly active singletnogo states.

If at a chromophore molecule there is an atom of a ytterbium energy tripletnogo level can be transmitted to a ytterbium with the subsequent flashing of fluorescence, thus the quantum yield singletnogo oxygen considerably decreases. As consequence, it leads

To lack or essential reduction of photodynamic effect. Phototoxicity decrease at formation itterbievyh complexes also has been shown for porfirinov in operations [14, 31].

YO

Drawing 35. The blanket plan of electronic transitions raised tetraariltetratsianoporfirazinov and formation singletnogo

Oxygen


By results of cellular experiments by the most perspective in a role of the potential agent for photodynamic therapy is Pz (I) PSHCH. For this photosensitizer comparison of photodynamic activity with activity of the preparations approved to clinical application has been spent. It is shown, that light toxicity Pz (I) PSHCH concerning tumoral crates is comparable with that of preparations fotosensj and fotoditazinj (drawing 36).


Thus, by results of examination of photophysical properties and the analysis of photodynamic activity concerning tumoral crates in culture Pz (I) can be considered as a potential photosensitizer for FDT, thus, thanking it "rotornym" to properties, monitoring of process FDT at cellular level on changes of photophysical properties entered fluorofora, related with vjazkostnymi in crate parametres can be carried out.

For acknowledgement of this hypothesis we had been spent an estimate of possibility of observation of change of the functional state of crates after photodynamic action according to a lifetime of excited state Pz (I). In drawing 37 the image of crates in medium with Pz (I) PSHCH, time-resolved of fluorescent microscopy gained with the help (FLIM) is given. In everyone piksile images the curve of signal attenuation of fluorescence which was approximated bieksponentsialnoj by dependence, measure of consent Pirsona х2 has been gained has made

Nearby 1,2 (drawing 37в). Thus a share short-lived builders ai (t ~ 210-220 ps) and long-living builders a22 ~ 1350-1400 ps) have made ~ 86 % and ~ 14 % accordingly. In connection with bieksponentsialnoj dependence the medial lifetime of an excited state (xav) (drawing 37) has been calculated. At reception of images the pseudo-colour palette which reflects a medial lifetime of an excited state fluorofora in each point is used.

And — the image of crates in the pseudo-colour palette coding Tav (a medial lifetime of an excited state). The size of the image

143x143 a micron.



By us it has been shown, that after photodynamic action (594 nanometers, 50 Dzh/sm) in the irradiated site are incremented a lifetime of excited state Pz (I) in a crate that can testify to magnification of viscosity of a microenvironment, and also about conformation changes of macromolecules to which it can potentially be related Pz (I) (drawing 38). Besides, the essential magnification of signal strength is observed. The last can be caused, first,

Magnification of a quantum yield of fluorescence, and secondly, infringement of integrity plasmatic and other membranes of a crate and receipt in cytoplasm of significant amounts fluorofora from a surrounding medium.

Drawing 38. Images of crates MCF-7 through various continuances

Time after photodynamic action, sensibilizirovannogo 5 microns Pz (I), and corresponding histograms of allocation (a black curve

— For not irradiated site, red — for irradiated). Method FLIM.

800 nm600-750 nanometer. The size of images 143x143 a micron.

Photodynamic action—594 nanometer, a dose of an irradiation 50 Dzh/see

Irradiated (from above) and not irradiated sites are parted by a dotted line


Thus, rotornye properties Pz (I) unclose new prospects in the field of examination of mechanisms of the answer of a crate on the photodynamic

Action, allowing by means of measuring of a lifetime of an excited state of a photosensitizer to spot change of viscosity of intracellular medium which, in turn, correlates with the functional state of a crate [179, 216, 260].

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A source: Shiljagina Natalia Jurevna. EXAMINATION TETRAARILTETRATSIANOPORFIRAZINOV As POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY And FLUORESCENT DIAGNOSTICS. 2014

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