This is the first study showing that singlet oxygen kinetics of topically applied photosensitizers coincides with the microarchitecture of skin, e.g., fissures and hair follicles. The kinetics indicate a chemical interaction of singlet oxygen with the skin, which allows differentiating between residual crème, e.g., in the follicular orifice, and photosensitizer penetrated into the skin. We show the feasibility of an easy-to-use fiber optic application providing the opportunity for in situ investigation, as well as a setup with focused optics for high-resolution two-dimensional scanning of singlet oxygen luminescence kinetics in skin samples. The results show that time-resolved singlet oxygen luminescence detection in tissue is a desirable tool for medical therapy, diagnostics, and evaluation of singlet oxygen interaction with biological environments.
LEDs have more and more influence on daily life as well as on scientific equipment. In this paper we want to report
results gained with the first LED powered setup for time resolved detection of the singlet oxygen luminescence in
solution as well as in cell suspension. The results show, that this setup can compete with the best laser powered setups
worldwide. The high sensitivity comes along with a superior long term stability and wavelength versatility. Setups based
on LED excitation can simplify the technical part of such measurement very much and reduce the costs, making this
technology available for a wider scientific community.
Singlet oxygen plays a crucial role in photo-dermatology and photodynamic therapy (PDT) of cancer. Its direct observation by measuring the phosphorescence at 1270 nm, however, is still challenging due to the very low emission probability. It is especially challenging for the time-resolved detection of singlet oxygen kinetics in vivo which is of special interest for biomedical applications. Photosensitized generation of singlet oxygen, in pig ear skin as model for human skin, is investigated here. Two photosensitizers (PS) were topically applied to the pig ear skin and examined in a comparative study, which include the amphiphilic pheophorbide-a and the highly hydrophobic perfluoroalkylated zinc phthalocyanine (F64PcZn). Fluorescence microscopy indicates the exclusive accumulation of pheophorbide-a in the stratum corneum, while F64PcZn can also accumulate in deeper layers of the epidermis of the pig ear skin. The kinetics obtained with phosphorescence measurements show the singlet oxygen interaction with the PS microenvironment. Different generation sites of singlet oxygen correlate with the luminescence kinetics. The results show that singlet oxygen luminescence detection can be used as a diagnostic tool, not only for research, but also during treatment. The detection methodology is suitable for the monitoring of chemical quenchers' oxidation as well as O2 saturation at singlet oxygen concentration levels relevant to PDT treatment protocols.
For the fist time worldwide we report high amplitude NIR-Luminescence signals, measured in pig skin. The measurements are achieved with a recently developed setup that was shown to provide superior performance for 1O2- luminescence detection in vitro. This setup has been adapted to allow now the detection of singlet oxygen signals in vivo. Pig ears were used for first measurements as a widely accepted in vivo model for human skin.
To determine factors that govern the uptake preference of photosensitizers in cellular organelles of human
adenocarcinoma cells, diarginyl-dialkoxy- and diarginyl-dimethoxyphenylporphyrins (TPPs) and two of their
corresponding indium(III) complexes were synthesized, characterized and incubated in androgen-sensitive human
prostate adenocarcinoma cells LNCaP. The porphyrins revealed properties that are of importance for phototherapy.
They are water-soluble, have their fourth Q-band absorbing at ≈ 650 nm, are taken up in relatively high concentrations in
LNCaP cells, and are phototoxic. Colocalization and phototoxicity studies revealed that all porphyrins localized
preferentially to the lysosomes and invoked cell death when excited with 650 nm light. Compared to the corresponding
methoxy-substituted TPPs, the diargininyl-dialkoxy-substituted porphyrins localized to a small extent in the
mitochondria. The corresponding In(III) chloride complexes that are slightly less water-soluble were also taken up in the
lysosomes of LnCaP cells. When the TPPs were compared to a pheophorbide derivative recently synthesized in our
laboratory, it was determined that the TPPs have a preference for lysosomal localization, whereas the pheophorbide
derivative co-localized to the mitochondria. Phototoxicity studies revealed that the longer chain dialkoxyTPPs were
more effective in cell killing and induced greater morphological changes typical of apoptotic cell death than the shorter
chain methoxy substituted porphyrins. The In(III) complexes seemed to be the most phototoxic. These results highlight
that the type, nature, and substitution pattern of the chromophore modulate the extent of apoptotic cell death and
influence cellular targeting.
The consumption of singlet oxygen quenchers during illumination of cells incubated with photosensitizers is a serious
issue for the determination of singlet oxygen kinetics in cells. Using LNCaP cells incubated with pheophorbide a it will
be shown, that already an illumination of 100 nJ/cell dramatically changes the value of the observables. This finding has
consequences for the spatial resolution of any time-resolved singlet oxygen measurement, since the radiative rate
constant of singlet oxygen in aqueous solutions is very small.
The estimation of PV-modules lifetime facilitates the further development and helps to lower risks for producers and
investors. One base for this extensive testing work is the knowledge of the degradation kinetics of encapsulating polymer
materials. Besides ethylen-vinylacetate copolymer (EVA), which is the prevalent material for encapsulation, new
materials like Poly-Vinyl-Butyral (PVB), and thermoplastic Poly-Urethan (TPU) become available and need the
assessment of their properties and the durability impact. In this context is it very important to identify the extent of
degradation caused by different parameters in order to identify the determining factor of polymer degradation as well as
potential interactions between different degradation processes.
To simulate long time degeneration processes accelerated aging under damp-heat and high-UV conditions was
performed on different EVA, TPU, and PVB samples. In this paper we report first results on measuring fluorescence
spectra from different encapsulation materials after accelerated ageing in dependence on time and aging procedure. Our
investigations clearly demonstrate that it is possible to follow damp-heat and UV induced aging processes of different
polymers used in PV-modules as encapsulation materials by luminescence detection.
The developments in porphyrin chemistry over the last decades give great advantages for the practical use of
porphyrin-based compounds. The properties of these compounds can be systematically tuned by rational utilization of
substituents on meso- and/or β-positions as well as by using different metal atoms in the center of the tetrapyrrole
macrocycle.
Recently we prepared novel mono- and bis-functionalized cycloketo-porphyrins (CKPors). In this work the results
of detailed spectroscopic investigations of these compounds are presented. It was found that a seven-membered ketone
exocycle remarkably influences the photophysical properties of the CKPor systems. For mono-functionalized CKPors it
results in strongly enhanced probability of intersystem crossing S1 → T1 with an ISC quantum yield up to 90%.
Moreover, the absorption of all CKPors undergoes a bathochromic shift and the Q-bands extinction is above two times
higher compared to that of H2TPP, what makes these compounds promising candidates for use as photosensitizers in
photodynamic therapy of tumors.
For the first time two NH-tautomers of nonsymmetrical CKPors were experimentally resolved at room
temperature using optical spectroscopic methods. It was found that the concentration of tautomer A with a lower
frequency of the S0,0 → S1,0 transition is higher than that one of tautomer B at room temperature, and becomes dominant
with cooling down. In contrast - and as it is expected - only one optical active species was observed for non-symmetrical
CKPor with a central Zn(II) atom as well as for symmetrical bis-CKPor.
KEYWORDS: Luminescence, Molecules, Quantum efficiency, Oxygen, Fullerenes, Dendrimers, Chromophores, Absorption, Energy transfer, Molecular energy transfer
The photophysical properties of DAB-dendrimers from 1st to 4th generation as well as Diaminohexane - all
substituted with the in maximum achievable quantity of pheophorbide a (Pheo) molecules were studied in comparison
with a novel hexapyropheophorbide a - fullerene hexaadduct (FHP6) and a fullerene [6:0]-hexaadduct which carries
twelve pyropheophorbide a units (FHP12) using both steady-state and time-resolved spectroscopic methods. It was
found that neighboring dye molecules covalently linked to one DAB- or fullerene moiety due to the length and high
flexibility of carbon chains could stack with each other. This structural property is the reason for the possibility of
formation different types of energy traps, which were resolved experimentally. The dipole-dipole resonance F&diaero;rster
energy transfer between the dye molecules coupled to one complex caused a very fast and efficient delivery of the
excitation to a trap. As result the fluorescence as well as the singlet oxygen quantum yields of the different complexes
were reduced with increasing number of dye molecules per complex. Nevertheless in every case the singlet oxygen
generation was less influenced then the fluorescence quantum yield, exposing the complex to a non-negligible amount of
excited oxygen in the singlet state. While the fullerene complexes turned out to be stable under these conditions, the
DAB-dendrimer-backbones were completely fragmented to small rudiments carrying just one or a small number of dye
molecules.
The pharmacokinetics of 132-hydroxy-bacteriopheophorbid-a methyl ester (132-OH-BPME) and octa-(alpha) -butyloxy-zinc phthalocyanine (8-(alpha) -bo-Zn-Pc) were studied in mice bearing Lewis lung carcinoma. Absorption spectroscopy was used to measure the photosensitizers concentrations. High 132- OH-BPME and 8-(alpha) -bo-Zn-Pc uptakes were recorded in the parenchymatous organs (liver, lung), with 8-(alpha) -bo-Zn-Pc long retention. The malignant tissues accumulated 132-OH- BPME of more than 20 folds than 8-(alpha) -bo-Zn-Pc at all incubation times. 8-(alpha) -bo-Zn-Pc concentrations in the skin and the muscle were lower than 132-OH-BPME concentrations at any interval period, but also, the 8- (alpha) -bo-Zn-Pc retained longer until 168 h. PDT with 132-OH-BPME will be more effective than with 8-(alpha) -bo- Zn-Pc where it has more concentrations in the malignant tissues.
The photodynamic therapy (PDT) of cancer is based on the reaction of dyes, light and oxygen in tumorous tissues. Currently mainly two types of photosensitizers [Photofrin II and Hematoporphirine derivatives (HPD)] are used in clinical investigations. But they are still far from being ideal for this purpose as they do not have the required specificity and the absorption maxima do not lie in the ideal region of 760 nm (maximal transmission for human tissue). Because of their absorption maxima, the high extinction coefficient and the good rate of singlet oxygen generation phthalocyanines (PCs) seem to be good photosensitizers for the photodynamic therapy of cancer. At this time the main problem for the use of PCs is the leak of solubility in physiological media. By partial sulfonation it is in fact possible to increase the hydrophilic attributes, but not seriously the selectivity to tumorous tissue. For these reasons it is absolutely necessary to synthesize monofunctionalized PCs, so that covalent coupling to carrier systems (e.g. monoclonal antibodies) are possible. By the synthesis of PCs via coupling to modified polystyrene as a carrier, it is possible, after linking a phthalodinitrile with a spacer (alkylchain C4 and longer) to the polymer to prepare exact defined monofunctionalized PCs.
The influence of spacers with different length (6, 10, or 13 C-atoms) on complex formation between (beta) -cyclodextrin dimers and t-butylphenoxy substituted Zn-phthalocyanine was studied by computer simulations. In complexes with C10-spacer the length of the sensitizer with end groups allowed an insertion in all cases. The dye fitted well into dimers with bent geometry. In the other cases (C6, C13) the length ratio of dimer and sensitizer was inappropriate.
Various tetrapyrrolic compounds are well-known photosensitizers in PDT. One of the important unsolved problems in PDT research is the selective accumulation of such compounds in tumor tissues. In this context different carrier systems for efficacious and directed transport of the dyes to the tumor tissue are still under investigation. From these investigations new problems have arisen like change of the sensitizer's photoactivity and photostability. As a result the relation between energy and electron transfer processes (that means the relative efficacy of sensitization via type I and II) during illumination can change. The photophysical properties of Pheoporbide a in different sensitizer-carrier systems (antibodies and liposomes), are investigated.
The phthalocyanines form a continuously growing family of potential photosensitizers. Their chemical and photophysical properties, and therefore their photodynamic activities, are influenced by the type of the central metal ion as well as the type and localization of substituents. Plasma and tissue distribution kinetics are also dependent on the chemical features of a dye molecule. We therefore investigated the pharmacokinetics of newly synthesized octa-(alpha) -butyloxy-zincphthalocyanine (8-(alpha) -bo-ZnPc) on the Lewis lung carcinoma in order to evaluate the application possibilities of this compound in experimental PDT treatments. The zinc complex was chosen on the basis of the known effect of this metal on the photochemical and photophysical properties of phthalocyanines. The pharmacokinetics of octa-(alpha) -butyloxy-zincphthalocyanine were studied on the Lewis lung carcinoma by absorption spectroscopy in mice. Afterwards a regression analysis was performed using the absorption spectroscopic data.
Hydrophilic silicon phthalocyanines derivatives with diaxial polyethyleneglycolmonomethylether ligand with different chain lengths were synthesized. Water soluble SiPc(OmPEG 5000)2 shows a completely monomeric UV/VIS spectrum in water. No influence of the ligand's chain length on photophysical properties, e.g., singlet oxygen quantum yield was found. First aspects of the photosensitizing properties were gained by photohemolyse experiments with human erythrocytes. SiPc(OmPEG 5000)2 is far more active than other water soluble phthalocyanines which are aggregated in water.
The newly synthesized potential photosensitizer octa-(alpha) -butyloxy-zinc phthalocyanine (8-(alpha) -bo-ZnPc) is characterized by a high absorption coefficient at the far red wavelength 735 nm and a good singlet oxygen quantum yield. The pharmacokinetics of 8- (alpha) -bo-ZnPc were studied on the Lewis lung carcinoma in mice after i.v. administration of 7.8 micrometers ole/kg body weight solubilized in PBS with 1% Tween 80 at 2, 6, 12, 24, 48 and 168 hour incubation intervals. The accumulated dye contents were chemically extracted from selected tissues and the concentrations were measured by absorption spectroscopy. The parenchymatous organs, liver and spleen, showed maximum 8-(alpha) -bo-ZnPc concentrations after 6 h incubation (33 nmole/g liver and 5 nmole/g spleen, respectively). An extensive uptake was detected in the lung where concentrations higher than 90 nmole/g tissue were measured in the 6, 12, 24, and 48 hour extracts. The malignant tissue did not accumulate higher 8-(alpha) -bo-ZnPc concentrations than 0.4 nmole/g tumor during the entire investigation period. The dye contents extracted from muscle, representing normal tumor surrounding tissue, and skin were even lower and never exceeded 0.11 nmole/g muscle (6 h) and 0.21 nmole/g skin (2 h), respectively.
In recent years a lot of new far red absorbing sensitizers for photodynamic therapy (PDT) were synthesized, like derivatives of bacteriopheophorbide. The possibility of increasing the accumulation in tumor tissue by coupling these dyes to carrier molecules is investigated. The photophysical properties of these dyes can be dramatically changed as a result of covalent binding to carrier molecules, different solvents, or biological surroundings. In heterogeneous systems 1O2 is strongly quenched. Therefore, its luminescence is difficult to detect. Another difficulty is the fact that especially far red absorbing sensitizers show fluorescence at the wavelength of 1O2-luminescence at 1270 nm, which can be several magnitudes higher than the luminescence of 1O2. The efficiency of singlet oxygen (1O2) generation is of major interest because 1O2 seems to play an important role in PDT. Time resolved luminescence measurements of 1O2 generated by dyes in micelles and liposomes in D2O were already made in our group. In order to investigate more complex systems and to avoid unwanted photochemistry we changed the excitation wavelength from UV (337 nm) to VIS/NIR(ND:YAG/OPO). The excitation power was increased and the detection limit was decreased. First measurements in red cell ghosts are presented.
Some photophysical properties (steady-state absorption, fluorescence and phosphorescence, fluorescence decay times and singlet oxygen quantum yields) of silicon phthalocyanines with methoxypolyethylene glycol (MPEG)-substituents of various chain length at the silicon atom [SiPc(OCH2CH2)n-OCH3; n equals 1,2,3,12], silicon phthalocyanine covalently bound to the water soluble polymer methoxypolyethylene glycol [SiPc(-O-MPEG 5000)2] and silicon naphthalocyanine [SiNc(CH2CH2OCH3)2] have been studied. The aim of these investigations was to get information about the influence of methoxypolyethylene-glycol-coupling on photophysical sensitizer parameters.
One of the most important problems for clinical use of photodynamic therapy is the selective accumulation of photosensitizer molecules by the tumor tissue. Possibly cyclodextrins could be used to solubilize and transport hydrophobic dyes to the target. Until now it has not been investigated to what extent the dye-cyclodextrin complexation alters the photophysical properties of the dye molecules important for their photodynamic activity. For this reason we investigated the behavior of three sensitizers belonging to different chemical classes: pheophorbide a, octa-(alpha) -butyloxy-Zn-phthalocyanine, and tetra-tert-butyl-Zn- naphthalocyanine in cyclodextrin solutions.
The pharmacokinetics of 132-hydroxy-bacteriopheophorbide a methyl ester (OH-BPME) was studied on Lewis lung carcinoma bearing mice by fluorescence spectroscopy. The sensitizer distribution in different organs and in the tumor was investigated in dependence on the incubation time. A very fast cleaning of the blood and the skin was observed. The results of our investigations suggest that PDT using OH-BPME could be effective at sensitizer incubation times of about 12 h.
Alexander Savitsky, K. Lopatin, Oleg Lobanov, N. Golubeva, Marina Poroshina, Elena Chernyaeva, L. Solovieva, Eugeny Lukyanets, Maria Galpern, B. Roder, Dirk Nather
Photodynamic therapy (PDT) of malignant tumors is based on the property of some photosensitizers to be accumulated by tumour tissues'. On subsequent illumination with light of the wavelength absorbed by these photosensitizers tumour cells can be destroyed. Hematoporphyrin derivative and similar agents presently have some negative features. First, these agents do not possess optimal absorption spectrum. In particular the absorbance in the red region is not very high. Second, the composition of these agents varies for different preparations and with the storage time. Third, they do not possess a sufficient selectivity for malignant cells. Furthermore, they are also accumulated in skin causing its damage upon exposure to light. Thus at present the developing of the effective PDT photosensitizers is of great interest. Recently some of this interest has been focussed on the phthalocyanines (PC). These dyes have sufficient molar extinction coefficient (<10 M1cm1) in red region of optical spectrum (650 700 nm). They are non-toxic. Some of them are effective photosensitizers and produce singlet oxygen. They are relatively easy synthesized and resistant to chemical degradation. PC can have different groups added to the external macrocycle perifery. These groups mainly determine chemical properties and solubility. They offer possibility to bind the dyes to any other molecules, for example, proteins. The properties of naphthalocyanines (NC) are rather similar to the ones of PC, but the most efficient absorption band is in region of 750 - 800 nm. An important problem is to increase the ratio between the level of photosensitizer in malignant and normal tissues. One of the possible solutions is the use of photoimmunotoxins, i.e. conjugates of photosensitizers with monoclonal antibodies (mAb) against cancer antigens or tumor marker. Experiments with immunoglobulin-hematoporphyrin2 and dimetoxyhematoporphyrin3 as well as chiorin e6 conjugates4 showed that they retained both photochemical properties of free dyes and immunological ones of antibodies.
Tetrapyrroles obtain very good photophysical properties making them efficient photosensitizers in photodynamic therapy. Most of them have high triplet quantum yields, long triplet lifetimes and triplet state energies suitable for singlet oxygen generation. These photophysical parameters differ depending on the chemical structure and interactions with the environment. Today a large variety of tetrapyrroles are synthetized or chemically separated from natural substances for investigation of their photophysical properties with the aim of using them in photodynamic therapy. In this paper, some general aspects of energy conversion and energy transfer of tetrapyrroles in relation to their photophysical and biophysical properties relevant for photodynamic activity are discussed. Essential optical methods for the investigation of these parameters, as well as the determination of segmental mobilities of sensitizer-carrier complexes, are discussed using the example of phthalocyanines.
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