APPLICATION OF REFLECTANCE CONFOCAL MICROSCOPY FOR EARLY DIAGNOSIS OF RADIATION-INDUCED ACUTE DERMATITIS IN RADIOSENSITIVE PATIENT: CASE STUDY.

According to the guidelines on cancer treatment up to 52% of newly diagnosed cancer patients should be treated with external beam radiotherapy. Ionizing radiation (IR)-induced skin injury (radiation dermatitis) occurs in up to 95% of radiotherapy patients and can manifest from mild erythema till necrosis and ulceration. Individual radiosensitivity was proposed to be an important factor for the development of adverse reactions to IR. Therefore, assessment of radiosensitivity could be useful in predicting and dealing with radiation injuries caused by both radiotherapy and accidental overexposure. Here, we present a case of early diagnosis of IR-induced skin lesions performed by reflectance confocal microscopy in comparison to clinical evaluation in a highly radiosensitive patient.

Spectroscopic assessment of endogenous porphyrins in a rheumatoid arthritis rabbit model after the application of ALA and ALA-Me.

The sensitization of inflamed knee tissues with endogenous porphyrins was studied by means of fluorescence spectroscopy in an experimental model of rabbit rheumatoid monoarthritis after intraarticular (i.a.) or intravenous (i.v.) injections of 5-aminolevulinic acid (ALA) or ALA methyl ester (ALA-Me). Fluorescence measurements in vivo on the skin of the inflamed knee joint showed the dominance of protoporphyrin IX (PpIX). The highest fluorescence intensity was registered 2h after i.a. injection of ALA and ALA-Me. Comparative analysis of the PpIX fluorescence spectra ex vivo revealed that more PpIX accumulated in the tissues of the inflamed joint than in the tissues of the control joint, and that ALA-Me induced about five times more PpIX in the inflamed synovium than ALA. Meanwhile, the cartilages of the inflamed and control knee joints also accumulated water-soluble porphyrins. Thus, in vivo and ex vivo spectroscopic assessment of endogenous porphyrins in rabbit rheumatoid arthritis tissues implied that the injection of ALA is more appropriate for the diagnostics of inflammation due to the higher PpIX fluorescence intensity on the skin surface, while ALA-Me is more appropriate for the therapeutic applications due to the higher and more selective accumulation of PpIX in the inflamed synovium.

Stabilization of (CdSe)ZnS quantum dots with polypyrrole formed by UV/VIS irradiation initiated polymerization.

Polypyrrole formation initiated by UV/VIS irradiation and stabilization of (CdSe)ZnS quantum dots is reported. Presented results demonstrate that UV/VIS irradiation is slowly destructing Q-dots that decreases quantum yield and shifts peak of photoluminescence (PL) spectra to short wavelength range. The same Q-dot solution under UV/VIS light irradiation in the presence of pyrrole is more stable, there is no PL band shifts only PL intensity of Q-dot decrease. UV/VIS induced Ppy formation process in the presence/absence of Q-dots dissolved in toluene and phosphate buffer saline (PBS) not containing Q-dots was investigated. The increase in the optical absorbance in the range of 400 nm-600 nm was exploited for the monitoring of polypyrrole formation process. Results obtained proved that in the presence of UV/VIS formed polypyrrole destruction of Q-dots is much slower if compared with bare Q-dots destruction under similar conditions. However, formed polypyrrole showed no positive effect on quantum yield of Q-dot, just the opposite-polypyrrole quenched Q-dot photoluminescence. Results presented and observation by other authors on pyrrole polymerization allow to predict that by UV/VIS induced polymerization formed polypyrrole is assembling polymeric layer on heterogeneous phase between Q-dot and toluene or water (in PBS solution). Established pi-pi conjugated chain of formed polypyrrole quenches photoluminescence of Q-dot because this polymer was formed in close proximity to Q-dot surface.

Spectroscopic studies of the human heart conduction system ex vivo: implication for optical visualization.

Fluorescence excitation and emission spectra of the heart tissues specimens have been measured ex vivo with the aim of finding out the optical differences characteristic for the human heart conduction system (the His bundle) and ventricular myocardium. The optimal conditions enhancing the spectral differences between the His bundle and myocardium were found by recording the fluorescence signal in the range from 420 nm to 465 nm under the excitation at wavelengths starting from 320 nm to 370 nm. In addition, the spectral differences between the His bundle and the connective tissue, which is often present in the heart, could be displayed by comparing the ratios of fluorescence intensities being measured at above 460 nm under the preferred excitation of elastin and collagen. The left and right branches of the His bundle were visualized ex vivo in the interventricular septum of the human heart under illumination at 366 nm.

Electroporation of transplantable tumour for the enhanced accumulation of photosensitizers.

The aim of this study was to verify whether electroporation could increase the accumulation of the hydrophilic photosensitizers: aluminium phthalocyanine tetrasulphonate (AlPcS(4)) and chlorin e(6) (C e(6)) in tumour tissue. The experiment was performed in vivo using hybrid mice (C57Bl/CBA) bearing hepatoma A22 (MH-A22) tumours transplanted in the right haunch. The time dependence of the fluorescence intensity of administered photosensitizers was measured after the ordinary and electrically stimulated delivery. The obtained fluorescence spectroscopy results implied the tumour being affected by an electrical field in a way, which led to a higher accumulation of both photosensitizers (AlPcS(4) and C e(6)) in the periphery of the tumour and it superficial layer. Our pilot study suggests that electroporation could be considered as a useful procedure seeking for the more effective application of photodynamic tumour treatment.

Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light.

Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.

Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study.

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

New potent sensitizers for photodynamic therapy: 21-oxaporphyrin, 21-thiaporphyrin and 21,23-dithiaporphyrin induce extensive tumor necrosis.

New sensitizers for photodynamic therapy (PDT) are reported. These compounds, namely 21-thiaporphyrin, 21,23-dithiaporphyrin and 21-oxaporphyrin, reveal some of the properties required for such therapy. Their physicochemical, chemical and pharmacological features meant that we could use them in the treatment of transplantable BFS1 fibrosarcoma in Balb/c mice. New sensitizers and the well-known chlorin e6 (Ce6) were used in doses of 2.5, 5.0, 7.5 and 10.0 mg/kg body weight, given intraperitoneally and followed by light irradiation, the total light doses being 50, 100 and 150 J/cm(2) within 24 h after injection. The effectiveness of new sensitizers in PDT was evaluated with in terms of tumor necrosis intensity, the survival time of treated animals, the rate of tumor response (complete/partial/no response), and skin photosensitivity. These results were compared to results obtained in analogous conditions after Ce6-PDT. Distribution studies revealed that the highest concentration of new compounds occurred within 24 h after injection. The results of these experiments confirmed that 21-thiaporphyrin, 21,23-dithiaporphyrin and 21-oxaporphyrin can be considered as potent tumor photosensitizers that do not exert any unwanted effects, primarily skin photosensitization. We suggest that these porphyrins are possible sensitizers to be applied in clinical PDT.

Effects of photodynamic therapy in combination with Adriamycin.

The combination of photodynamic therapy (PDT) and Adriamycin (ADM) was studied in the animal model system. Photohem (PH) was used as a photosensitizer. Mice bearing carcinoma epidermoides LL of the lung received PH once at a dose 10 mg/kg and after 24 h ADM was injected i.p. at a dose 3 mg/kg and tumors were illuminated with laser light after three different time intervals, 15 min, 3 and 24 h. To evaluate the effect of PDT and PDT combined with ADM the intensity of lipid peroxidation in tumor tissue and in blood serum was determined using the thiobarbituric acid assay. PDT induces an increase of malondialdehyde (MDA) concentration in tumor tissue as well as in blood serum. When PDT is combined with ADM, the MDA level in tumor tissue is similar to the level of this product as in the PDT alone. No enhancement of the efficiency of the combined treatment was observed at these experimental conditions. This is also confirmed by the tumor growth dynamics, survival time of animals and flow cytometric DNA analysis of tumor cells. For the successful combination of PDT with chemotherapy it is suggested to apply the drugs at the regimen which will allow to avoid the interaction between two agents since the ground state interaction between PH and ADM is stated spectroscopically. It should lead to the conclusion that the sequence of the combination of two treatment modalities is an important factor for synergistic effect.

Phototransformation of sensitisers: 3. Implications for clinical dosimetry.

Spectroscopic studies of aqueous solutions of haematoporphyrin-type sensitisers reveal that photobleaching during eposure to light is followed by the formation of stable red-absorbing photoproducts. Experiments in model systems (sensitisers bound to human serum albumin or in a suspension of resealed erythrocyte 'ghosts') and in tumour tissue show that similar photomodification takes place in all investigated environments. Loss of total absorption and emission intensities is accompanied by an increase of absorption in the red spectral region (630-650 nm) which is used for the treatment of tumours because of the deeper penetration of light into tissues. This should be taken into account when the duration of illumination is chosen to reach an appropriate photodynamic dose using Hp-type sensitisers in the photodynamic treatment of tumours.

Spectroscopic studies of photobleaching and photoproduct formation of porphyrins used in tumour therapy.

The illumination of haematoporphyrin, meso-tetraphenylporphyrin tetrasulphonate and haematoporphyrin derivative in aqueous solution causes two simultaneously occurring processes: photodegradation and the formation of stable photoproducts absorbing in the red spectral region. In the case of haematoporphyrin and its derivatives, these photoproducts have an absorption maximum around 640 nm (photoproduct 640). The former process, which is detected as the bleaching of the porphyrin absorption spectrum as well as a decrease in the fluorescence intensity, is slightly dependent on the solution pH and becomes dominant when the formation of the photoproduct reaches saturation. For the most part, the photodegradation can be explained as the opening of the porphyrin ring, leading to an increase in light absorbance in the UV region. The formation of photoproduct 640 is closely related to the aggregation state of the porphyrins, and shows a distinct dependence on the medium pH. The effectiveness of photoproduct 640 formation strongly increases in neutral and alkaline solutions, whereas the porphyrins are photostable below pH 5. The spectroscopic features of the photoproducts of haematoporphyrin and haematoporphyrin derivative, with absorption bands in the visible region, are similar to those of chlorin and/or porphyrin-chlorin linked systems. On the basis of these spectroscopic studies, it is suggested that photoproduct 640 is a chlorin-type molecule formed predominantly from the aggregates of porphyrins when photo-oxidation and photoreduction are in competition.

Fluence-rate-dependent photosensitized oxidation of NADH.

The photosensitizing activity of dimethoxyhaematoporphyrin, excited by a laser pulse at 532 nm (YAG-Nd3+), was investigated using reduced nicotinamide adenine dinucleotide (NADH) as a substrate. The photo-oxidative modification of NADH was monitored by measuring the absorbance at 340 nm. The use of nanosecond pulses (15 and 0.5 ns) resulted in photosensitized NADH oxidation which depended on the fluence but not on the fluence rate up to a peak fluence rate of 10(7) W cm-2. At higher fluence rates a decrease in NADH photo-oxidation was observed, as well as on irradiation with picosecond pulses (35 ps). Stern-Volmer assay of the quenching by sodium azide revealed a decrease in quenching efficiency with increasing peak fluence rate. Oxidation of NADH was not suppressed by the addition of 20 mM sodium azide at peak fluence rates above 6 x 10(9) W cm-2. This observation, as well as the significant bleaching of dye absorption, indicates excitation of the photosensitizer into higher lying excited singlet states and the involvement of processes other than photodynamic action.

Phototransformations of porphyrins in aqueous and micellar media.

Porphyrin photoproducts with absorption maxima at 640 and 660 nm are formed in aqueous and micellar solutions during light exposure. Changes in the dimethoxyhaematoporphyrin (DMHp) Soret band on irradiation suggest that the photoproduct (640 nm) formation is conditioned by the "sandwich"-type self-associates. The formation of the photoproduct (660 nm) in micellar haematoporphyrin (Hp) and photosan-3 (PS) solutions and its formation in small amounts in phosphate-buffered solution (PBS), is related to the presence of covalently linked porphyrin structures and/or interaction with surfactant molecules. PS is the most photostable of the three investigated porphyrins. Its photostability is probably due to the presence of covalently linked "sandwich"-type aggregates.

Fluorescence spectra of hematoporphyrin and hematoporphyrin-diacetate aggregates in buffer solution.

Different fluorescence spectra are observed on preferential excitation of monomer and aggregate species of hematoporphyrin and hematoporphyrin-diacetate, respectively. From these measurements we obtain the fluorescence spectrum of aggregate species in buffer solution, which shows two maxima, at 628 nm and around 700 nm. The fluorescence intensity in the region between these maxima is very sensitive to environmental changes. It is suggested that the changes in the fluorescence of hematoporphyrin in vivo as compared with that in buffer solution is associated with both hematoporphyrin-substrate interactions and an enhanced contribution from aggregate species.

Light-induced transformations of hematoporphyrin diacetate and hematoporphyrin.

Illumination of hematoporphyrin diacetate (HP-Diac) and hematoporphyrin (HP) in phosphate-buffered solutions causes the formation of a stable photoproduct absorbing at 636 nm. Simultaneously the photo-oxidation of HP-Diac and HP takes place. These phototransformations depend on the illumination dose and the concentration of the HP-Diac or HP solution, and cause qualitatively the same spectral changes independently of the light source used.