Giedre Streckyte

Photobleaching of protoporphyrin IX in cells incubated with 5‐aminolevulinic acid

Protoporphyrin IX (Pp IX) is the main photosensitizer in photochemotherapy with 5‐aminolevulinic acid (ALA). Pp IX is photolabile and the present work shows that 70–95% of Pp IX in cells is degraded by clinically relevant light exposures (40–200 J cm−2 at 630 nm). During light exposure a small yield of photoprotoporphyrin, which is also photolabile, is formed. A substantial fraction of Pp IX in cells incubated with ALA is bound to proteins. During light exposure these binding sites are destroyed, those close to tryptophan residues being the most sensitive. The rate of photodegradation of Pp IX in the cells is dependent on the initial concentration of Pp IX. The degradation mechanisms are therefore not only first order processes. Different degradation rates appear to be related to different types of binding sites. During light exposure, Pp IX molecules appear to move to different binding sites, evidently sites that are more vital for cell survival. Thus, the yield of photoinactivation of the cells, as measured per emitted photon of Pp IX fluorescence, increased during light exposure.

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.

Phototransformations of sensitizers 2. Photoproducts formed in aqueous solutions of porphyrins

Abstract The photodestruction of porphyrins results in a loss of absorption and emission intensity. Photoproducts are formed which absorb in the UV and red spectral regions and exhibit an emission maximum at 545 nm. Photoinduced modification leaving the porphyrin macrocycle intact gives rise to photoproducts which absorb at 640 and 660 nm. These photoproducts were separated by thin layer chromatography. An analysis of the spectroscopic properties of the separated fractions showed that the two photoproducts with absorption bands at 640 and 660 nm are chlorin- and bacteriochlorin-type molecules respectively. The appearance of a photoproduct with an emission maximum at 545 nm is probably caused by the formation of bilirubin-type molecules during the intermediate stage of photodestruction.

Phototransformations of sensitizers 1. Significance of the nature of the sensitizer in the photobleaching process and photoproduct formation in aqueous solution

Abstract Comparative spectroscopic studies of haematoporphyrin, haematoporphyrin derivative, dimethoxyhaematoporphyrin, Photofrin II, Photosan-3, meso-tetraphenylporphine tetrasulphonate, chlorin e6 and aluminium phthalocyanine tetrasulphonate have been performed. The photosensitizers studied are not photostable and are bleached during illumination. The rate constants of absorption bleaching, measured in pH 7.2 phosphate buffer, indicate that, of the investigated sensitizers, chlorin e6 is the least photostable and aluminium phthalocyanine tetrasulphonate is the most photostable. Simultaneous with photobleaching, the formation of red-absorbing photoproducts is observed for haematoporphyrin-like sensitizers. The efficiency of photobleaching and the formation of photoproducts in aqueous solution seems to be conditioned by the aggregation state and chemical structure of the photosensitizer. Spectroscopic changes induced during illumination have important consequences for the dosimetry of photosensitized tumour therapy.

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.

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.

A non-covalent complex of quantum dots and chlorin e6: efficient energy transfer and remarkable stability in living cells revealed by FLIM

A Forster resonance energy transfer (FRET) system of semiconductor quantum dots and porphyrins represents a new promising photosensitizing tool for the photodynamic therapy of cancer. In this work, we demonstrate the ability of a non-covalent complex formed between commercial lipid-coated CdSe/ZnS quantum dots (QD) bearing different terminal groups (carboxyl, amine or non-functionalized) and a second-generation photosensitizer, chlorin e6 (Ce6) to enter living HeLa cells with maintained integrity and perform FRET from two-photon excited QD to bound Ce6 molecules. Spectroscopic changes, the highly efficient FRET, observed upon Ce6 binding to QD, and remarkable stability of the QD–Ce6 complex in different media suggest that Ce6 penetrates inside the lipid coating close to the inorganic core of QD. Two-photon fluorescence lifetime imaging microscopy (FLIM) on living HeLa cells revealed that QD–Ce6 complexes localize within the plasma membrane and intracellular compartments and preserve high FRET efficiency (∼50%). The latter was confirmed by recovery of QD emission lifetime after photobleaching of Ce6. The intracellular distribution pattern and FRET efficiency of QD–Ce6 complexes did not depend on the charge of QD terminal groups. Given the non-covalent nature of the complex, its exceptional stability in cellulo can be explained by a combination of hydrophobic interactions and coordination of carboxyl groups of Ce6 with the ZnS shell of QD. These findings suggest a simple route to the preparation of QD-photosensitizer complexes featuring efficient FRET and high stability in cellulo without using time-consuming conjugation protocols.

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

Abstract 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/cm2 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.

Complex of water-soluble CdSe/ZnS quantum dots and chlorin e6: interaction and FRET

In this work we present the spectral study on complex formation between CdSe/ZnS-amino (620 nm) quantum dots (QDs) and second-generation photosensitizer chlorin e6 (Ce6). In the presence of QDs, significant changes in the absorption and fluorescence properties of Ce6 were observed. The fluorescence spectrum of bound Ce6 molecules displayed the new intensive fluorescence band at 670 nm, similar to that observed in organic solvent chloroform. With an increase in QDs:Ce6 molar ratio up to 1:20, the spectrum of QDs-Ce6 showed the decrease in intensity of QDs fluorescence band at 620 nm, while the intensity of fluorescence band at 670 nm was increasing simultaneously. The fluorescence excitation spectrum of bound Ce6 molecules exposed a contribution of QDs spectrum. The fluorescence decay measurements of QDs-Ce6 solution displayed the shortening of QDs fluorescence lifetime at increasing Ce6concentration. We conclude, that Ce6 molecules interact with the hydrophobic groups of QDs coating, which results in the changes of spectral properties of Ce6. Upon binding, Ce6 molecules are located close enough to the shell/core of QDs for Förster resonance energy transfer (FRET) from QDs to Ce6 to occur.

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.