Saulius Bagdonas

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.

Phototransformations of 5-Aminolevulinic Acid–induced Protoporphyrin IX in vitro: A Spectroscopic Study¶

Abstract 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 chlorin-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 blueshifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

The bystander effect in photodynamic inactivation of cells

Treatment of MDCK II cells with the lipophilic photosensitizer tetra(3-hydroxyphenyl)porphyrin and light was found to induce a rapid apoptotic response in a large fraction of the cells. Furthermore, the distribution of apoptotic cells in microcolonies of eight cells was found to be different from the binomial distribution, indicating that the cells are not inactivated independently, but that a bystander effect is involved in cell killing by photodynamic treatment. The observation of a bystander effect disagrees with the common view that cells are inactivated only by direct damage and indicates that communication between cells in a colony plays a role in photosensitized induction of apoptosis. The degree of bystander effect was higher for cells dying by necrosis than for cell dying by apoptosis.

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.

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.

The photosensitizing effect of the photoproduct of protoporphyrin IX.

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm(2) light at 635 nm, the photoproduct--the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.

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.

Excitation relaxation and structure of TPPS4 J-aggregates

The energy relaxation kinetics and the structure of the J-aggregates of water-soluble porphyrin 5,10,15,20tetrasulphonatophenyl porphine (TPPS4) were investigated in aqueous medium by means of time-resolved fluorescence spectroscopy and confocal laser-scanning fluorescence microscopy. The excitation of the J-aggregates, at excitation intensities higher than B10 15 photons/cm 2 per pulse, results in a remarkable decrease of the fluorescence quantum yield and in the appearance of an additional, non-exponential energy relaxation channel with a decay constant that depends on the excitation intensity. This relaxation mechanism was attributed to the exciton single–singlet annihilation. The exciton lifetime in the absence of the annihilation was calculated to be B150 ps. Using exciton annihilation theory, the exciton migration within the J-aggregates could be characterized by determining the exciton diffusion constant (1.870.9) 10 � 3 cm 2 /s and the hopping time (1.270.6) ps. Using the experimental data, the size of the J-aggregate could be evaluated and was seen to yield at least 20 TPPS4 molecules per aggregate. It was shown by means of confocal fluorescence laser scanning microscopy that TPPS4 does self-associate in polyvinyl alcohol (PVA) at acidic pH forming molecular macro-assemblies on a scale of B1mm in PVA matrices. r 2002 Elsevier Science B.V. All rights reserved.

Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison with incubation as well as the limited transfer of quantum dots from vesicles into the cytosol and vice versa support the endocytotic origin of the natural uptake of quantum dots. Quantum dots with proteins adsorbed from the culture medium had a different fate in the final stage of accumulation from that of the protein-free quantum dots, implying different internalization pathways.