Beata Čunderlíková

Acid-base properties of chlorin e6: relation to cellular uptake.

Chlorins are attractive compounds for photodynamic therapy because of their high absorption in the red spectral region. In this study, the absorbance, fluorescence excitation and fluorescence emission spectra of chlorin e6 have been recorded as functions of pH in phosphate-buffered saline (PBS) solution with and without fetal calf serum (FCS). For pure PBS solutions, variation of the pH of the solution results in a shift of both the absorption and the fluorescence spectrum as well as in a decrease of the fluorescence intensity. Spectrophotometric and fluorimetric titration curves, based on observed changes, have been plotted. There is an indication of aggregate formation at low pH values (pH < 5). The presence of 5% FCS results in a shift of the titration curve, from an inflection point at about 6.5 to one at about 7.6. Pronounced spectral changes of the fluorescence emission spectra of protein-bound chlorin e6 (change of spectral shape, decrease of peak intensity) are also observed. The partition coefficients in the 1-octanol-water system increase with decreasing pH. Thus, relatively more of the drug is incorporated in the octanol phase at low pH. Cellular uptake of chlorin e6 in the presence of serum is significantly higher at pH 6.7 as compared with that at 7.3 and 7.6. We conclude that a change in the pH value of the surrounding medium leads to a change in the lipophilicity of chlorin e6. Such a change is likely to influence its binding to the serum proteins as well as its interaction with the plasma membrane of cells and may also be related to the selective tumor uptake of the drug.

pH effects on the cellular uptake of four photosensitizing drugs evaluated for use in photodynamic therapy of cancer

The difference in extracellular pH in malignant as compared to normal healthy tissues has been proposed to contribute to selective uptake of photosensitizers in tumors. Hematoporphyrin IX (HpIX), disulfonated meso-tetraphenylporphine (TPPS(2a)), meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin (mTHPC) were chosen to examine the pH dependence of their cellular drug uptake. The study was performed in the pH range 6.5-8.0 and showed that significantly higher amounts of the drug are taken up by T-47D cells at low pH values only in the case of HpIX. The pH value of the incubation medium did not influence the cellular uptake of mTHPP, mTHPC and TPPS(2a) significantly. The present work indicates that tumor selectivity of dyes, which get more lipophilic with decreasing pH value, may be related to the low extracellular pH value.

pH-Dependent Spectral Properties of HpIX, TPPS2a, mTHPP and mTHPC¶

Abstract Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso-tetraphenylporphine [TPPS2a], meso-tetra(3-hydroxyphenyl)porphine [mTHPP] and meso-tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0–8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values <5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs.

Solvent effects on photophysical properties of merocyanine 540

Abstract Absorption and corrected fluorescence emission spectra of merocyanine 540 (MC 540) were recorded in solvents with different physico-chemical parameters – dielectric constant e r , refractive index n , dipolarity/polarizability π * , hydrogen-bonding ability α . Hypsochromic shifts of their maxima positions did not obey Lippert–Mataga equation. However, a correlation with π * and α was observed. Relative quantum yields φ f and rate constants of nonradiative deactivation processes k nr of the dye in different solvents based on steady-state measurements were estimated. Dependence on hydrogen-bonding ability was indicated – with increasing α , φ f decreased and k nr increased. The interaction between MC 540 and solvent molecules is discussed in terms of the different interactions contributing to the solvent stabilization of merocyanine dyes. Our results indicate that hydrogen bonding may contribute to solvent stabilization of MC 540.

Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.

Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples

Objectives:  Urinary bladder urothelial carcinoma is diagnosed by a combination of cystoscopy and biopsy, with cytology as a valuable additional technique. The accuracy of cytological diagnosis depends on the experience of the cytologist and can inevitably vary from one cytologist to another. There is a need for an easy, reliable and objective diagnostic method. In the present study a new method was designed for the detection of bladder cancer cells in urine.

pH-Dependent Modification of Lipophilicity of Porphyrin-type Photosensitizers

Abstract Structural modifications of photosensitizers (changes in protonation, ionic state and aggregation state) under different environmental conditions should be precisely determined to understand the interaction of the photosensitizers with biological systems. In the present study partition coefficients of hematoporphyrin IX (HpIX), disulfonated meso-tetra-phenylporphine, meso-tetra(3-hydroxyphenyl)porphine (mTHPP) and meso-tetra(3-hydroxyphenyl)chlorin in the 1-octanol–phosphate buffer system were determined in the pH region 4.0–8.0. Only the partition coefficients of HpIX and mTHPP were found to be pH dependent. Computer processing of fluorimetric titration data was applied to estimate pKa values of the imino nitrogens of mTHPP. Monoprotonated species of mTHPP seem to be unstable or nonexistent. The possibility that both imino nitrogens of this dye are protonated according to a common pKa is proposed. The pKa value of the imino nitrogens of mTHPP was found to be 2.99 ± 0.04 after the application of a model taking aggregation of the drug into account. The contributions of various aqueous ionic species of mTHPP as functions of pH were calculated and compared with partition coefficients.

Interaction of merocyanine 540 with cations of physiological solutions

Abstract The effect of H+, Na+, Ca2+, Mg2+ on merocyanine 540 (MC 540) absorption and fluorescence spectra was determined. While MC 540 showed two absorption peaks in water (at 535 nm, the monomer peak; at 503 nm, the dimer peak) the addition of salts to MC 540 aqueous solutions resulted in the formation of a new absorption peak at 517 nm and the disappearance of both MC 540 “water” bands. MC 540 fluorescence spectra showed only one peak in pure water (at 573 nm) as well as in salt and HCl aqueous solutions (at 571 nm). The addition of salts did not change the shape of the fluorescence spectra but substantially quenched the fluorescence intensity. The effect of salts reflected principally differences in their cationic charge. These spectral changes were discussed in terms of interaction between the dye anion MC 540− and the aqueous proton H+ followed by dimerization of the protonated merocyanine dye. This assumption was supported by the low pH form of MC 540. A pK value of 1.6 between the neutral protonated form and the anionic form of MC 540 was determined.

pH dependence of merocyanine 540 absorption and fluorescence spectra

Abstract The effect of pH on the absorption and fluorescence spectra of merocyanine 540 has been determined. At neutral pH, a double peaked (at 500 and 534 nm) absorption band and a single fluorescence peak (at 577 nm) of anionic merocyanine 540 were recorded. The spectral shape and the peak positions were almost unchanged over the pH range 1.7–7.6 at a temperature of 23 ± 2δC. Decreasing the pH from 5.5 to 1.7 caused only a reversible diminution of the absorption spectra and quenching of the fluorescence intensity without the appearance of any other new spectral band. The dye was seen to precipitate with an increase in acidity. Increasing the pH above 7.6 in aqueous solutions of merocyanine 540 resulted in the irreversible formation of a new, broad band in both the absorption (at 390 nm) and fluorescence (at 500 nm) spectra. These irreversible changes could be due to an attack on the merocyanine molecules by hydroxyl ions.

Issues to be considered when studying cancer in vitro

Various cancer treatment approaches have shown promising results when tested preclinically. The results of clinical trials, however, are often disappointing. While searching for the reasons responsible for their failures, the relevance of experimental and preclinical models has to be taken into account. Possible factors that should be considered, including cell modifications during in vitro cultivation, lack of both the relevant interactions and the structural context in vitro have been summarized in the present review.