The effect of pH and surfactant on the aggregation behavior of chlorin p6: a fluorescence spectroscopic study

Steady state and time-resolved fluorescence properties of chlorin P6, a potential drug for photodynamic therapy, have been investigated as functions of pH. A decrease in pH of the medium has been shown to cause protonation of the ionizable carboxylic acid side chain, leading to an increase in hydrophobicity and consequent aggregation. The aggregates dissociate on further protonation. The dissociation is explained in terms of formation of cations and their mutual repulsion. A synchronous fluorescence spectroscopic study revealed the presence of two anionic forms in equilibrium at physiological pH, with a shift in the equilibrium on slight decrease in the pH. The anionic nature of chlorin P6 in aqueous solutions at physiological pH has been confirmed by complexation with surfactants. The nature of the charge on the headgroups of the surfactants has been found to govern the formation of chlorin-surfactant complexes.     

Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo.

Several parameters of chlorin e6 and its derivative chlorin e6 ethylenediamide have been investigated as these compound are potential sensitizers for photodynamic therapy. A study carried out to compare the cellular uptake of the pigments indicates that chlorin e6 ethylenediamide possesses an enhanced affinity for tumour cells and cellular membranes. Comparison of the uptake in induced sarcoma shows that chlorin e6 ethylenediamide is a much better tumour localizer than chlorin e6. The efficiency of phototherapy with chlorin e6 ethylenediamide is higher than that with chlorin e6. These data show the influence of the substitution of the carboxyl groups in chlorin e6 by ester and amide groups on the photosensitizing properties of the pigments.     

Near-infrared photosensitizer based on a cycloimide derivative of chlorin p6: 13,15-N-(3'-hydroxypropyl)cycloimide chlorin p6

The 13,15-N-(3'-hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell-killing activity. It is 43-, 61- and 110-fold more active than chlorin p6, 3-formyl-3-devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002-0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 +/- 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3- to 4-fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 +/- 5 min. The time for 50% release of CIC from the cells is 60 +/- 10 min. A 10-fold decrease in CIC intracellular penetration at 22 degrees C proves that temperature-sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2-1.6 microM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic-type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near-IR photosensitizer.     

Photodynamic therapy with chlorin e6. A morphologic study of tumor damage efficiency in experiment.

Morphological changes in rat sarcoma M-1 after photodynamic treatment with chlorin e6 were studied. The frequency of necrosis appearance and the depth of its spreading in tumor tissue were evaluated after intraperitoneal injection of chlorin e6 in doses of 1-10 mg kg-1 and subsequent irradiation by a krypton laser with light energy density 22.5-135 J cm-2, using the method of vital staining with Evans blue. It was found that the antitumoral effect of photodynamic treatment was strengthened by increasing the dose of the agent and light and reduced by increasing the time interval between chlorin e6 injection and light irradiation. The treatment being given in the parameters mentioned produced a depth of tumor necrosis which varied from 4.0 mm to 16.6 mm. The mechanisms of tumor tissue damage after photodynamic treatment in vivo are discussed.     

Comparative study of photodynamic properties of 13,15-N-cycloimide derivatives of chlorin p6

Comparative study of 13,15-[N-(2-hydroxyethyl)]cycloimide chlorin p6 (2), 13,15-(N-acetoxy)cycloimide chlorin p6 (3), 13,15-(N-hydroxy)cycloimide chlorin p6 methyl ester (4) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (5) together with the previously investigated 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (1) was performed. The dependence of the key photodynamic properties of 1-5 on the introduced substituents was analyzed. The photoinduced cell-killing activity of 4 is 100- and 280-fold higher than that of chlorin p6 and Photogem, respectively, as estimated on A549 human lung adenocarcinoma cells. The activity is reduced eight times in the order 4 > 5 > 1 > 2 > 3. The intracellular accumulation of 1-5 occurs in cytoplasm in a monomeric form bound to the lipids of cellular membranes. This form of 1, 2, 3, 4 and 5 is characterized by the high quantum yield of singlet oxygen generation, which depends on the introduced substituents, 0.66, 0.59, 0.35, 0.51 and 0.73, respectively. The photostability is two-fold less for 1 and four-fold less for 2, 3 and 5 than for 4. The rates of cellular uptake and efflux of 1-5 vary widely, thus providing the way to optimize the pharmacological properties of the photosensitizer (PS) using the respective substituents. Modifying the substituents, 1-5 were targeted to different cellular organelles. The enhanced accumulation in the Golgi apparatus and mitochondria complemented with diffuse staining of intracellular membranous structures is a property of 1-4. Compound 5 accumulates selectively in the lipid droplets and stains weakly perinuclear structures. Temperature-sensitive mechanisms of transport are responsible for the 1-4 uptake. Diffusion can play a role in the internalization of 5 but not of 1-4. Endocytosis via caveolae, clathrin-dependent and adenosine triphosphate-dependent pathways are not noticeably involved in the 1-5 internalization. Independently from their intracellular localization 1, 4 and 5 are highly efficient near-IR PS, which induce predominantly an apoptotic type of cell death under conditions providing ca 50% level of phototoxicity and necrosis at the 100% level of phototoxicity.     

Phase-resolved fluorescence study of mono-L-aspartyl chlorin E6

The properties of a photosensitizer for photodynamic therapy, mono-L-aspartyl chlorin e6 (NPe6), were investigated using phase-resolved fluorescence. NPe6 was analyzed in water solution at concentrations ranging from 3.13x10(-7) to 8.00x10(-5) M. The photophysical parameters of the lowest singlet excited state of NPe6 molecules were experimentally determined. It was confirmed that NPe6 molecules were in the isolated molecular state at concentrations below 1.00x10(-5) M. It was also confirmed that the fluorescence in this concentration range was ascribable to the electronic transition of isolated NPe6 molecules from the lowest singlet excited state to the ground state. At concentrations above 1.00x10(-5) M, some of the NPe6 molecules formed dimers in water solution, which caused a red shift of the fluorescence spectrum and an enhancement of fluorescence in the 700-750 nm wavelength region. Semiempirical molecular orbital calculation revealed that the sodium aspartate attached to the tetrapyrrole ring through the ethanoic group was remarkably bent with respect to the tetrapyrrole plane. This bending appeared to hinder the formation of NPe6 dimers at concentrations up to 1.00x10(-5) M.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin, pheophorbide a and chlorin e6 in ethanol and aqueous solution

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.     

Tissue distribution and in vivo photosensitizing activity of 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester

Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.     

Interaction of chlorin p6 with bovine serum albumin and photodynamic oxidation of protein

The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern-Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62+/-0.27 x 10(5)M(-1) and 1.086+/-.019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein carbonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroperoxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H2O was replaced by D2O. The results show that chlorin p6 induced photodynamic oxidation of BSA was mainly mediated by singlet oxygen.     

HPLC study of chlorin e6 and its molecular complex with polyvinylpyrrolidone

Several HPLC methods with UV detection were developed for qualitative and quantitative analysis of chlorin e(6) and photosensitizer Photolon either in the free form or upon pre-derivatization (methylation) under reversed- and normal-phase conditions. Optimum analysis conditions providing the best resolution of analytes were found at acidic pH where polar groups are completely protonated. The separation was performed by gradient elution with mobile phases of 0.08% trifluoroacetic acid and acetonitrile on an XTerra RP(18) column. The method was specific, accurate and precise, allowing the analysis of chlorin e(6) in the presence of numerous degradation products useful in the manufacturing process and quality control of chlorin e(6) and Photolon.     

The interplay of hydrophobic and electrostatic effects in the surfactant-induced aggregation/deaggregation of chlorin p6

The aggregation/deaggregation of chlorin p6 with the surfactants CTAB, SDS, and TX 100 have been studied by using absorption, fluorescence, and light scattering techniques. The ionic surfactants are found to cause aggregation of fluorophore at submicellar concentrations. The aggregates dissolve at higher surfactant concentrations to yield micellized monomers. This is rationalized by the interplay of electrostatic and hydrophobic effects. A prominent pH effect is observed in the ionic surfactant induced aggregation process as the charge on the fluorophore is controlled by the pH of the medium. Interestingly, the neutral TX-100 also induces aggregation of chlorin p6 at low concentrations, indicating that hydrophobic effects by themselves can cause aggregation unless there is a hindrance by repulsive electrostatic effects.     

Design and synthesis of novel water-soluble amino acid derivatives of chlorin p6 ethers as photosensitizer

Eight new water-soluble amino acid derivatives of chlorin p₆ ethers 6a-h were designed and synthesized using purpurin-18 (2) as key intermediate. All target compounds exhibited better phototoxicity than talaporfin and the most phototoxic compound 6d showed IC₅₀ values of 0.20 μmol/L against A549 cell and 0.41 μmol/L against B16-F10 cell, which represented 31- and 24-fold increase of PDT antitumor efficacy compared to talaporfin.     

The pH dependent Raman spectroscopic study of caffeine

First of all the surface enhanced Raman spectroscopy (SERS) and normal Raman spectra of caffeine aqueous solution were obtained at different pH values. In order to obtain the detailed vibrational assignments of the Raman spectroscopy, the geometry of caffeine molecule was optimized by density functional theory (DFT) calculation. By comparing the SERS of caffeine with its normal spectra at different pH values; it is concluded that pH value can dramatically affect the SERS of caffeine, but barely affect the normal Raman spectrum of caffeine aqueous solution. It can essentially affect the reorientation of caffeine molecule to the Ag colloid surface, but cannot impact the vibration of functional groups and chemical bonds in caffeine molecule.     

Anomalous pH dependent stability behavior of surfactant-free nonpolar oil drops in aqueous electrolyte solutions

Recent advances in atomic force microscopy (AFM) force measurement techniques have allowed the direct measurement and theoretical interpretation of the interaction between a liquid droplet and a solid surface or between two liquid droplets. In this study, we investigated the interaction across an aqueous thin film between fluorocarbon (perfluoropentane) droplets, hydrocarbon (tetradecane) droplets, and a droplet and a flat mica surface in the absence of stabilizers. It was found that even at a relatively elevated electrolyte concentration of 0.1 M NaNO3, depending on the solution pH, interactions between two identical droplets or a droplet and a mica surface could be repulsive. A simple theoretical analysis of the magnitude and range of these interactive forces suggests that the DLVO theory cannot explain the observed behavior. The measured force behavior is discussed in the context of ion adsorption, and the arising charging effects, at the bare oil-water interface.     

Toward understanding the high PDT efficacy of chlorin e6-polyvinylpyrrolidone formulations: photophysical and molecular aspects of photosensitizer-polymer interaction in vitro

It is recognized that chlorin e6-polyvinylpyrrolidone (Ce6-PVP) formulations are characterized by a high efficacy in photodynamic therapy of malignant tumors. Currently, a commercially available formulation of this type is Photolon((R)) (Fotolon((R))) with Ce6:PVP=1:1 (w/w) and the weight-average molecular weight of PVP is 1.2x10(4). To gain a better understanding of the role played by PVP in Ce6-PVP formulations, we carry out experiments on IR and UV-VIS absorption, steady-state and time-resolved fluorescence, time-resolved triplet-triplet absorption, octanol-water partitioning, and solubility of chlorin e6 in buffer solutions at pH 6.3, 7.4, and 8.5 in presence of PVP with Ce6:PVP ratios ranging from 1:0 to 1:1000 (w/w) for PVP samples with weight-average molecular weights of 8x10(3), 1.2x10(4), and 4.2x10(4). We show that Ce6 interacts with PVP by forming molecular complexes via hydrophobic interactions and determine the Ce6-PVP binding constant, as well as the mean number of PVP monomers per binding site. We find that complexation of Ce6 with PVP prevents Ce6 aggregation in aqueous media and leads to an enhancement of Ce6 fluorescence quantum yield, while keeping the quantum yield of the intersystem crossing essentially unchanged. Possible scenarios of how the presence of PVP can favorably affect the PDT efficacy of chlorin e6 in Ce6-PVP formulations are discussed.     

Pharmacokinetics of Tetra (m-hydroxyphenyl)chlorin in Human Plasma and Individualized Light Dosimetry in Photodynamic Therapy

The pharmacokinetics of the photosensitizer 5,10,15,20-tetra( m -hydroxyphenyl) chlorin(mTHPC) was investigated in the plasma of 20 patients by absorption and fluorescence spectroscopy. The temporal behavior was characterized by a rapid decrease in concentration during the first minutes after intravenous injection of 0.15 mg/kg mTHPC. A minimum concentration in the plasma was reached after about 45 min. The drug concentration then increased again, attaining a maximum after about 10 h, after which it decreased again with a halflife of about 30 h. Irradiation tests in the oral cavity at different time intervals after the injection revealed that the tissue re-action was only partially correlated with the mTHPC plasma level. The tissue response was stronger at later drug-light intervals (1–4 days) than during the first hours after injection even though the mTHPC plasma concentration was higher at the shorter times. Relative mTHPC concentrations were also measured in the mucosae of the oral cavity, the esophagus and the bronchi of 27 patients by light-induced fluorescence spectroscopy using an optical fiber-based spectrometer. These measurements were performed prior to photodynamic therapy (PDT), 4 days after injection of the photosensitizer. Highly significant linear correlations were found between the relative mTHPC concentrations in the mucosae of these three organs. Likewise, the plasma levels of mTHPC measured just before PDT were significantly correlated with the mTHPC concentrations in the three types of mucosae mentioned above. These results indicate that mTHPC plasma levels measured just before PDT can be used for PDT light dosimetry.     

pH dependent stability of aqueous suspensions of graphene with adsorbed weakly ionisable cationic polyelectrolyte

Stable graphene suspensions were prepared through ultrasonic exfoliation followed by surface modification with the cationic polyelectrolyte poly(ethyleneimine) (PEI). The stability of the suspensions was found to be dependent upon the pH of the solution and the molecular weight of the PEI adsorbed. For the graphene sheets with adsorbed PEI with a molecular weigh of 600 Da, the particles were stabilised through an increased electrostatic repulsion at low pH inferred from in an increase in the measured zeta potential of the particles. However, the graphene with higher molecular weight PEI (70 kDa) was stable over a comparatively larger pH range through a combination of electrostatic repulsion at low pH and steric repulsion at elevated pH. Thus, solution conditions allowing the control of the colloidal sized graphene particles can be easily tuned through judicious management of solution conditions as well as polymer layer properties.