Evaluation of cytotoxic effect of photodynamic therapy in combination with electroporation in vitro

Under the influence of electric pulses cells undergo membrane electroporation (EP), which results in increased permeability of the membrane to exogenous compounds. EP is applied in oncology as a method to enhance delivery of anticancer drugs. For that reason it was essential to combine photodynamic tumor therapy (PDT)--the cancer treatment method based on the use of photosensitizers that localize selectively in malignant tumors and become cytotoxic when exposed to light, and EP, with the aim to enhance the delivery of photosensitizers into the tumor and therefore to increase the efficacy of PDT. Thus, the aim of study was to evaluate the cytotoxic effect of PDT in combination with EP. A Chinese hamster lung fibroblast cell line (DC-3F) was used. The cells were affected by photosensitizers chlorin e(6) (C e(6)) at the dose of 10 mug/ml and aluminium phthalocyanine tetrasulfonate (AlPcS4) at the dose of 50 microg/ml. Immediately after adding of photosensitizers the cells were electroporated with 8 electric pulses at 1200 V/cm intensity, 0.1 ms duration, 1 Hz frequency. Then, after 20 min of incubation the cells were irradiated using a light source--a visible light passing through a filter (KC 14, emitted light from 660 nm). The fluence rate at the level of the cells was 3 mW/m(2). Cytotoxic effect on cells viability was evaluated using MTT assay. Our in vitro data showed that the cytotoxicity of PDT in combination with EP increases fourfold on the average. Based on the results we suggest that EP could enhance the effect of PDT.     

Photodynamic properties of amphiphilic derivatives of aluminum tetrasulfophthalocyanine

Photodynamic therapy (PDT) is a promising treatment modality that has recently been accepted in clinics as a curative or palliative therapy for cancer and other nonmalignant conditions. Phthalocyanines (Pc) are attractive photosensitizers for PDT because of their enhanced photophysical and photochemical properties. The overall charge and solubility of Pc play a major role in their potential usefulness for PDT. A series of amphiphilic derivatives of tetrasulfonated aluminum Pc (AlPcS4) was prepared by substituting one of the four sulfonate groups with aliphatic side chains of 4, 8, 12 and 16 carbon atoms. The photodynamic properties of the derivatives were compared with those of AlPcS4 and the adjacent disulfonated aluminum Pc. Parameters studied included reversed-phase high-performance liquid chromatography (HPLC) retention times, capacity to generate singlet oxygen (1O2), in vitro cell uptake and phototoxicity, as well as PDT response of transplantable EMT-6 tumors in mice. The monomerized AlPcS4 derivatives showed similar or higher capacities to generate 1O2 as compared with the parent AlPcS4 as measured from relative L-tryptophan photooxidation yields. A549 cell uptake of the AlPcS4 derivatives decreased in the following order: AlPcS4(C16) > AlPcS4(C12) > AlPcS4(C8) > AlPcS4(C4). Human low-density lipoprotein at high concentrations (40 micrograms/mL) completely prevented uptake, whereas at 4 micrograms/mL uptake was decreased for the more lipophilic compounds and yet remained unaffected for the more hydrophilic dyes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, A549 cell survival was assessed; it showed that photocytotoxic activity varied directly with the HPLC retention times, i.e. more hydrophilic compounds were less phototoxic. As 1O2 yields were similar for the four substituted AlPcS4 derivatives, it was postulated that the increased cytotoxic activity was caused by enhanced subcellular localization as a result of the long aliphatic side chains. These amphiphilic compounds proved to be photodynamically potent against the EMT-6 mouse mammary tumor model implanted in Balb/c mice. At dye doses of 0.2 mumol/kg and a fluence of 400 J/cm2 complete tumor regression was observed with no morbidity. The substitution of AlPcS4 with long aliphatic chains on the macrocycle greatly enhances its photodynamic efficacy both in vitro and in vivo.     

Spectroscopic evidence of monomeric aluminium phthalocyanine tetrasulphonate in aqueous solutions

Aqueous solutions of aluminium phthalocyanine tetrasulphonate (AlPcS(4)) were investigated by means of absorption and fluorescence spectroscopy. The absorption spectrum of AlPcS(4) is independent of concentration in a wide range (from 10(-8) to 10(-4) M). The fluorescence spectrum measured with a standard setup is strongly dependent on AlPcS(4) concentration, and the fluorescence maximum is gradually red-shifted with increasing concentration. Calculations that take into account reabsorption of fluorescence (inner-filter effect) fit the experimental observations at low concentrations (up to 10(-6) M). Disagreement between the calculations and spectra recorded at higher concentrations (above 10(-5) M) shows that the reabsorbed light may be reemitted as fluorescence. The influence of inner-filter effects on the spectral shape was demonstrated by the experiments where a fibre-optic front-face fluorescence setup was applied: Under such conditions the shape of the fluorescence spectra for a high concentration (10(-3) M) coincided with that of a low concentration (10(-8) M). In conclusion, the present spectroscopic results show that AlPcS(4) does not form aggregates and is a very stable compound in aqueous solutions.     

ACTION SPECTRA OF PHTHALOCYANINES WITH RESPECT TO PHOTOSENSITIZATION OF CELLS

Human carcinoma cells (NHIK 3025 cells) and Chinese hamster cells (V79 cells) were incubated with AlPcS1, AlPcS2 and AlPcS4, phthalocyanines with different lipophilicity but with similar photochemical properties when in monomeric solutions. The absorption- and fluorescence spectra of the dyes in the cells were recorded as well as their action spectra with respect to sensitizing cells to photoinactivation. These spectra show that under the present conditions AlPcS1 is strongly aggregated in both cell lines; AlPcS2 is aggregated in V79 cells but much less so in NHIK 3025 cells. A main finding is that the shapes of the action spectra are similar to that of the fluorescence excitation spectra, but not to the absorption spectra, indicating that the photosensitizing effects of the dyes are mainly due to their monomeric fraction in the cells. AlPcS2 and AlPcS4 localize intracellularly mainly in lysosomes while AlPcS1 was found to be more diffusely distributed in cells. As measured per quantum of fluorescence emitted, AlPcS1 and AlPcS2 are more efficient sensitizers than AlPcS4. The difference in efficiency between AlPcS2 and AlPcS4 is supposedly due to a different localization pattern on the suborganelle level.     

Evaluation of different photosensitizers for use in photochemical gene transfection

Many potentially therapeutic macromolecules, e.g. transgenes used in gene therapy, are taken into the cells by endocytosis, and have to be liberated from endocytic vesicles in order to express a therapeutic function. To achieve this we have developed a new technology, named photochemical internalization (PCI), based on photochemical reactions inducing rupture of endocytic vesicles. The aim of this study was to clarify which properties of photosensitizers are important for obtaining the PCI effect improving gene transfection. The photochemical effect on transfection of human melanoma THX cells has been studied employing photosensitizers with different physicochemical properties and using two gene delivery vectors: the cationic polypeptide polylysine and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Photochemical treatment by photosensitizers that do not localize in endocytic vesicles (tetra[3-hydroxyphenyl]porphyrin and 5-aminolevulinic acid-induced protoporphyrin IX) do not stimulate transfection, irrespective of the gene delivery vector. In contrast, photosensitizers localized in endocytic vesicles stimulate polylysine-mediated transfection, and amphiphilic photosensitizers (disulfonated aluminium phthalocyanine [AlPcS2a] and meso-tetraphenylporphynes) show the strongest positive effect, inducing approximately 10-fold increase in transfection efficiency. In contrast, DOTAP-mediated transfection is inhibited by all photochemical treatments irrespective of the photosensitizer used. Neither AlPcS2a nor Photofrin affects the uptake of the transfecting DNA over the plasma membrane, therefore photochemical permeabilization of endocytic vesicles seems to be the most likely mechanism responsible for the positive PCI effect on gene transfection.     

Photodynamic therapy using topically applied hypericin: comparative effect with methyl-aminolevulinic acid on UV induced skin tumours

Photodynamic therapy (PDT) is a treatment option particularly well-suited for superficial (pre)malignant skin lesions due to the skin's accessibility to light. In the present study, the efficacy of topical hypericin-PDT was evaluated using a mouse model for actinic keratosis. For comparison, similar experiments were conducted with methyl-aminolevulinic acid (Me-ALA). Small skin tumours (1-2 mm) were induced in hairless mice by chronic UV irradiation. After topical application of hypericin (0.1% in gelcream for 24 h) or Me-ALA (Metvix? for 4 h), the lesional/non-lesional skin surface fluorescence ratio was determined and fluorescence microscopy was used to study the skin penetration of the photosensitizers. The antitumour activity of topical PDT (20 mW cm(-2), 40 J cm(-2)) was evaluated by measurement of the lesional diameters. Moreover, biopsies were taken at various time points after PDT for histological evaluation of the therapy. Our results demonstrate that after topical application of hypericin and Me-ALA, tumour selectivity is limited in mouse skin. The microscopic distribution of hypericin fluorescence showed an accumulation in the stratum corneum and low fluorescence levels in the rest of the lesions, whereas the distribution of PpIX in the skin was more homogenous. Topical hypericin-PDT was found to be less efficient (44% total lesional clearance) as compared to Me-ALA-PDT (80% total lesional clearance). Full lesional necrosis was observed in responsive lesions, and the atypical cells of actinic keratosis were replaced by normal keratinocytes 3 weeks later, both after hypericin-PDT and Me-ALA-PDT.     

Indirect detection of photosensitizer ex vivo

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0-5 microg x ml(-1)) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg x kg(-1) iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg x kg(-1)), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.     

Room-temperature long-lived triplet excited states of naphthalenediimides and their applications as organic triplet photosensitizers for photooxidation and triplet-triplet annihilation upconversions

Naphthalenediimide (NDI) derivatives with 2,6- or 2,3,6,7-tetrabromo or amino substituents were prepared. N,N'-dialkyl-2,6-dibromo NDI (compound 2) and N,N'-dialkyl-2,3,6,7-tetrabromo NDI (compound 4) show phosphorescence emission at 610 or 667 nm, respectively. Phosphorescence was never observed for NDI derivatives. Conversely, N,N'-dialkyl-2,6-dibromo-3,7-diamino NDI (compound 5) shows strong absorption at 526 nm and fluorescence at 551 nm, and no phosphorescence was observed. However, nanosecond time-resolved transient difference absorption spectroscopy confirmed that the triplet excited state of 5 was populated upon photoexcitation. 2,3,6,7-Tetraamino NDI (6) shows fluorescence, and no triplet excited state was populated upon excitation. The compounds were used as singlet oxygen ((1)O(2)) photosensitizers for the photooxidation of 1,5-dihydroxylnaphthalene (DHN). We found that 5 is more efficient than the conventional photosensitizer, such as Ir(ppy)(2)(bpy)[PF(6)]. The compounds were also used as organic triplet photosensitizers for triplet-triplet annihilation based upconversions. An upconversion quantum yield up to 18.5% was observed.     

Preparation, phototoxicity and biodistribution studies of anti-carcinoembryonic antigen monoclonal antibody-phthalocyanine conjugates

Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of a conjugated dye. Aluminum tetrasulfophthalocyanine (AlPcS4) was covalently coupled to a 35A7 MAb directed against carcinoembryonic antigen (CEA) via a five-carbon spacer chain (A1) to yield conjugates with a molar ratio ranging from 5 to 16 mol of AlPcS4 per mol of 35A7 MAb. Conjugates were labeled with radioiodine for characterization. The immunoreactivity of the conjugates, determined in a direct binding assay on CEA coupled to sepharose, was not modified by the coupled AlPcS4A1 molecules. In vivo, these conjugates were evaluated in nude mice bearing human colon carcinoma xenografts (T380). 35A7 MAb-(AlPcS4A1)5, 35A7 MAb-(AlPcS4A1)12 and 35A7 MAb-(AlPcS4A1)16 conjugates displayed a tumor uptake of 35 +/- 5.0%, 40 +/- 5.7% and 32 +/- 3.3% of the injected dose per gram of tumor tissue, respectively, corresponding to an uptake of 97%, 104% and 91% as compared to that of the unconjugated 35A7 MAb. In each experimental group, the tumor-to-normal tissue ratios obtained with the conjugates were almost identical to those obtained with unconjugated 35A7 MAb. Average values of 1.8, 7 and about 30 were obtained for blood, liver and muscle, respectively. Phototoxic efficacy of the 35A7 MAb-(AlPcS4A1)12 conjugate was demonstrated in vitro on the LoVo cell line giving a 91% growth inhibition for a 2.50 micrograms/mL AlPcS4A1 concentration. We conclude that these conjugates demonstrate clear in vivo tumor-seeking capacity and in vitro photocytotoxic properties. Such conjugates could thus be promising candidate drugs for clinical photodynamic therapy of cancers expressing CEA.     

Receptor-mediated targeting of phthalocyanines to macrophages via covalent coupling to native or maleylated bovine serum albumin

Targeted delivery of aluminum tetrasulfophthalocyanine (AlPcS4) to the scavenger receptor of macrophages, via coupling to maleylated bovine serum albumin (mal-BSA), was explored as a means to improve photodynamic efficacy. The AlPcS4 was covalently coupled to BSA (9:1 molar ratio) via one or two sulfonamide-hexanoic-amide spacer chains, followed by treatment with maleic anhydride to yield the mal-BSA-phthalocyanine conjugates. The latter were tested for singlet oxygen production, receptor-mediated cell uptake and phototoxicity toward J774 cells of macrophage origin and nonphagocytic EMT-6 cells. Cell uptake of 125I-mal-BSA showed specific binding for J774 cells but not for EMT-6 cells. Competition studies of the conjugates with 125I-mal-BSA showed that coupling of AlPcS4 to BSA resulted in recognition of the conjugate by the scavenger receptor, whereas coupling to mal-BSA further enhanced its binding affinity. This suggests that affinity for the scavenger receptor is related to the overall negative charge of the protein. Phototoxicity of the conjugates toward J774 cells paralleled their relative affinity, with mal-BSA-AlPcS4 coupled via two spacer chains showing the highest activity. The conjugates were less phototoxic toward the EMT-6 cell line. The activities in both cell lines of all conjugated AlPcS4 preparations were, however, lower than that of the free disulfonated AlPcS2. Possible implications for the in vivo use of protein-photosensitizer conjugates to target selectively various macrophage-associated disorders is discussed.     

Internalization of aggregated photosensitizers by tumor cells: subcellular time-resolved fluorescence spectroscopy on derivatives of pyropheophorbide-a ethers and chlorin e6 under femtosecond one- and two-photon excitations

Amphiphilic sensitizers self-associate in aqueous environments and form aggregated species that exhibit no or only negligible photodynamic activity. However, amphiphilic photosensitizers number among the most potent agents of photodynamic therapy. The processes by which these sensitizers are internalized into tumor cells have yet to be fully elucidated and thus remain the subject of debate. In this study the uptake of photosensitizer aggregates into tumor cells was examined directly using subcellular time-resolved fluorescence spectroscopy with a high temporal resolution (20-30 ps) and high sensitivity (time-correlated single-photon counting). The investigations were performed on selected sensitizers that exhibit short fluorescence decay times (< 50 ps) in aggregated form. Derivatives of pyropheophorbide-a ether and chlorin e6 with varying lipophilicity were used for the study. The characteristic fluorescence decay times and spectroscopic features of the sensitizer aggregates measured in aqueous solution also could be observed in A431 human endothelial carcinoma cells administered with these photosensitizers. This shows that tumor cells can internalize sensitizers in aggregated form. Uptake of aggregates and their monomerization inside cells were demonstrated directly for the first time by means of fluorescence lifetime imaging with a high temporal resolution. Internalization of the aggregates seems to be endocytosis mediated. The degree of their monomerization in tumor cells is strongly influenced by the lipophilicity of the compounds.     

EVALUATION OF SULFONATED ALUMINUM PHTHALOCYANINES FOR USE IN PHOTOCHEMOTHERAPY. A STUDY ON THE RELATIVE EFFICIENCIES OF PHOTOINACTIVATION

The cellular photosensitivity caused by aluminum phthalocyanines sulfonated to different degrees (AlPcSn) has been investigated. The phototoxic effect increased with decreasing number of sulfonate groups on the macrocycle, with the exception of AlPcS1 which was less phototoxic than AlPcS2 but more phototoxic than AlPcS3 and AlPcS4. The tendency of the AlPcSns to aggregate in our cellular system increased with increasing lipophilicity of the sensitizers. The aggregates had little or no photosensitizing activity. The low efficiency of cell inactivation caused by AlPcS1 can be explained by the highly aggregated state of this sensitizer in the cells. AlPcS2 and AlPcS3 induced a lower degree of cell inactivation per fluorescing quantum and per quantum absorbed by monomeric species than did AlPcS2 and AlPcS1. AlPcS4 and AlPcS3 are therefore suggested to be in different intracellular locations than AlPcS2 and AlPcS1.     

Photodynamic Therapy: Tumor Targeting with Adenoviral Proteins

A brief summary of the mechanisms involved in photodynamic therapy (PDT) and the role of delivery vehicles for photosensitizer targeting is addressed. Phthalocyanines (Pc) have been coupled to adenovirus type 2 capsid proteins including the hexon, the penton base and the fiber to enhance their target selectivity. Adenovirus penton base proteins contain the arginine-glycine-aspartic acid peptidic sequence (RGD) motif known to bind with great affinity and high specificity to integrin receptors, expressed by several types of cancer. Tetrasulfonated aluminum phthalocyanine (AlPcS4) was covalently coupled to the various capsid proteins via one or two caproic acid spacer chains (A1 or A2) in 7:1 up to 66:1 molar ratios. The capacity of the bioconjugates for singlet oxygen production, as measured by an L-tryptophan oxidation assay, was strongly reduced, likely reflecting scavenging by the carrier. Cell adsorption and in vitro photocytotoxicity assays were carried out using the A549 and HEp2 human cell lines expressing integrin receptors, and one murine, the EMT-6 cell line, which lacks receptors for the RGD sequence. The AlPcS4A2-protein complexes induced greater cytotoxicity as compared to the analogous AlPcS4A1 preparations. The penton base-AlPcS4A2 derivative was the more phototoxic for all cell lines tested. Tumor response studies using Balb/c mice with EMT-6 tumor implants demonstrated that the free AlPcS4A2 induced complete tumor regression at a dose of 1 mumol/kg and 400 J/cm2, which is comparable to the activity of the known AlPcS2adj. A mixture of adenovirus type 2 soluble proteins covalently labeled with AlPcS4A2 required 0.5 mumol/kg to induce the same response with the same light dose, suggesting that the high affinity RGD/receptor complex is able to target Pc for PDT.     

MITOTIC INHIBITION BY PHENYLPORPHINES AND TETRASULFONATED ALUMINIUM PHTHALOCYANINE IN COMBINATION WITH LIGHT

This work relates to studies on modes of phototoxicity by tetrasulfonated aluminium phthalocyanine (AlPcS4), tetrahydroxy- and monosulfonated meso-tetraphenylporphines (3-THPP and TPPS1) on culture cells. Toxicity at moderate light exposures appears to be related to inhibition of microtubule function. Treatment of human cervix carcinoma cells of the line NHIK 3025 incubated for 18 h with the sensitizers and exposed to light inhibits multiplication for the first hours after light exposure, a significant fraction of the cells accumulating in mitosis. For the first hours after treatment, the mitotic cells were always mainly found in metaphase; generally seen as c-metaphases and three-group metaphases. During this time, anaphase and telophase cells were absent or greatly reduced in number. Indirect immunofluorescence staining of beta-tubulin showed that the spindle apparatus of mitotic cells was perturbed in all cases. The accumulation in mitosis was more extensive after treatment with AlPcS4 and light than after treatment with 3-THPP or TPPS1 and light. This may be related to the great difference in the lipophilic properties of these sensitizers; i.e. AlPcS4 being highly water soluble while TPPS1 and 3-THPP are lipophilic sensitizers. The lipophilicity of several sensitizers has been measured by two different methods, the partition between an aqueous and a lipophilic phase (Triton X-114) and the binding strength to a reverse phase column. The results show that the measured relative lipophilicity of the sensitizers may be influenced by the method of analysis.     

The role of the low density lipoprotein receptor pathway in the delivery of lipophilic photosensitizers in the photodynamic therapy of tumours

Lipoproteins are now recognized as major blood carriers of many hydrophobic porphyrins and related chromophores which are being investigated as possible photosensitizers in the photodynamic therapy of tumours. In vitro and in vivo studies have demonstrated the role of the low density lipoprotein (LDL) receptor pathway in the delivery of photosensitizers to tumour cells and its importance in porphyrin accumulation by tumours. Lysosomes, which are involved in the cellular processing of LDL, are important intracellular targets in the LDL-porphyrin-induced phototoxicity. The use of the LDL receptor pathway as a tool for enhancing the selectivity of photosensitizer delivery to tumour cells appears to be a promising field of research in the photodynamic therapy of tumours.     

Light-induced apoptosis involves a defined sequence of cytoplasmic and nuclear calcium release in AlPcS4-photosensitized rat bladder RR 1022 epithelial cells

Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose-dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate-labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-L-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.     

Synthesis, photophysical and photochemical properties of poly(oxyethylene)-substituted zinc phthalocyanines

The synthesis, photophysical and photochemical properties of the tetra- and octa-poly(oxyethylene)substituted zinc (II) phthalocyanines are reported for the first time. The new compounds have been characterized by elemental analysis, IR, 1H and 13C NMR spectroscopy, electronic spectroscopy and mass spectra. General trends are described for photodegradation, singlet oxygen, triplet state and fluorescence quantum yields, and triplet and fluorescence lifetimes of these compounds in dimethylsulfoxide (DMSO). Photophysical and photochemical properties of phthalocyanine complexes are very useful for PDT applications. The effects of the substituents on the photophysical and photochemical parameters of the zinc(II) phthalocyanines (3a, 5a and 6a) are also reported. The singlet oxygen quantum yields (Phi(Delta)), which give an indication of the potential of the complexes as photosensitizers in applications where singlet oxygen is required (Type II mechanism) ranged from 0.60 to 0.72. Thus, these complexes show potential as Type II photosensitizers. The fluorescence of the complexes was quenched by benzoquinone (BQ).     

Studies of mixed surfactant solutions of cationic dimeric (gemini) surfactant with nonionic surfactant C12E6 in aqueous medium

The interaction between the alkanediyl-alpha,omega-type cationic gemini surfactant, [(C(16)H(33)N(+)(CH(3))(2)(CH(2))(4)N(+)(CH(3))(2)C(16)H(33))2Br(-)], 16-4-16 and the conventional nonionic surfactant [CH(3)(CH(2))(10)CH(2)(OCH(2)CH(2))(6)OH], C(12)E(6) in aqueous medium has been investigated. The critical micelle concentrations of different mixtures have been measured by surface tension using a du Nouy tensiometer in aqueous solution at different temperatures (303, 308, and 313 K). Maximum surface excess (Gamma(max)) and minimum area per molecule (A(min)) were evaluated from a surface tension vs log(10)C (C is concentration) plot. The cmc value of the mixture was used to compute beta(m), the interaction parameter. The beta(sigma), the interaction parameter at the monolayer air-water interface, was also calculated. We observed synergism in 16-4-16/C(12)E(6) system at all concentration ratios. The micelle aggregation number (N(agg)) has been measured using a steady state fluorescence quenching method at a total surfactant concentration approximately 2 mM at 25 degrees C. The micropolarity and the binding constant (K(sv)) of mixed systems were determined from the ratio of intensity of peaks (I(1)/I(3)) of the pyrene fluorescence emission spectrum. The micellar interiors were found to be reasonably polar. We also found, using Maeda's concept, that the chain-chain interactions are very important in this system.     

Effect of fluoride anions on gramicidin photoinactivation sensitized by sulfonated aluminum phthalocyanines.

Interaction of potent photodynamic agents, sulfonated aluminum phthalocyanines (AlPcSn where n is a number of sulfonic groups), with biological membranes was studied here using model systems: sensitized photoinactivation of gramicidin channels in planar lipid bilayers and adsorption on lipid monolayers. Fluoride anions known to form complexes with aluminum were found to inhibit both the adsorption of aluminum phthalocyanines on lipid monolayers, as measured with a Langmuir trough by surface pressure and surface potential changes, and photodynamic efficacy of the dyes, as studied by gramicidin channel photoinactivation. The similar effects were caused by the alkalinization of the medium. Fluoride anions appeared to be much more effective in the case of AlPcS4 as compared to AlPcS3. The suppression of the photodynamic potency of aluminum phthalocyanines was attributed to desorption of the dyes from lipid bilayers induced by fluoride or hydroxyl ions. With AlPcS4 an enhancement of the dye aggregation leading to a decrease in the sensitizing activity was probably involved in the fluoride effect as revealed by absorption and fluorescence spectral measurements. Capillary electrophoresis was employed to understand the mechanism of the dye desorption. The results of these experiments indicated that the reduction in the membrane affinity was associated with an increase in the negative charge of the dye molecules due to the binding of fluoride or hydroxyl ions.     

Singlet oxygen production by Peptide-coated quantum dot-photosensitizer conjugates

Peptide-coated quantum dot-photosensitizer conjugates were developed using novel covalent conjugation strategies on peptides which overcoat quantum dots (QDs). Rose bengal and chlorin e6, photosensitizers (PSs) that generate singlet oxygen in high yield, were covalently attached to phytochelatin-related peptides. The photosensitizer-peptide conjugates were subsequently used to overcoat green- and red-emitting CdSe/CdS/ZnS nanocrystals. Generation of singlet oxygen could be achieved via indirect excitation through F?rster (fluorescence) resonance energy transfer (FRET) from the nanocrystals to PSs, or by direct excitation of the PSs. In the latter case, by using two color excitations, the conjugate could be simultaneously used for fluorescence imaging and singlet oxygen generation. Singlet oxygen quantum yields as high as 0.31 were achieved using 532-nm excitation wavelengths.