Synthesis, characterization, and cellular investigations of porphyrin- and chlorin-indomethacin conjugates for photodynamic therapy of cancer.

Indomethacin is a potent non-steroidal anti-inflammatory drug (NSAID) with a strong selective inhibitor activity towards cyclooxygenase-2 (COX-2), an enzyme that is highly overexpressed in various tumour cells, being involved in tumourigenesis. Concomitantly, porphyrins have gained much attention as promising photosensitizers (PSs) for the non-invasive photodynamic therapy (PDT) of cancer. Herein, we report the design, and determine the singlet oxygen generation capacity and in vitro cellular toxicity of porphyrin- and chlorin-indomethacin conjugates (P2-Ind and C2-Ind). Both the conjugates were obtained in high yields and were characterized by 1H, 19F and 13C NMR as well as by high resolution mass spectrometry. The singlet oxygen generation properties were assessed by the 1,3-diphenylisobenzofuran singlet oxygen trap method, which showed that C2 and C2-Ind are the best singlet oxygen photosensitizers. In addition, it was found that the presence of indomethacin did not influence the singlet oxygen generation of porphyrin or chlorin. Cytotoxicity studies of the conjugate in human HEp2 cells revealed that the porphyrin- and chlorin-indomethacin conjugates have similar dark cytotoxicities, while chlorin C2 was shown to be the most phototoxic. Despite having lower cellular uptake than C2-Ind after 24 hours, chlorin C2 had a broad localization in HEp2 cells while the chlorin-indomethacin conjugate C2-Ind could be detected in the form of small aggregates. DFT calculations were performed to shed light on the reaction energy involved in the formation of the indomethacin conjugates and to compare the relative stability of selected isomers in solution. Moreover, the calculated energy of their first excited triplet state structures confirmed their use as suitable photosensitizers to generate singlet oxygen for PDT.

Synthesis, Characterization, and Evaluation of Near-IR Boron Dipyrromethene Bioconjugates for Labeling of Adenocarcinomas by Selectively Targeting the Epidermal Growth Factor Receptor.

A series of five boron dipyrromethene (BODIPY) bioconjugates containing an epidermal growth factor receptor (EGFR)-targeted pegylated LARLLT peptide and/or a glucose or biotin ethylene diamine group were synthesized, and the binding capability of the new conjugates to the extracellular domain of EGFR was investigated using molecular modeling, surface plasmon resonance, fluorescence microscopy, competitive binding assays, and animal studies. The BODIPY conjugates with a LARLLT peptide were found to bind specifically to EGFR, whereas those lacking the peptide bound weakly and nonspecifically. All BODIPY conjugates showed low cytotoxicity (IC50 > 94 µM) in HT-29 cells, both in the dark and upon light activation (1.5 J/cm2). Studies of nude mice bearing subcutaneous human HT-29 xenografts revealed that only BODIPY conjugates bearing the LARLLT peptide showed tumor localization 24 h after intravenous administration. The results of our studies demonstrate that BODIPY bioconjugates bearing the EGFR-targeting peptide 3PEG-LARLLT show promise as near-IR fluorescent imaging agents for colon cancers overexpressing EGFR.

Synthesis and investigation of phthalocyanine-biotin conjugates.

An isothiocyanato-functionalized phthalocyanine (Pc) was synthesized in good yield from the corresponding amine-substituted Pc. This Pc reacted with ethanolamine, biotin hydrazine, and biotin diethylamine under mild conditions (room temperature in DMF or DMSO in the presence of TEA) to produce the corresponding thiourea products in 60-75% yields. All Pcs showed intense Q absorptions in DMF around 677 nm, emissions centered at 683 nm, and fluorescence quantum yields in the range 0.18-0.27. The Pcs were phototoxic to human carcinoma HEp2 cells (IC50 ~ 7 at 1.5 J/cm2) and localized in multiple organelles, including the lysosomes, Golgi and ER. One biotin-Pc conjugate was injected via tail vein into nude mice bearing HT-29 tumors and demonstrated selective localization in the tumor tissue.

Syntheses and PDT activity of new mono- and di-conjugated derivatives of chlorin e6.

Syntheses of three new chlorin e6 conjugates for PDT of tumors are reported. One of the new compounds 17 is conjugated with lysine at the 131-position, but the others are mono-conjugated 14 or diconjugated 15 with the non-amino acid species ethanolamine. Cellular experiments with the three new compounds and previously synthesized non-amino acid 152-conjugates (7-10), 131-monoconjugates 14, 16, and a 131,152-diconjugate 12 are reported. In vitro cytotoxicity experiments show that the 131-conjugates are more toxic than the 152-conjugates, and the most toxic derivative (dark- and photo-toxicity) is the 131-ethylenediamine conjugate 11. The most useful PDT photosentitizers appear to be the ethanolamine derivatives, conjugated at the 152- and the 131,152-positions; these show high phototoxicity but relatively low dark toxicity compared with 11, and also the highest dark/photo cytotoxicity ratios.

Synthesis and Spectroscopic and Cellular Properties of Near-IR [a]Phenanthrene-Fused 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacenes.

A new synthetic method to build aryl-fused 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) is reported. The intramolecular cyclization step was completed in a short time (1-2 h) and in high yields (>90%), due to the intrinsic rigid structural conformation of the precursor BODIPY and the high reactivity of its 1,7-bromo groups. The [a]phenanthrene-fused BODIPYs 4a-c were characterized by NMR spectroscopy, HRMS, DFT calculations, and, in the case of 4a, by X-ray crystallography. Spectroscopic studies show that 4a-c strongly absorb and emit in the NIR spectral region, in the range 642-701 nm. In addition, BODIPYs 4b and 4c exhibit no toxicity in the light or dark in HEp2 cells and accumulate intracellularly in a time-dependent manner, mainly in the cell endoplasmic reticulum. These results suggest the potential use of [a]phenanthrene-fused BODIPYs as NIR bioimaging probes.

Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR Overexpressing Cells.

Regioselective functionalization of 2,3,5,6,8-pentachloro-BODIPY 1 produced unsymmetric BODIPY 5, bearing an isothiocyanate group suitable for conjugation, in only four steps. The X-ray structure of 5 reveals a nearly planar BODIPY core with aryl dihedral angles in the range 47.4-62.9°. Conjugation of 5 to two EGFR-targeting pegylated peptides, 3PEG-LARLLT (6) and 3PEG-GYHWYGYTPQNVI (7), under mild conditions (30 min at room temperature), afforded BODIPY conjugates 8 and 9 in 50-80% isolated yields. These conjugates showed red-shifted absorption and emission spectra compared with 5, in the near-IR region, and were evaluated as potential fluorescence imaging agents for EGFR overexpressing cells. SPR and docking investigations suggested that conjugate 8 bearing the LARLLT sequence binds to EGFR more effectively than 9 bearing the GYHWYGYTPQNVI peptide, in part due to the lower solubility of 9, and its tendency for aggregation at concentrations above 10 µM. Studies in human carcinoma HEp2 cells overexpressing EGFR demonstrated low dark and photo cytotoxicities for BODIPY 5 and the two peptide conjugates, and remarkably high cellular uptake for both conjugates 8 and 9, up to 90-fold compared with BODIPY 5 after 1 h. Fluorescence imaging studies in HEp2 cells revealed subcellular localization of the BODIPY-peptide conjugates mainly in the Golgi apparatus and the cell lysosomes. The low cytotoxicity of the new conjugates and their remarkably high uptake into EGFR overexpressing cells renders them promising imaging agents for cancers overexpressing EGFR.

Targeting of the epidermal growth factor receptor with mesoporphyrin IX-peptide conjugates.

The synthesis and in vitro evaluation of four mesoporphyrin IX-peptide conjugates designed to target EGFR, over-expressed in colorectal and other cancers, are reported. Two peptides with known affinity for EGFR, LARLLT (1) and GYHWYGYTPQNVI (2), were conjugated to mesoporphyrin IX (MPIX, 3) via one or both the propionic side chains, directly (4, 5) or with a triethylene glycol spacer (7, 8). The conjugates were characterized using NMR, MS, CD, SPR, UV-vis and fluorescence spectroscopies. Energy minimization and molecular dynamics suggest different conformations for the conjugates. SPR studies show that conjugate 4, bearing two LARLLT with no PEG spacers, has the greatest affinity for binding to EGFR, followed by conjugate 7 with two PEG and two LARLLT sequences. Molecular modeling and docking studies suggest that both conjugates 4 and 7 can bind to monomer and dimer EGFR in open and closed conformations. The cytotoxicity and cellular targeting ability of the conjugates were investigated in human HEp2 cells over-expressing EGFR. All conjugates showed low dark- and photo-toxicities. The cellular uptake was highest for conjugates 4 and 8 and lowest for 7 bearing two LARLLT linked via PEG groups, likely due to decreased hydrophobicity. Among the conjugates investigated 4 is the most efficient EGFR-targeting agent, and therefore the most promising for the detection of cancers that over-express EGFR.

Synthesis and in Vitro Studies of a Series of Carborane-Containing Boron Dipyrromethenes (BODIPYs).

A series of seven BODIPYs functionalized with ortho-carborane groups at the 8(meso) or 3/5(α) position were synthesized and characterized by NMR, HRMS, HPLC, and in the cases of 2b and 5b, by X-ray analysis. The BODIPYs exhibited low dark toxicity and phototoxicity toward human glioma T98G cells, and their cellular uptake varied significantly, with 5b accumulating the most and 7 the least. All BODIPYs localized mainly within the cell ER. The BODIPYs showed higher permeabilities than lucifer yellow across human hCMEC/D3 brain endothelial cell monolayers as the BBB model. Among this series, 1b showed the highest BBB permeability (Pe = 16.4 × 10(-5) cm/s), probably as a result of its lower MW (366 Da) and favorable hydrophobicity (log P = 1.5). The combination of low cytotoxicity, amphiphilicity, high boron content, high cellular uptake, and moderate BBB permeability renders these compounds promising boron delivery agents for the BNCT of brain tumors.

Syntheses and cellular investigations of di-aspartate and aspartate-lysine chlorin e(6) conjugates.

Chlorin e6 is a tricarboxylic acid degradation product of chlorophyll a. Four chlorin e6 bis(amino acid) conjugates were regioselectively synthesized bearing two aspartate conjugates in the 13(1),17(3)- and 15(2),17(3)-positions, or at the 13(1),15(2)via an ethylene diamine linker. One additional conjugate bearing two different amino acids, lysine at 13(1)via an ethylene diamine linker and an aspartate at 15(2)via a ß-alanine linker was also synthesized. The cytotoxicity and uptake of four di(amino acid) chlorin e6 conjugates were investigated in human HEp2 cells, and compared with chlorin e6. The most cytotoxic and most taken up conjugates were the zwitterionic 13(1),15(2)-disubstituted conjugates 28 and 33; these also localized in multiple organelles. In contrast, the anionic 13(1),17(3)- and 15(2),17(3)-di-aspartyl chlorin e6 conjugates 12 and 13 showed low dark cytoxicity and lower phototoxicity compared with chlorin e6.

Synthesis, spectroscopic, and in vitro investigations of 2,6-diiodo-BODIPYs with PDT and bioimaging applications.

A series of five mono-styryl and their corresponding symmetric di-styryl-2,6-diiodo-BODIPYs containing indolyl, pyrrolyl, thienyl or tri(ethylene glycol)phenyl groups were synthesized using Knoevenagel condensations. The yields for the condensation reactions were improved up to 40% using microwave irradiation (90°C for 1h at 400W) due to lower decomposition of BODIPYs upon prolonged heating. The spectroscopic, structural (including the X-ray of a di-styryl-2,6-diiodo-BODIPY) and in vitro properties of the BODIPYs were investigated. The extension of π-conjugation through the 3,5-dimethyls of the known phototoxic 2,6-diiodo-BODIPY 1 produced bathochromic shifts in the absorption and emission spectra, in the order of 63-125nm for the mono-styryl- and 128-220nm for the di-styryl-BODIPYs in DMSO. The largest red-shifts were observed for the indolyl-containing BODIPYs while the largest fluorescence quantum yields were observed for the tri(ethyleneglycol)phenylstyryl-BODIPYs. Among this series, only the mono-styryl-BODIPYs were phototoxic (IC50=2-15µM at 1.5J/cm(2)), and were observed to localize preferentially in the cell ER and mitochondria. On the other hand, the di-styryl-BODIPYs were found to have low or no phototoxicity (IC50>100µM at 1.5J/cm(2)). Among this series of compounds BODIPY 2a shows the most promise for application as photosensitizer in PDT.

Tetrakis(p-Carboranylthio-Tetrafluorophenyl)Chlorin (TPFC): Application for Photodynamic Therapy and Boron Neutron Capture Therapy.

Carboranyl-containing chlorins have emerged as promising dual sensitizers for use in both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT), by virtue of their known tumor affinity, low cytotoxicity in dark conditions, and their strong absorptions in the red region of the optical spectrum. Tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC) is a new synthetic carboranyl-containing chlorin of high boron content (24% by weight). To evaluate TPFC's applicability as sensitizer for both PDT and BNCT, we performed an in vitro and in vivo study using F98 rat glioma cells and F98 rat glioma-bearing brain tumor models. For the in vivo BNCT study, we used boronophenylalanine (BPA), which is currently used in clinical BNCT studies, via intravenous administration (i.v.) and/or used TPFC via convection-enhanced delivery (CED), a method for local drug infusion directly into the brain. In the in vitro PDT study, the cell surviving fraction following laser irradiation (9J/cm2) was 0.035 whereas in the in vitro BNCT study, the cell surviving fraction following neutron irradiation (thermal neutron=1.73×1012 n/cm2) was 0.04. In the in vivo BNCT study, the median survival time following concomitant administration of BPA (i.v.) and TPFC (CED) was 42days (95% confidence interval; 37-43days). © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC): application for photodynamic therapy and boron neutron capture therapy.

Carboranyl-containing chlorins have emerged as promising dual sensitizers for use in both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT), by virtue of their known tumor affinity, low cytotoxicity in dark conditions, and their strong absorptions in the red region of the optical spectrum. Tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC) is a new synthetic carboranyl-containing chlorin of high boron content (24% by weight). To evaluate TPFC's applicability as sensitizer for both PDT and BNCT, we performed an in vitro and in vivo study using F98 rat glioma cells and F98 rat glioma-bearing brain tumor models. For the in vivo BNCT study, we used boronophenylalanine (BPA), which is currently used in clinical BNCT studies, via intravenous administration (i.v.) and/or used TPFC via convection-enhanced delivery (CED), a method for local drug infusion directly into the brain. In the in vitro PDT study, the cell surviving fraction following laser irradiation (9 J/cm(2) ) was 0.035 whereas in the in vitro BNCT study, the cell surviving fraction following neutron irradiation (thermal neutron = 1.73 × 10(12) n/cm(2) ) was 0.04. In the in vivo BNCT study, the median survival time following concomitant administration of BPA (i.v.) and TPFC (CED) was 42 days (95% confidence interval; 37-43 days).

Synthesis and in vitro evaluation of BBB permeability, tumor cell uptake, and cytotoxicity of a series of carboranylporphyrin conjugates.

A series of tri[(p-carboranylmethylthio)tetrafluorophenyl]porphyrin conjugates of linear and branched polyamines, glucose, arginine, tri(ethylene glycol), and Tyr-D-Arg-Phe-ß-Ala (YRFA) peptide were synthesized. These conjugates were investigated for their BBB permeability in human hCMEC/D3 brain endothelial cells, and their cytotoxicity and uptake were assessed using human glioma T98G cells. For comparison purposes, a symmetric tetra[(p-carboranylmethylthio)tetrafluorophenyl]porphyrin was also synthesized, and its crystal structure was obtained. All porphyrin conjugates show low dark cytotoxicity (IC50>400 µM) and low phototoxicity (IC50>100 µM at 1.5 J/cm2) toward T98G cells. All conjugates were efficiently taken up by T98G cells, particularly the cationic polyamine and arginine conjugates, and were localized in multiple cellular organelles, including mitochondria and lysosomes. All compounds showed relatively low in vitro BBB permeability compared with that of lucifer yellow because of their higher molecular weight, hydrophobicity, and tendency for aggregation in solution. Within this series, the branched polyamine and YRFA conjugates showed the highest permeability coefficient, whereas the glucose conjugate showed the lowest permeability coefficient.

Synthesis, spectroscopic, and cellular properties of α-pegylated cis-A2B2- and A3B-types ZnPcs.

A series of pegylated cis-A2B2- or A3B-type ZnPcs, substituted on the α-positions with tri(ethylene glycol) and hydroxyl groups, were synthesized from a new bis-phthalonitrile. A clamshell-type bis-phthalocyanine was also obtained as a byproduct. The hydroxyl group of one ZnPc was alkylated with 3-dimethylaminopropyl chloride to afford a pegylated ZnPc functionalized with an amine group. All mononuclear ZnPcs were soluble in polar organic solvents, showed intense Q absorptions in DMF, and had fluorescence quantum yields in the range 0.10-0.23. The clamshell-type bis-phthalocyanine adopts mainly open shell conformations in DMF, and closed clamshell conformations in chloroform. All ZnPcs were highly phototoxic to human carcinoma HEp2 cells, particularly the amino-ZnPc mainly protonated under physiological conditions, which showed the highest phototoxicity (IC50 = 0.5 µM at 1.5 J/cm2) and dark cytotoxicity (IC50 = 22 µM), in part due to its high cellular uptake. The ZnPcs localized in multiple organelles, including mitochondria, lysosomes, Golgi and ER.

Spectroscopic, computational modeling and cytotoxicity of a series of meso-phenyl and meso-thienyl-BODIPYs.

A series of twenty-two BODIPY compounds were synthesized, containing various meso-phenyl and meso-thienyl groups, and their spectroscopic and structural properties were investigated using both experimental and computational methods. Further functionalization of the BODIPY framework via iodination at the 2,6-pyrrolic positions was explored in order to determine the effect of these heavy atoms on the photophysical and cytotoxicity of the meso-aryl-BODIPYs. BODIPYs bearing meso-thienyl substituents showed the largest red-shifted absorptions and emissions and reduced fluorescence quantum yields. The phototoxicity of the BODIPYs in human carcinoma HEp2 cells depends on both the presence of iodines and the nature of the meso-aryl groups. Six of the eleven 2,6-diiodo-BODIPYs investigated showed at least a sevenfold enhancement in phototoxicity (IC50 = 3.5-28 µM at 1.5 J/cm(2)) compared with the non-iodinated BODIPYs, while the others showed no cytotoxicity, while their singlet oxygen quantum yields ranged from 0.02 to 0.76. Among the series investigated, BODIPYs 2a and 4a bearing electron-donating meso-dimethoxyphenyl substituents showed the highest phototoxicity and dark/phototoxicity ratio, and are therefore the most promising for application in PDT.

Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain.

Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing SKBR-3 and BT-474 cells. Furthermore, cellular uptake of conjugate of BODIPY was studied in the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the conjugate. Inhibition of protein-protein interaction and downstream signaling effect of compound 5-1 was also studied by proximity ligation assay and Western blot analysis. Results suggested that compound 5-1 inhibit protein-protein interactions of HER2-HER3 and phosphorylation of HER2 in a time-dependent manner.

Aluminum, gallium, germanium, copper, and phosphorus complexes of meso-triaryltetrabenzocorrole.

5,10,15-Triaryltetrabenzocorrole complexes of aluminum, gallium, germanium, and phosphorus were synthesized by coordination of these metal ions in the preformed triaryltetrabenzocorrole macrocycle, opening a way to the investigation of different metal complexes. The UV-vis spectra of these derivatives exhibit a red shift and broadening of all absorption bands because of the π-extended aromatic system and distortion of the molecular framework. The electrochemical and photophysical behaviors of the free base and the metal complexes of meso-triaryltetrabenzocorrole were investigated and characterized.

Synthesis and cellular studies of polyamine conjugates of a mercaptomethyl-carboranylporphyrin.

Seven polyamine conjugates of a tri(p-carboranylmethylthio)tetrafluorophenylporphyrin were prepared in high yields by sequential substitution of the p-phenyl fluoride of tetrakis(pentafluorophenyl)porphyrin (TPPF), and investigated as boron delivery agents for boron neutron capture therapy (BNCT). The polyamines used were derivatives of the natural-occurring spermine with different lengths of the carbon chains, terminal primary amine groups and, in two of the conjugates, additional aminoethyl moieties. A tri(polyethylene glycol) conjugate was also synthesized for comparison purposes. The polyamine conjugates showed low dark cytotoxicity (IC(50) >400 µM) and low phototoxicity (IC(50) >40 µM at 1.5 J/cm(2)). All polyamine conjugates, with one exception, showed higher uptake into human glioma T98G cells (up to 12-fold) than the PEG conjugate, and localized preferentially in the cell ER, Golgi and the lysosomes. Our results show that spermine derivatives can serve as effective carriers of boronated porphyrins for the BNCT of tumors.

Synthesis and biological investigations of a ZnPc-antiCEA bioconjugate for imaging of colorectal cancer.

Two zinc(II) phthalocyanines (ZnPcs) were conjugated with a monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA), using an in situ activated carboxylic acid on the ZnPcs. The bioconjugate with the highest ZnPc/MAb ratio of 3 was investigated in vitro for its ability to target and fluorescently label human colorectal HT-29 cells. The ZnPc-CEA MAb 2 was observed to efficiently target HT-29 cells, about 37 times more than unconjugated ZnPc. Furthermore, in the presence of a 4-fold excess of unlabelled anti-CEA antibody, the fluorescence signal of 2 was reduced by ~90% showing that the targeting is CEA-mediated. These studies further confirm the high specificity of Pc-antibody conjugates for antigens over-expressed on tumor cells and warrant further investigations of these immunoconjugates and their derivatives for imaging of colorectal cancer.

Syntheses and Photodynamic Activity of Pegylated Cationic Zn(II)-Phthalocyanines in HEp2 Cells.

Di-cationic Zn(II)-phthalocyanines (ZnPcs) are promising photosensitizers for the photodynamic therapy (PDT) of cancers and for photoinactivation of viruses and bacteria. Pegylation of photosensitizers in general enhances their water-solubility and tumor cell accumulation. A series of pegylated di-cationic ZnPcs were synthesized from conjugation of a low molecular weight PEG group to a pre-formed Pc macrocycle, or by mixed condensation involving a pegylated phthalonitrile. All pegylated ZnPcs were highly soluble in polar organic solvents but were insoluble in water; they have intense Q absorptions centered at 680 nm and fluorescence quantum yields of ca. 0.2 in DMF. The non-pegylated di-cationic ZnPc 6a formed large aggregates, which were visualized by atomic force microscopy. The cytotoxicity, cellular uptake and subcellular distribution of all cationic ZnPcs were investigated in human carcinoma HEp2 cells. The most phototoxic compounds were found to be the α-substituted Pcs. Among these, Pcs 4a and 16a were the most effective (IC(50) ca. 10 µM at 1.5 J/cm(2)), in part due to the presence of a PEG group and the two positive charges in close proximity (separated by an ethylene group) in these macrocycles. The ß-substituted ZcPcs 6b and 4b accumulated the most within HEp2 cells but had low photocytoxicity (IC(50) > 100 µM at 1.5 J/cm(2)), possibly as a result of their lower electron density of the ring and more extended conformations compared with the α-substituted Pcs. The results show that the charge distribution about the Pc macrocycle and the intracellular localization of the cationic ZnPcs mainly determine their photodynamic activity.