THE EFFECTS OF PHOTODYNAMIC THERAPY USING DIFFERENTLY SUBSTITUTED ZINC PHTHALOCYANINES ON VESSEL CONSTRICTION, VESSEL LEAKAGE AND TUMOR RESPONSE

Abstract— The effects of four different zinc phthalocyanines were studied during and after photodynamic therapy (PDT). Measurements of vessel constriction, vessel leakage, tumor interstitial pressure, eicosanoid release, and tumor response of chondrosarcoma were made in Sprague-Dawley rats. Animals were injected intravenously with 1 μmol/ kg of mono-, di-, or tetrasulfonated zinc phthalocyanine, or 1 μmol/kg of a zinc phthalocyanine substituted with four tertiary butyl groups. Tissues were exposed to 400 J/cm² 670 nm light 24 h after photosensitizer injection. An additional group of animals was given indomethacin before treatment. The use of the monosulfonated and tertiary butyl substituted zinc phthalocyanines in PDT caused the release of specific eicosanoids, caused vessel constriction, and induced venule leakage and increases in tumor interstitial pressure. Tumor cures of 27% and 7% were observed. Photodynamic therapy using the disulfonated zinc phthalocyanine did not induce vessel constriction or the release ofeicosanoids, however; tumor cure was 43%. The use of thc tetrasulfonated zinc phthalocyanine caused intermediate effects between the mono- and disulfonated compounds. The administration of indornethacin to animals completely inhibited the effects of PDT using the monosulfonated compound but had minimal effects on treatment using the disulfonated compound. This suggests that the monosulfonated and disulfonated compounds act by different mechanisms of destruction.     

SULFONATED PHTHALIMIDOMETHYL ALUMINUM PHTHALOCYANINE: THE EFFECT OF HYDROPHOBIC SUBSTITUENTS ON THE in vitro PHOTOTOXICITY OF PHTHALOCYANINES

The photocytotoxicity of sulfonated phthalimidomethyl aluminum phthalocyanine, a more hydrophobic photosensitizer as compared to phthalocyanine substituted with sulfonate groups only, was investigated. Inclusion of 1-2 phthalimidomethyl groups into disulfonated aluminum phthalocyanine, resulted in increased partition coefficients between n-octanol and water, and a six-fold increase in both cellular uptake and photocytotoxicity towards Chinese hamster lung fibroblast cells (line V-79). Reducing the number of phthalimidomethyl groups, or increasing the degree of sulfonation, lead to a decrease in the partition coefficient, cellular uptake, and phototoxicity. The quantum yield of singlet oxygen was comparable for all dyes tested in this series, indicating that no significant change in this photophysical parameter resulted from phthalimidomethylation. These results suggest that the addition of 1-2 phthalimidomethyl groups to disulfonated aluminum phthalocyanine improves cellular uptake, but, as the relative efficiency of cell killing was not effected, the intracellular distribution on photosensitive molecules may not be modified.     

Immobilized phthalocyanines of magnesium,aluminum, and zinc in photodynamic treatment of mesenchymal stromal cells

Phthalocyanines of magnesium, aluminum, and zinc immobilized on nano-sized silica and poly-N-vinylpyrrolidone in aqueous solutions were synthesized. Photochemical activity of the immobilized metal complexes was assessed by generation of singlet oxygen. Nontoxic concentrations of the new photosensitizers were determined in vitro. A comparative analysis of the efficiency of photodynamic therapy (PDT) was performed for immobilized phthalocyanines using mesenchymal stromal cells as a cell model. Aluminum phthalocyanine immobilized on nano-sized silica displayed the highest cell tropism. Irradiation of phthalocyanine-loaded cells resulted in generation of active singlet oxygen and subsequent apoptotic cell death. The use of immobilized phthalocyanines allowed decreasing the effective concentration (dose) of photosensitizer and enhancing the PDT cytotoxicity.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—VIII. CELLULAR DISTRIBUTION INV–79 CHINESE HAMSTER CELLS AND PHOTOTOXICITY OF SELECTIVELY SULFONATED ALUMINUM PHTHALOCYANINES

Abstract— Water soluble chloro aluminum phthalocyanines sulfonated to different degrees are studied for phototoxicity and cellular distribution inV–79 Chinese hamster cells. The more hydrophobic disulfonated dyes, with sulfonate substituents on adjacent benzyl groups of the phthalocyanine ring structure, exhibited the best cell penetrating properties and the highest phototoxicity. Fluorescence microscopy revealed that the dye was uniformly distributed in the cytoplasm but absent in the nucleus. The greater cell membrane penetrating properties of the lower as compared to the higher sulfonated dyes are attributed to the amphiphilic nature of the former.     

Anticancer Photodynamic Therapy Properties of Sulfur‐Doped Graphene Quantum Dot and Methylene Blue Preparations in MCF‐7 Breast Cancer Cell Culture

Photodynamic therapy (PDT) is a field with many applications including chemotherapy. Graphene quantum dots (GQDs) exhibit a variety of unique properties and can be used in PDT to generate singlet oxygen that destroys pathogenic bacteria and cancer cells. The PDT agent, methylene blue (MB), like GQDs, has been successfully exploited to destroy bacteria and cancer cells by increasing reactive oxygen species generation. Recently, combinations of GQDs and MB have been shown to destroy pathogenic bacteria via increased singlet oxygen generation. Here, we performed a spectrophotometric assay to detect and measure the uptake of GQDs, MB and several GQD‐MB combinations in MCF‐7 breast cancer cells. Then, we used a cell counting method to evaluate the cytotoxicity of GQDs, MB and a 1:1 GQD:MB preparation. Singlet oxygen generation in cells was then detected and measured using singlet oxygen sensor green. The dye, H₂DCFDA, was used to measure reactive oxygen species production. We found that GQD and MB uptake into MCF‐7 cells occurred, but that MB, followed by 1:1 GQD:MB, caused superior cytotoxicity and singlet oxygen and reactive oxygen species generation. Our results suggest that methylene blue's effect against MCF‐7 cells is not potentiated by GQDs, either in light or dark conditions.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES-XL PHOTOTOXICITY OF SULFONATED ALUMINUM NAPHTHALOCYANINES TOWARDS V-79 CHINESE HAMSTER CELLS

The phototoxicity of sulfonated aluminum naphthalocyanines towards V-79 Chinese hamster cells is investigated. The disulfonated naphthalocyanine exhibits similar photostability, but better cell penetrating properties than the tetrasulfonated dyes. The capacity of the naphthalocyanines to generate singlet oxygen is comparable to that of the corresponding phthalocyanines. However, in contrast to the phthalocyanine dyes, the sulfonated aluminum naphthalocyanines show very little phototoxicity towards the V-79 cells, suggesting close association with non-vital cell constituents or extensive formation of photoinactive adducts and aggregates.     

A Phthalocyanine–Peptide Conjugate with High In Vitro Photodynamic Activity and Enhanced In Vivo Tumor‐Retention Property

A novel zinc(II) phthalocyanine conjugated with a short peptide with a nuclear localization sequence, Gly‐Gly‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐Val, was synthesized by click chemistry and a standard Fmoc solid‐phase peptide synthesis protocol. The conjugate was purified by HPLC and characterized with UV/Vis and high‐resolution mass spectroscopic methods. Both this compound and its non‐peptide‐conjugated analogue are essentially non‐aggregated in N,N‐dimethylformamide and can generate singlet oxygen effectively with quantum yields (Φ_Δ) of 0.84 and 0.81, respectively, relative to unsubstituted zinc(II) phthalocyanine (Φ_Δ=0.56). Conjugation of the peptide sequence, however, can enhance the cellular uptake, efficiency in generating intracellular reactive oxygen species, and photocytotoxicity of the phthalocyanine‐based photosensitizer against HT29 human colorectal carcinoma cells. The IC₅₀ value of the conjugate is as low as 0.21 μM . In addition, the conjugate shows an enhanced tumor‐retention property in tumor‐bearing nude mice. After 72 h post‐injection, the dye concentration in the tumor was significantly higher than that in other organs. The results suggest that this phthalocyanine–peptide conjugate is a highly promising photosensitizer for photodynamic therapy.     

A comparative study of the cellular uptake and photodynamic efficacy of three novel zinc phthalocyanines of differing charge

Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF-1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly improved activity over PHP and m-THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m-THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low-level light exposure, and this was charge dependent. The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zinc [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low-level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.     

Comparison between non-peripherally and peripherally tetra-substituted zinc (II) phthalocyanines as photosensitizers: Synthesis,spectroscopic, photochemical and photobiological properties

A series of zinc phthalocyanines tetra-α-substituted with 4-(butoxycarbonyl) phenoxy groups (1a) or 4-carboxylphenoxy groups (2a) or 4-(2-carboxyl-ethyl)phenoxy groups (3a), and the corresponding tetra-β-substituted (1–3b) analogues, have been synthesized and characterized. The effects of the position of substituents at the phthalocyanine skeleton on their spectroscopic, photochemical and photobiological properties have been revealed. When compared with the tetra-β-substituted phthalocyanines, the corresponding tetra-α-substituted analogues exhibit a less aggregating trend in the cellular growth medium, a slightly higher singlet oxygen quantum yield and higher photo-stability in DMF, and a comparable cellular uptake. As a result, the tetra-α-substituted zinc phthalocyanines exhibit a higher photocytotoxicity toward MGC803 human gastric carcinoma cells than the tetra-β-substituted counterparts. Among all these compounds, phthalocyanine 2a shows the highest photodynamic activity, which may mainly be due to its non-aggregated nature in cellular culture medium and high cellular uptake.     

Synthesis,Supramolecular Behavior,and In Vitro Photodynamic Activities of Novel Zinc(II) Phthalocyanines “Side‐strapped” with Crown Ether Bridges

Two new tetra‐ or di‐α‐substituted zinc(II) phthalocyanines 5 and 6 have been prepared through a “side‐strapped” method. In the molecules, the adjacent benzene rings of the phthalocyanine core are linked at α‐position through a triethylene glycol bridge to form a hybrid aza‐/oxa‐crown ether. The tetra‐α‐substituted phthalocyanine 5 shows an eclipsed self‐assembly property in CH₂Cl₂ and the effect on the di‐α‐substituted analogue 6 is significantly weakened. Furthermore, the crown ethers of these compounds can selectively complex with Fe³⁺ or Cu²⁺ ion in DMF, leading to formation of J‐aggregated nano‐assemblies, which can be disaggregated in the presence of some organic or inorganic ligands, such as triethylamine, tetramethylethylenediamine, CH₃COO^?, or OH^?. In addition, both compounds are efficient singlet oxygen generators with the singlet oxygen quantum yields (Φ_Δ) of 0.54‐0.74 in DMF relative to unsubstituted zinc(II) phthalocyanine (Φ_Δ=0.56). They exhibit photodynamic activities toward HepG2 human hepatocarcinoma cells, but the compound 6 , which has more than 40‐fold lower IC₅₀ value (0.08 μM ) compared to the analogue 5 (IC₅₀=3.31 μM ), shows remarkablely higher in vitro photocytotoxicity due to its significantly higher cellular uptake and singlet oxygen generation efficiency. The results suggest that these compounds can serve as promising multifunctional materials both in (opto)electronic field and photodynamic therapy.     

Highly Efficient Singlet Oxygen Generators Based on Ruthenium Phthalocyanines: Synthesis,Characterization and in vitro Evaluation for Photodynamic Therapy

The synthesis of ruthenium(II) phthalocyanines (RuPcs) endowed with one carbohydrate unit—that is, glucose, galactose and mannose—and a dimethylsulfoxide (DMSO) ligand at the two axial coordination sites, respectively, is described. Two series of compounds, one unsubstituted at the periphery, and the other one bearing eight PEG chains at the isoindole meta-positions, have been prepared. The presence of the axial DMSO unit significantly increases the phthalocyanine singlet oxygen quantum yields, related to other comparable RuPcs. The compounds have been evaluated for PDT treatment in bladder cancer cells. In vitro studies have revealed high phototoxicity for RuPcs unsubstituted at their periphery. The phototoxicity of PEG-substituted RuPcs has been considerably improved by repeated light irradiation. The choice of the axial carbohydrate introduced little differences in the cellular uptake for both series of photosensitizers, but the phototoxic effects were considerably higher for compounds bearing mannose units.     

Synthesis of a Photostable Near‐Infrared‐Absorbing Photosensitizer for Selective Photodamage to Cancer Cells

A new class of near‐infrared (NIR)‐absorptive (>900 nm) photosensitizer based on a phenothiazinium scaffold is reported. The stable solid compound, o‐DAP, the oxidative form of 3,7‐bis(4‐methylaminophenyl)‐10H‐phenothiazine, can generate reactive oxygen species (ROS, singlet oxygen and superoxide) under appropriate irradiation conditions. After biologically evaluating the intracellular uptake, localization, and phototoxicity of this compound, it was concluded that o‐DAP is photostable and a potential selective photodynamic therapy (PDT) agent under either NIR or white light irradiation because its photodamage is more efficient in cancer cells than in normal cells and is without significant dark toxicity. This is very rare for photosensitizers in PDT applications.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES-IX. PHOTOSENSITIZATION OFV–79 CHINESE HAMSTER CELLS ANDEMT–6 MOUSE MAMMARY TUMOR BY SELECTIVELY SULFONATED ZINC PHTHALOCYANINES

Abstract— Zinc phthalocyanines sulfonated to different degrees are tested for their ability to sensitizeV–79 Chinese hamster cells andEMT–6 mouse mammary tumors to red light. In vitro , the lower sulfonated derivatives were the most active with the exception of the poorly water-soluble monosulfonated dye. An isomeric mixture of tetrasulfonated derivatives obtained via direct sulfonation was ten times more active than the homogeneous tetrasulfo derivative prepared via the condensation of sulfophthalic acid. In vivo , the latter dye was completely inactive, whereas the remainder of the sulfonated preparations exhibited a similar structure-activity pattern as observed with theV–79 cells in vitro . The disulfonated zinc phthalocyanine showed the best tumoricidal activity in the series and also appeared to be a more efficient photosensitizer of cell inactivation and tumor cure than the aluminum or gallium complexes as well as hematoporphyrin derivative preparations. No significant differences in skin phototoxicity were observed among the various dyes.     

Probing Supramolecular Interactions between a Crown Ether Appended Zinc Phthalocyanine and an Ammonium Group Appended to a C60 Derivative

Self‐assembly driven by crown ether complexation of zinc phthalocyanines equipped with one 18‐crown‐6 moiety and fullerenes bearing an ammonium head group afforded a novel donor–acceptor hybrid. In reference experiments, fullerenes containing a Boc‐protected amine functionality have been probed. The circumvention of zinc phthalocyanine aggregation is important for the self‐assembly, which required the addition of pyridine. From absorption and fluorescence titration assays, which provided sound and unambiguous evidence for mutual interactions between the electron donor and the electron acceptor within the hybrids, association constants in the order of 8.0×10⁵ m ^?1 have been derived. The aforementioned is based on 1:1 stoichiometries, which have been independently confirmed by Job's plot measurements. In the excited state, which has been examined by transient absorption experiments, intermolecular charge separation evolves from the photoexcited zinc phthalocyanine to the fullerene subunit and leads to short‐lived charge‐separated states. Interestingly, photoexcitation of zinc phthalocyanine dimers/aggregates can also be followed by an intermolecular charge separation between vicinal phthalocyanines. These multicomponent supramolecular ensembles have also been shown by in‐depth electrospray ionization mass spectrometry (ESI‐MS) studies, giving rise to the formation and detection of a variety of non‐covalently linked species.     

Preparation and photodynamic activities of silicon(IV) phthalocyanines substituted with permethylated β-cyclodextrins

A series of silicon(IV) phthalocyanines linked to two permethylated β‐cyclodextrin moieties through different spacers at the axial positions have been synthesized and characterized. The effects of these spacers on the photophysical properties and in vitro photodynamic activities have also been examined. Having two bulky hydrophilic substituents, all of these compounds are soluble and essentially nonaggregated in DMF and even in aqueous media. The fluorescence and singlet oxygen quantum yields in DMF are lower for the analogue with the shortest separation between the amino group in the spacer and the phthalocyanine ring. The fluorescence quantum yield of this compound increases in water probably due to protonation of the amino group, which inhibits the reductive quenching process. This series of compounds also exhibit photocytotoxicity toward HT29 human colon adenocarcinoma and HepG2 human hepatocarcinoma cells with IC₅₀ values in the range of 0.04–1.32 μM . The analogue with an α,ω‐aminohydroxypentyl linker shows the highest potency, which can be ascribed to its high cellular uptake and high efficiency in generating intracellular reactive oxygen species. This compound also shows preferential localization in the lysosome, induces cell death mainly through apoptosis, and inhibits the growth of tumor in vivo. The results suggest that it is a promising photosensitizer for photodynamic therapy.     

Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells

Abstract Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.     

Photophysicochemical and fluorescence quenching studies of benzyloxyphenoxy-substituted zinc phthalocyanines

Photochemical and photophysical measurements were conducted on peripheral and non-peripheral tetrakis- and octakis(4-benzyloxyphenoxy)-substituted zinc phthalocyanines (1, 2 and 3). General trends are described for photodegradation, and fluorescence quantum yields, triplet lifetimes and triplet quantum yields as well as singlet oxygen quantum yields of these compounds in dimethylsulphoxide (DMSO) and toluene. The fluorescence of the complexes is quenched by benzoquinone (BQ), and fluorescence quenching properties are investigated in DMSO and toluene. The effects of the solvents on the photophysical and photochemical parameters of the zinc(II) phthalocyanines (1, 2 and 3) are also reported. Photophysical and photochemical properties of phthalocyanine complexes are very useful for PDT applications.     

Semiconductor quantum dots for photodynamic therapy

The applicability of semiconductor QDs in photodynamic therapy (PDT) was evaluated by studying the interaction between CdSe QDs with a known silicon phthalocyanine PDT photosensitizer, Pc4. The study revealed that the QDs could be used to sensitize the PDT agent through a fluorescence resonance energy transfer (FRET) mechanism, or interact directly with molecular oxygen via a triplet energy-transfer process (TET). Both mechanisms result in the generation of reactive singlet oxygen species that can be used for PDT cancer therapy.     

Glycosylated zinc(II) phthalocyanines as efficient photosensitisers for photodynamic therapy. Synthesis, photophysical properties and in vitro photodynamic activity

Treatment of 3- or 4-nitrophthalonitrile with 1,2:5,6-di-O-isopropylidene-alpha-d-glucofuranose or 1,2:3,4-di-O-isopropylidene-alpha-d-galactopyranose in the presence of K(2)CO(3) gave the corresponding glycosubstituted phthalonitriles. These precursors underwent self-cyclisation, or mixed-cyclisation with the unsubstituted phthalonitrile, to afford the tetra- or mono-glycosylated zinc(ii) phthalocyanines, respectively. As shown by absorption spectroscopy, these compounds were not significantly aggregated in organic solvents, giving a weak to moderate fluorescence emission. Upon irradiation these compounds could sensitise the formation of singlet oxygen in DMF, with quantum yields in the range of 0.40-0.66. The in vitro photodynamic activities of these compounds against HepG2 human hepatocarcinoma and HT29 human colon adenocarcinoma cells were also studied. The mono-glycosylated phthalocyanines exhibited significantly higher photocytotoxicity compared with the tetra-alpha-glycosylated analogues, having IC(50) values down to 0.9 muM. The tetra-beta-glycosylated counterparts were essentially inactive. The lower photocytotoxicities of the tetra-glycosylated phthalocyanines are in line with their lower cellular uptake and/or higher aggregation tendency as reflected by weaker intracellular fluorescence, and lower efficiency at generating intracellular reactive oxygen species. For the mono-glycosylated phthalocyanines, the higher uptake can be attributed to their hydrophilic saccharide units, which increase the amphiphilicity of the macrocycles.     

Phthalocyanine‐Assembled Nanodots as Photosensitizers for Highly Efficient Type I Photoreactions in Photodynamic Therapy

Owing to their unique, nanoscale related optical properties, nanostructures assembled from molecular photosensitizers (PSs) have interesting applications in phototheranostics. However, most nanostructured PS assemblies are super‐quenched, thus, preventing their use in photodynamic therapy (PDT). Although some of these materials undergo stimuli‐responsive disassembly, which leads to partial recovery of PDT activity, their therapeutic potentials are unsatisfactory owing to a limited ability to promote generation reactive oxygen species (ROS), especially via type I photoreactions (i.e., not by ¹O₂ generation). Herein we demonstrate that a new, nanostructured phthalocyanine assembly, NanoPcA, has the ability to promote highly efficient ROS generation via the type I mechanism. The results of antibacterial studies demonstrate that NanoPcA has potential PDT applications.