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.     

THE PHOTOKILLING OF BLADDER CARCINOMA CELLS in vitro BY PHENOTHIAZINE DYES

The potential photodynamic therapy photosensitizers Methylene Blue, Azure C, Methylene Violet, Thionine, Methylene Green, Haematoporphyrin, Nile Blue A, chloroaluminium phthalocyanine and bis-aluminium phthalocyanine were examined for their photoeffects and dark toxicity against a human superficial bladder carcinoma cell-line. By examination of [3H]thymidine uptake into dye-treated cells after irradiation with a copper-vapour pumped dye laser, it was found that Methylene Blue was the most phototoxic and dark toxic of all the dyes tested, suggesting that the dye might be of some use as a topically applied photodrug for use in photodynamic therapy of superficial or early-recurring carcinomas.     

Photophysical properties of [60]fullerenes and phthalocyanines embedded in ordered mesoporous silica films annealed at various temperatures

The photophysical properties of fullerene and/or phthalocyanine dyes embedded in ordered mesoporous silica films and the influence of annealing temperature on the nature of the immobilized dye molecules has been investigated using photoluminescence (PL) and diffuse reflectance (DR) studies. The PL and DR studies show that fullerene (C60) and/or zinc phthalocyanine (ZnPc) molecules incorporated into transparent mesoporous silica films, via either sol-gel or grafting routes, exist predominantly in monomeric form. Careful choice of annealing temperature, between 25 and 225 degrees C, can further enhance monomeric dispersion. For C60-containing films, monomeric dispersion of fullerene was observed for annealing temperatures up to 175 degrees C for sol-gel derived films and 225 degrees C for grafted films. Both sol-gel and grafted ZnPc-containing films showed evidence of monodispersed phthalocyanine for annealing temperatures up to 225 degrees C. In general, annealing temperatures in the range 125-175 degrees C were found to yield optimal monodispersion of the dye molecules. When both C60 and ZnPc were incorporated into the silica films, no evidence of interaction between the dyes, i.e., charge-transfer transitions or the formation of fullerene/phthalocyanine charge-transfer complexes, was observed. This suggests that embedded fullerene and phthalocyanine molecules may be used for the preparation of solid-state optical limiters, based on reverse saturable absorption, where monomeric dispersion of the dye molecules is important.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES—VI. PHOTOOXIDATION OF L-TRYPTOPHAN BY SELECTIVELY SULFONATED GALLIUM PHTHALOCYANINES: SINGLET OXYGEN YIELDS AND EFFECT OF AGGREGATION

Abstract— The photosensitized oxidation of L-tryptophan by gallium phthalocyanines sulfonated to different degrees is studied as a function of both substrate and sensitizer concentrations in water and 50% MeOH-H₂O solutions. The maximum quantum yield of singlet oxygen was found to be nearly 0.5 for all sulfonated gallium complexes. The effect of adding sulfonate groups in the phthalocyanine backbone is to change the tendency of dye molecules to dimerize or aggregate in a particular solvent. A shift in the chemical equilibrium away from the monomeric state, which occurs at high dye concentrations and at lower degrees of dye sulfonation, results in a reduced photochemical yield. The variation of quantum yields in different solvent systems and at several wavelengths is similarly accounted for by the fraction of light absorbed by the productive monomer state.     

DISTRIBUTION AND ELIMINATION OF PHOTOFRIN II IN MICE

The distribution and elimination of [14C]PII, the radioisotopically-labeled equivalent of the mixture of porphyrins known as Photofrin II used in the photodynamic treatment of solid tumors, were determined in tumor-free and SMT-F tumor-bearing DBA/2 Ha-DD mice. Following i.p. injection, drug was absorbed from the peritoneum with a half-life of about 1 h; elimination from plasma was rapid, declining about 1.4 logs in concentration over 48 h following i.v. administration. However, some [14C]-activity was still detectable after 75 days. Normal tissues take up the drug within about 7.5 h after administration, with peak concentrations distributed as follows: liver, adrenal gland, urinary bladder greater than pancreas, kidney, spleen greater than stomach, bone, lung, heart greater than muscle much greater than brain. Only skeletal muscle, brain, and skin located contralaterally to subcutaneously implanted SMT-F tumors had peak [14C]-activities lower than tumor tissue; skin overlying SMT-F tumors showed concentrations not significantly different (P greater than 0.3) from tumor. After 75 days all tissues examined retained some fraction of [14C]-activity, ranging from 16% for kidney to 61% for spleen, of the initial peak tissue levels. The primary route of elimination of Photofrin II was through the bile-gut pathway, with greater than 59% of the administered [14C]-activity recovered in the feces, and only about 6% in the urine, over 192 h. HPLC analyses of fecal extracts showed that mostly monomeric and other low molecular weight porphyrin components of Photofrin II were eliminated. The higher molecular weight oligomeric fractions of Photofrin II were retained in liver and spleen up to 14 days after injection.     

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.     

Synthesis of fluorescent rhodamine dyes using an extension of the Heck reaction

Both benzo[b]thiophene and indole containing rhodamine dyes were synthesized according to a new pathway requiring a Heck-type coupling and a Pictet-Spengler reaction. The electron-rich indolic dye was shown to have noteworthy absorption and emission maximum.     

Calix[4]arene-functionalized naphthalene and perylene imide dyes

[reaction: see text] Calix[4]arenes bearing one, two, or four 1,8-naphthyl imide groups at the wide rim and bis-calix[4]arenes connected via perylene-bisimide dye spacers have been synthesized. The low-temperature NMR spectrum of the tetranaphthylimide suggests, in agreement with a crystal structure, a C2-symmetrical pinched cone conformation stabilized via face-to-face pi-pi interactions between opposite naphthylimide groups. UV-vis and fluorescence studies have been carried out for the perylene bis-calix[4]arene dyes.     

PHARMACOKINETIC AND TISSUE DISTRIBUTION STUDIES OF THE PHOTOSENSITIZER bis(DI-ISOBUTYLOCTADECYLSILOXY)SILICON 2,3-NAPHTHALOCYANINE (isoBOSINC) IN NORMAL AND TUMOR-BEARING RATS

Abstract Bis (di-isobutyl octadecylsiloxy)silicon 2,3-naphthalocyanine ( iso BOSINC) is a representative of a group of naphthalocyanine derivatives with spectral and photophysical properties that make them attractive candidates for photodynamic therapy (PDT). Tissue distributions were studied in normal and in tumor-bearing rats as a function of time following intravenous injection of iso BOSINC as a suspension in 10% Tween 80 in saline. The dose studied was 0.25 mg/kg of body weight. The compound iso BOSINC was isolated from several tissues and organs, as well as tumors and peritumoral muscles and skin, and quantitated by a high-performance liquid chromatographic technique. The tumor model, an N -(4-[5-nitro-2-furyl)-2-thiazolyl)formamide (FANFT)-induced urothelial cell carcinoma, was transplanted into the hind legs of Fischer 344 rats. The dye was retained in tumors at higher concentrations than in all tissues and organs examined, except for spleen and liver. The highest concentration ratio of dye in tumor versus peritumoral muscle (24.5) occurred 9 h after injection. Serum clearance of iso BOSINC showed similar kinetic behavior for both groups of rats, with a t_1/2 of elimination of ∼ 10 h. At 7 and 14 days postinjection, the levels of dye found in testes were generally higher than in most other tissues, except spleen and liver. Concentrations of iso BOSINC were either very low or not detectable in rat brain. Trace amounts of the dye were excreted in the urine, and by day 14 approximately 17% of the dose was accounted for in the feces. The significant levels of the drug in tumors, as well as the excellent ratios of tumor-to-muscle concentration observed, have promising implications for PDT of tumors.     

Structure-Photodynamic Activity Relationships of a Series of 4-Substituted Zinc Phthalocyanines

Abstract— Radioiodinated zinc phthalocyanine including [¹²⁵I]ZnPcI₄ and differently sulfonated [⁶⁵Zn]ZnPcS (ZnPcS₄, ZnPcS₃, ZnPcS₂ and ZnPcS_1.75, a mixture of adjacent di and 25% mono) were prepared in order to study cell uptake and release kinetics in EMT-6 cells. The same compounds were evaluated for their in vitro phototoxicity and the biological parameters were compared to partition coefficients to arrive at quantitative structure-activity relationships (QSAR). At 1 μM in 1% serum, at 37°C, all dyes showed rapid cell uptake during the first hour followed by a slow accumulation phase. After 24 h, the highest cellular concentration was observed with the lipophilic ZnPcI₄, followed by the amphiphilic ZnPcS₂ and ZnPcS_1.75. The hydrophilic ZnPcS₄ and ZnPcS₃ showed lower uptake. Dye release from dyeloaded cells during incubation in dye-free medium could reach up to 60% and was shown to depend mainly on the amount of drug incorporated rather than the type of compound. These results suggest that care should be taken in interpreting dye toxicity data, which involve in vitro cell manipulations in dye-free medium, particularly during in vitro/in vivo protocols. The EMT-6 cell survival after 1 h or 24 h incubation with 1 μM dye in 1% serum followed by exposure to red light was assessed by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. Photocytotoxicities correlated inversely with the tendencies of the dyes to aggregate. Increased dye uptake by the cells also correlated with their activities, except for the lipophilic ZnPcI₄, which showed the highest cell uptake but little phototoxicity. The QSAR between phototoxicity and the log of the partition coefficients (phosphate-buffered saline and n-octanol) gave a parabola with optimal partition values corresponding to the adjacent sulfonated ZnPcS₂.     

EPR investigation of the origin of the magnetic field effect on the dimerization of aluminum(III) and gallium(III) phthalocyanine chlorides in film structures

The origin of the magnetic field effect on the dimerization of aluminum(III) and gallium(III) phthalocyanine chloride films is revealed. The EPR technique is employed to demonstrate that the dimerization of the above coordination compounds occurs via a free radical pathway with the formation of phthalocyanine and OH radicals, thus resulting in the appearance of the magnetic moment in the studied compounds.     

Fluorescence lifetime properties of near-infrared cyanine dyes in relation to their structures

Structurally diverse near-infrared (NIR) absorbing polymethine dyes were prepared and their fluorescence lifetimes (FLT) were evaluated in relation to their structural features. Comparative FLT analysis based on the modification of methine chain length and heterocyclic system showed that indolium or benzo[e]indolium heptamethine dyes exhibited longer FLT than the benzo[c,d]indolium trimethine dye. Modification of heterocyclic system alone with an intact chain length showed that indolium-based heptamethine dyes showed approximately 30% longer FLT than the benzo[e]indolium-based dyes. In general, the FLT of polymethine dyes increased from polar to non-polar solvents. In addition, correlation study between the theoretical and the experimental FLT for indocyanine green (ICG) suggests that the lack of structural rigidity for these cyanine dyes is primarily responsible for the loss of the excited state energy via non-radiative pathway.     

THE PREPARATION OF RADIOLABELED PORPHYRINS and THEIR USE IN STUDIES OF PHOTODYNAMIC THERAPY

Abstract— We tested water-soluble sulfonated phthalocyanine and three metal chelate derivatives for their tumoricidal effect on the EMT-6 mammary tumor in mice exposed to red light. The metal-free sulfophthalocyanine had little effect, whereas the aluminum complex and the lower sulfonated fraction of the gallium complex exhibited tumoricidal activity similar to hematoporphyrin-based photosensitizer (Photofrin II). The higher sulfonated fractions of the gallium complex were less active as compared to the lower sulfonated fraction. The cerium complex was the most active sensitizer in terms of dye and light doses required to induce tumor necrosis and cure but also showed the highest phototoxicity towards healthy skin. These results suggest that sulfonated phthalocyanines will offer a new alternative in photodynamic therapy of light-accessible neoplasms.     

Template synthesis of cyanine dye H-aggregates on nanostructured [6,6]-phenyl C61-butyric acid methyl ester substrates

A novel and general method for the synthesis of H-aggregates is presented. H-aggregates can be precipitated from solutions of cyanine dyes in nanostructured [6,6]-phenyl C(61)-butyric acid methyl ester (PCBM) template films. H-aggregates synthesized that way have a narrow line shape width comparable to the smallest H-bands reported for thin films so far. We demonstrate that H-aggregates can be induced in different cyanine dyes (trimethine-, pentamethine- and heptamethine dyes). Aggregation follows a universal pathway, the concentration dependence of aggregation for different dyes can be scaled to fall onto one master curve. According to our model, H-aggregates have the form of 3D-crystals that form within cavities of the template. The template substrate is fabricated by means of a phase separation process of PCBM with a specific cyanine dye and consecutive selective dissolution of the dye. The dimensions of the template cavities have a major influence on aggregation.     

Preparation of colored latex containing oil-soluble dyes with high dye content by mini-emulsion polymerization

Polymer particles containing oil-soluble dyes (colored latex particles) were prepared by mini-emulsion polymerization. Copper phthalocyanine dyes and styryl dyes were used as oil-soluble dyes. Highly hydrophobic dyes played the role of hydrophobe by themselves and enabled the full incorporation of dyes in the latex without additional hydrophobes. Two phthalocyanine dyes having similar color were blended in a mini-emulsion polymerization so that the resulting colored latex showed enough strong color depth as a colorant. Colored latexes with high dye content (more than 30 wt% for phthalocyanine dye system and 40 wt% for styryl one) and with particle size less than 100 nm were obtained.     

Concomitant increase in putrescine incorporation with transferrin uptake into rat reticulocytes.

Concomitant increase in putrescine incorporation with transferrin uptake into rat reticulocytes was found. [14C]Putrescine incorporation occurred in the presence of transferrin at 37 degrees C. The subcellular distribution of incorporated [14C]putrescine showed that the incorporation in the plasma membrane time dependently increased. Both 125I-labeled transferrin uptake and [14C]putrescine incorporation decreased when ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid was added, while it increased with the addition of A23187.     

In viva PHOTODYNAMIC EFFECTS OF PHTHALOCYANINES IN A SKIN-FOLD OBSERVATION CHAMBER MODEL: ROLE OF CENTRAL METAL ION AND DEGREE OF SULFONATION

Six sulfonated metallophthalocyanines, chelated with either aluminum or zinc and sulfonated to different degrees, were studied in vivo for their photodynamic activity in a rat skin-fold chamber model. The chamber, located on the back of female WAG/Rij rats, contained a syngeneic mammary carcinoma implanted into a layer of subcutaneous tissue. Twenty-four hours after intravenous administration of 2.5 μmol/kg of one of the dyes, the chambers received a treatment light dose of 600 J/cm² with monochromatic light of 675 nm at a power density of 100 mW/ cm². During light delivery and up to a period of 7 days after treatment, vascular effects of tumor and normal tissue were scored. Tumor cell viability was determined by histology and by reimplantation of the chamber contents into the skin of the same animal, either 2 h after treatment or after the 7 day observation period. Vascular effects of both tumor and subcutaneous tissue were strongest with dyes with the lowest degree of sulfonation and decreased with increasing degree of sulfonation. Tumor regrowth did not occur with aluminum phthalocyanine mono- and disulfonate and with zinc phthalocyanine monosulfonate. With the protocol that was used, complete necrosis without recovery was only observed when reimplantation took place at the end of the 7 day follow-up period. Reimplantation 2 h after treatment always resulted in tumor regrowth. At this interval, the presence of viable tumor cells was confirmed histologically. In general tumor tissue vasculature was more susceptible to photodynamic damage than vasculature of the normal tissue. The effect on the circulation of both tumor and normal tissue increased with decreasing degree of sulfonation. Based on this study, the photodynamic effects using the six sulfonated metallophthalocyanines on the vasculature can be ranked from high to low as: AlPcS₂= ZnPcS₁ > AIPcS₁ > AIPcS₄ > ZnPcS₂ > ZnPcS₄.     

Design and synthesis of novel antimicrobial acyclic and heterocyclic dyes and their precursors for dyeing and/or textile finishing based on 2-N-acylamino-4,5,6,7-tetrahydro-benzo[b]thiophene systems

A series of novel polyfunctionalized acyclic and heterocyclic dye precursors and their respective azo (hydrazone) counterpart dyes and dye precursors based on conjugate enaminones and/or enaminonitrile moieties were synthesized. The dyes and their precursors are based on 2-cyano-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-acetamide, 2-ethoxycarbonyl-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-acetamide or 2-phenylcarbamoyl-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-acetamide systems as precursors. The latter compounds were used to synthesize polyfunctional thiophene-, thiazole-, pyrazole, pyridine-, pyrimidine-, oxazine-, as well as acyclic moieties. The dyes and dye precursors were characterized by elemental analysis and spectral methods. All dyes and their precursors were screened in vitro and evaluated for both their antibacterial and antifungal activities. MIC data of the novel dye systems and their respective precursors showed significant antimicrobial activity against most tested organisms. Some compounds exhibited comparable or even higher efficiency than selected standards. Dyes were applied at 5% depth for disperse dyeing of nylon, acetate and polyester fabrics. Their spectral characteristics and fastness properties were measured and evaluated.     

SULFOPHTHALOCYANINES FOR PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD PRODUCTS: EFFECT OF STRUCTURAL MODIFICATIONS

Abstract— Transmission of infectious diseases through blood transfusions is well known. Ultraviolet irradiation, solvents and detergents provide a means of sterilizing noncellular blood components. However these harsh methods are not applicable to cellular blood products. Recently, attempts have been made to sterilize biological fluids using photodynamic treatment and phthalocyanine (Pc) dyes have been advanced as photosensitizers for this purpose. We have evaluated a series of water-soluble Pc, chelated with different central metal ions, substituted to different degrees with sulfonato and r-butyl groups, for their effectiveness to reduce virus infectivity in red blood cell suspensions. Vaccinia virus cytopathogenicity was determined by endpoint serial dilutions in the CV-1 cell line. Anti-viral activity increased with the central metal ion in the following order: Ga(III) < Al(III) < Zn(II), and varied inversely with the degree of sulfonation. Furthermore, addition of a t -butyl group onto the trisulfonated dyes (PcS₃[ t -Bul) resulted in a 5–40-fold increase in anti-viral potency, suggesting that amphiphilicity enhances the photodynamic activity of the dye. Strong anti-viral photosensitizing properties cannot be the sole selection criterion. Of equal importance is the preservation of blood component integrity. Accordingly, the photohemolytic activity of the dyes was evaluated using the rate of hemolysis as a parameter and a toxicity index was defined. Among the most active dyes, the AlPcS₃( t -Bu) complex exhibited the most favorable anti-viral properties combined with a low toxicity index. Our results suggest that trisulfophthalocyanines, bearing an additional t -butyl group to enhance amphiphilicity, are particularly promising dyes for photodynamic blood sterilization.     

Cationic Zr-based metal-organic framework via post-synthetic alkylation for selective adsorption and separation of anionic dyes

Adsorption and separation of toxic organic dyes are of great importance in wastewater treatment and dye recycling. In this work, cationic metal-organic framework MIL-140C–2NMe⁺ with triangular hydrophobic channels was prepared in which methyl groups were added to the pyridyl sites of the ligand [2,2'-bipyridine]-5,5'-dicarboxylic acid (H₂bpydc) via post-synthetic alkylation reaction. MIL-140C–2NMe⁺ can be used as an efficient adsorbent for the selective adsorption and separation of anionic dyes in the aqueous mixture of cationic/anionic dyes. Specifically, the adsorption capacities of MIL-140C–2NMe⁺ for anionic methyl orange can reach 310 mg/g in 10 min. With a facile doctor-blading process, we have also polymerized the MIL-140C–2NMe⁺ nanocrystals and polyvinylidene fluoride (PVDF) polymer to fabricate a flexible and self-supporting mixed matrix membrane (MMM), which can selectively capture and separate the anionic organic dyes from the binary dye mixtures.