METALLOPURPURINS and LIGHT: EFFECT ON TRANSPLANTABLE RAT BLADDER TUMORS and MURINE SKIN

Purpurins are modified chlorins with photodynamic properties. Their strong absorption in the red region of the visible spectrum makes them candidates for use in photodynamic cancer therapy. A series of metal derivatives of the free base purpurins have been synthesized and shown to cause tumor necrosis in transplantable tumors when exposed to visible light. In the following set of experiments, the effects of two metallo-derivatives (tin and zinc) of two purpurins, octaethylpurpurin (NT2) and etiopurpurin (ET2), and light on the N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide transplantable tumors in Fischer CDF(F344)/CrlBr rats were studied. The photodynamic activity was assessed by a short term assay using tumor dry weight 12 days after purpurin-PDT as a criterion of response. From these experiments it appears that SnET2 greater than SnNT2 greater than ZnET2 greater than ZnNT2 in photodynamic activity. SnET2 was further characterized by attempting to determine the time interval after systemic injection at which maximum therapeutic effect occurred. These studies shown that 24 h after metallopurpurin injection was the optimum time for treatment of tumors with visible light. In a final set of experiments, the effect of solar light on the skin of hairless mice injected with SnET2 was found to be much less injurious than with hematoporphyrin derivative.     

TIN ETIOPURPURIN DICHLORIDE-SENSITIZED LIPID PHOTOOXIDATION OF ERYTHROCYTE MEMBRANES

Tin(IV) etiopurpurin dichloride (SnET2 x 2Cl) is a photosensitizer which has been shown to be an effective photodynamic agent for the treatment of transplantable animal tumors in vivo. The purpose of this study was to understand the effect of SnET2 x 2Cl on membrane lipid peroxidation. When erythrocyte membranes were exposed to visible light in the presence of SnET2 x 2Cl, lipid peroxidation was observed. An accumulation of lipid hydroperoxides and an increase in lipid fluorescence were also observed. Thin layer chromatography of lipid extracts from photooxidized membrane revealed photoperoxide products derived from phospholipid. Investigations into the mechanism(s) of lipid peroxidation by SnET2 x 2Cl and light-sensitized membranes were also performed. Results indicate that singlet oxygen (1O2) plays a major role in lipid peroxidation.     

In situ COMPARISON OF 665 nm and 633 nm WAVELENGTH LIGHT PENETRATION IN THE HUMAN PROSTATE GLAND

Abstract— The depth of treatment in photodynamic therapy (PDT) of tumors varies with the wavelength of light activating the photosensitizer. New generation photosensitizers that are excited at longer wavelengths have the potential for increasing treatment depths. Tin ethyl etiopurpurin (SnET2), a promising second-generation photosensitizer is maximally activated at 665 nm, which may be significantly more penetrating than 633 nm light currently used with porphyrins in PDT. The penetration of 665 nm and 633 nm wavelength red light in the prostate gland was compared in 11 patients undergoing prostatic biopsies for suspected prostatic cancer. Interstitial optical fibers determined the light attenuation within the prostate gland. Of the 11 patients, 7 had dual wavelength and 4 had single wavelength studies. The mean attenuation coefficients, μ_eff, for 665 nm and 633 nm wavelength light were 0.32 ± 0.05 mm^-1 and 0.39 ± 0.05 mm^-1, respectively, showing a statistically significant difference (P = 0.0003). This represented a 22% increase in the mean penetration depth and at 10 mm from the delivery fiber there was 1.8 times as much 665 nm light fluence than 633 nm. The mean μ_eff at 665 nm for benign and malignant prostate tissue were similar ( P = 0.42), however, there was significant interpatient variation (μ_eff ranging from 0.24 to 0.42 mm^-1) reflecting biological differences of therapeutic importance. The enhanced light fluence and penetration depth with 665 nm light should allow significantly larger volumes of prostatic tissue to be treated with SnET2-mediated PDT.     

PHOTODYNAMIC THERAPY: EFFECT ON THE ENDOTHELIAL CELL OF THE RAT AORTA

Previous studies in our laboratory have demonstrated that photodynamic therapy (PDT) of experimental bladder tumors leads to rapid destruction of the endothelial lining within the tumor microvasculature. Endothelial cell death during PDT may be a consequence of direct cell injury resulting from retention of photosensitizer within the endothelial cell or, alternatively, result from intravascular activation of circulating photosensitizer with subsequent indirect endothelial damage. In the experiments described here, we investigated the possibility that photosensitizer retained within the endothelial cell was sufficient to cause endothelial cell injury in the absence of circulating drug. The experimental model was rat aorta photosensitized in vivo via the intravenous injection of tin(II) etiopurpurin dichloride (SnET2), and subsequent in situ or in vitro (in explant culture) light (670 nm) treatment from an argon pumped dye laser. Damage to the lining of the aorta was assessed morphometrically by determining the areal density of silver stained endothelial cells. Results indicate that purpurin SnET2-PDT directly damages the endothelial lining.     

Effects of ursodeoxycholic acid on photodynamic therapy in a murine tumor model

We previously reported that the efficacy of photodynamic therapy (PDT) in cell culture was enhanced by ursodeoxycholic acid (UDCA), a nontoxic bile acid. In this study, we examined the ability of UDCA to promote tumor control by PDT in the mouse, using the radiation-induced fibrosarcoma tumor and the photosensitizing agent tin etiopurpurin (SnET2). These experiments revealed that the addition of UDCA to a PDT protocol promoted inhibition of tumor growth, a phenomenon unrelated to either altered SnET2 biodistribution or the level of vascular shutdown during irradiation. These results indicate that UDCA acts solely by promoting direct tumor cell kill by PDT.     

Rapid Initiation of Apoptosis by Photodynamic Therapy

Abstract— Photodynamic therapy (PDT) of neoplastic cell lines is sometimes associated with the rapid initiation of apoptosis, a mode of cell death that results in a distinct pattern of cellular and DNA fragmentation. The apoptotic response appears to be a function of both the sensitizer and the cell line. In this study, we examined photodynamic effects of several photosensitizers on murine leukemia P388 cells. Two drugs, a porphycene dimer (PcD) and tin etiopurpurin (SnET2), which localized at lysosomal sites, were tested at PDT doses that resulted in 50% loss of viability (LD₅₀), measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. An oligonucleosomal pattern of DNA degradation was observed within 1 h after irradiation. Neither sensitizer antagonized PDT-mediated internucleosomal DNA cleavage by the other. Very high PDT doses with either agent abolished this rapid internucleosomal cleavage. Exposure of cells to high concentrations of either sensitizer in the dark also resulted in rapid DNA fragmentation to nucle-osomes and nucleosome multimers; this effect was not altered by the antioxidant 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), although the latter could protect cells from cytotoxicity and apoptotic effects caused by LD₅₀ PDT doses. Photodamage from two cat-ionic sensitizers, which localized at membrane sites, caused rapid DNA cleavage to 50 kb particles; however, no further fragmentation was detected after 1 h under LD₁₀, LD₅₀ or LD₉₅ PDT conditions. Moreover, the presence of either cationic sensitizer inhibited the rapid internucleosomal cleavage induced by SnET2 or PcD photodamage. The site of photodynamic action may therefore be a major determinant of the initiation and rate of progression of apoptosis.     

Activation of mouse macrophages by in vivo and in vitro treatment with a cyanine dye, lumin.

The cyanine photosensitizer, lumin, is a potent macrophage activating agent: 4 days after administration of small amounts of lumin to mice (20-40 ng mouse-1), peritoneal macrophages exhibited a greatly enhanced Fc-mediated ingestion activity; higher doses (more than 3000 ng mouse-1) did not have this effect. The in vitro photodynamic activation of macrophages in mouse peritoneal cells exposed to white fluorescent light (3 J m-2 s-1) was also studied in media containing various concentrations of lumin. A short light exposure (45 J m-2) with 10 ng lumin ml-1 produced a maximum ingestion activity of macrophages. Lumin has absorption peaks at 670 and 760 nm. Therefore we designed experiments in which peritoneal cells were exposed to a red fluorescent light (emission, 660 nm; 0.5 J m-2 s-1). In a medium containing 3 ng lumin ml-1 with 7.5 J m-2 of red light, a markedly enhanced ingestion activity of macrophages was observed. The photodynamic treatment of peritoneal macrophages alone did not stimulate phagocytic activity, but the photodynamic treatment of a mixture of non-adherent (B and T) cells and macrophages resulted in a greatly enhanced ingestion activity of macrophages. Thus non-adherent cells are required for the photodynamic activation of macrophages, implying that an activating factor is generated within the non-adherent cells and transmitted to the macrophages. This hypothesis was confirmed by the observation that co-cultivation of photodynamically treated non-adherent cells with untreated macrophages resulted in a greatly enhanced ingestion capacity.     

THE ROLE OF LIPOPROTEINS IN THE DISTRIBUTION OF TIN ETIOPURPURIN (SnET2) IN THE TUMOR-BEARING RAT

The role of plasma lipoproteins in the distribution of the photosensitizing agent tin etiopurpurin (SnET2) was examined in male rats bearing the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced tumor. Treatement with 17α-ethinyl estradiol resulted in the depletion of total plasma cholesterol by > 70% and a corresponding decrease in plasma lipoproteins. To both control and estradiol-treated animals, a therapeutic dose (1.5mg/kg) of SnET2 was administered and biodistribution measured 24 h later. Estradiol treatment was not associated with difference in the distribution of SnET2 to liver, skin or tumor, or in the pattern of affinity of SnET2 to plasma albumin and lipoprotein. These results indicate that a substantial decrease in circulating lipoprotein levels does not alter patterns of SnET2 biodistribution.     

Studies on the heme and H2-uptake reaction from Azotobacter vinelandii bacterial ferritin

Bacterial ferritin of Azotobacter vinelandii (AvBF) is directly able to pick electrons up for iron release from or transfer them for storage to a platinum electrode in the absence of mediator or other reducer. The ferritin containing the structure of heme-Co2+ in part shows weakened activity to electrode and decreases the rate of iron release greatly. A reversible reduction process of the ferritin is observed by the spectral change regularly ranging from 310 to 260 nm under mixed gases containing 98% H2 and 2% to O2. The activity of nitrogen fixation from the whole cell of A. vinelandii increases greatly by H2 reduction with potentials ranging from -397 to -425 mV vs. NHE, indicating two important roles of H2-uptake reaction of the ferritin in increasing activity of nitrogen fixation and in supplying iron to synthesize nitrogenase.     

Porphyrin bleaching and PDT-induced spectral changes are irradiance dependent in ALA-sensitized normal rat skin in vivo

Photobleaching kinetics of aminolevulinic acid-induced protoporphyrin IX (PpIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for changes in excitation wavelength optical properties using diffuse reflectance measured at the excitation wavelength. Loss of PpIX fluorescence was monitored during photodynamic therapy (PDT) performed using 514 nm irradiation. Bleaching in response to irradiances of 1, 5 and 100 mW cm-2 was evaluated. The results demonstrate an irradiance dependence to the rate of photobleaching vs irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence. The kinetics for the accumulation of the primary fluorescent photoproduct of PpIX also exhibit an irradiance dependence, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction mechanism in vivo, and they provide spectroscopic evidence that PDT delivered at low irradiance deposits greater photodynamic dose for a given irradiation fluence. We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.     

Synthesis, photophysical studies and anticancer activity of a new halogenated water-soluble porphyrin

A water-soluble halogenated porphyrin, namely 5,10,15,20-tetrakis(2-chloro-3-sulfophenyl)porphyrin (TCPPSO(3)H), was prepared and evaluated as sensitizer for photodynamic therapy (PDT). Photophysical properties of TCPPSO(3)H, such as high photostability, long triplet lifetime and high singlet oxygen quantum yield suggest high effectiveness of this class of halogenated porphyrins in PDT. TCPPSO(3)H is non-toxic in the dark and causes a significant photodynamic effect examined against MCF7 (human breast carcinoma), SKMEL 188 (human melanoma) and S91(mouse melanoma) cell lines upon red light irradiation (cutoff < 600 nm) at low light doses. Time-dependent cellular uptake of TCPPSO(3)H reached plateau at 120 min and was the highest for S91, 20% lower for MCF7 and 70% lower for SKMEL 188. Our results show that this halogenated water-soluble porphyrin is an efficient photosensitizer and reveal the potential of this class of compounds as PDT agents.     

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

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

Diiodinated Mono- and Dipyridylvinyl BODIPY Dyes: Photophysicochemical Properties,in vitro Antibacterial Studies,Molecular Docking and Theoretical Calculations

In this study, novel mono- and dipyridylvinyl boron dipyrromethene dyes are prepared to compare their photodynamic antimicrobial chemotherapy (PACT) activities against Staphylococcus aureus to the corresponding core dyes. Pyridylvinyl substitution at the 3- or 3,5-positions of a meso-4-bromophenylBODIPY core dye via a Knoevenagel reaction with an aromatic 2-bromopyridinecarboxaldehyde shifts the major BODIPY spectral band to longer wavelength. The extended π-conjugation red shifts the main spectral band into the 602–618 nm region in CHCl₃, THF, ethanol and DMSO after monopyridylvinyl substitution and to 685–704 nm after dipyridylvinyl substitution. An enhancement of the population of the T₁ state through the incorporation of iodine atoms at the 2,6-positions results in moderately high singlet oxygen quantum yields in DMSO. The π-extended dyes were found to have significantly lower PACT activities than the diiodinated core dye.     

Large Nonlinear Optical Activity of a Near-infrared-absorbing Bithiophene-based Polymer with a Head-to-head Linkage

Although the production of near-infrared (NIR)-absorbing organic polymers with an excellent nonlinear optical (NLO) response is vital for various optoelectronic devices and photodynamic therapy, the molecular design and relevant photophysical investigation still remain challenging. In this work, large NLO activity is observed for an NIR-absorbing bithiophene-based polymer with a unique head-to-head linkage in the NIR region. The saturable absorption coefficient and modulation depth of the polymer are determined as ∼−3.5×10⁵ cm GW⁻¹ and ∼32.43%, respectively. Notably, the polymer exhibits an intrinsic nonlinear refraction index up to ∼−9.36 cm² GW⁻¹, which is six orders of magnitude larger than that of CS₂. The maximum molar-mass normalized two-photon absorption cross-section (σ₂/M) of this polymer can be up to ∼14 GM at 1200 nm. Femtosecond transient absorption measurements reveal significant spectral overlap between the 2PA and excited state absorption in the 1000–1400 nm wavelength range and an efficient triplet quantum yield of ∼36.7%. The results of this study imply that this NIR-absorbing polymer is promising for relevant applications.     

Studies on the synthesis of two hydrophilic hypocrellin derivatives with enhanced absorption in the red spectral region and on their photogeneration of O2*- and O2(1(delta)g)

To improve hydrophilicity and photoactivity of the new type of photosensitizer, hypocrellin, two new derivatives were synthesized through a mild reaction method between hypocrellion B (HB) and ethanolamine in tetrahydrofuran (THF) and their molecular structures were characterized by IR, NMR, MS and UV-Vis spectrometry. In the molecular structures of the two derivatives, the peri-hydroxylated perylenquinone structure of the parent HB is preserved and their photoresponses at 600-900 nm (the red spectral region) are enhanced markedly (the molar absorption coefficients at 650 nm for the two new derivatives are EAHB1 log epsilon = 3.72 and EAHB2 log epsilon = 3.91, respectively. In contrast, the parent compound HB exhibits little absorption at 650 nm). Electron paramagnetic resonance (EPR) spin trapping measurement and a 9,10-diphenylanthracene (DPA) bleaching method were employed to investigate the photodynamic action of two chemicals in the presence of oxygen. The quantum yields of O2(1(delta)g) generation of EAHB1 and EAHB2 are 0.08 and 0.45, respectively; the relative quantum yields of (O2*-) generation of EAHB1 and EAHB2 are 0.15 and 0.76, respectively, with the parent compound HB as the standard.     

Structural characterization of a degradation product of tin ethyl etiopurpurin (SnET2): Comparison of the long‐range heteronuclear correlations detected using conventional GHMBC and IMPEACH‐MBC in conjunction with submicro NMR probe technology

During the terminal heat sterilization of the lipid emulsion final dose formulation of the photodynamic therapeutic (PDT) agent tin ethyl etiopurpurin (SnET2), a new degradant was observed at very low levels. The degradant, which was prone to photo‐instability, was isolated by preparative chromatography and subsequently characterized by mass spectrometry and NMR methods. Reproducible parent ion clusters were only observable via negative ion APCI methods. Because of the limited isolate sample, NMR characterization was done using 1.7 mm SMIDG (SubMicro Inverse‐Detection Gradient) NMR probe technology in conjunction with the accordion‐optimized IMPEACH‐MBC long‐range heteronuclear shift correlation experiment. The “static” 8 Hz optimization of the GHMBC experiment failed to allow the observation of a number of long‐range correlations that were of critical importance to the determination of the structure of the impurity. In contrast, all of the correlations required to assemble the structure were obtained from an IMPEACH‐MBC experiment optimized for long‐range heteronuclear couplings in the range from 2–10 Hz.     

Direct near-infrared luminescence detection of singlet oxygen generated by photodynamic therapy in cells in vitro and tissues in vivo

Singlet oxygen (1O2) is believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). Measurement of 1O2 near-infrared (NIR) luminescence at 1270 nm in biological environments is confounded by the strongly reduced 1O2 lifetime and probably has never been achieved. We present evidence that this is now possible, using a new NIR-sensitive photomultiplier tube. Time-resolved 1O2 luminescence measurements were made in various solutions of aluminum tetrasulphonated phthalocyanine (AlS4Pc) and Photofrin. Measurements were also performed on suspensions of leukemia cells incubated with AlS4Pc, and a true intracellular component of the 1O2 signal was clearly identified. Time-resolved analysis showed a strongly reduced 1O2 lifetime and an increased photosensitizer triplet-state lifetime in the intracellular component. In vivo measurements were performed on normal skin and liver of Wistar rats sensitized with 50 mg/kg AlS4Pc. In each case, a small but statistically significant spectral peak was observed at 1270 nm. The 1O2 lifetime based on photon count rate measurements at 1270 nm was 0.03-0.18 micros, consistent with published upper limits. We believe that these are the first direct observations of PDT-generated intracellular and in vivo 102. The detector technology provides a new tool for PDT research and possibly clinical use.     

Chelation with metal is not essential for antitumor photodynamic activity of sulfonated phthalocyanines

It is generally assumed that a central metal is essential for the efficiency of phthalocyanines in photodynamic therapy (PDT) of cancer. Contrary to the set opinion, the results of the present study indicate that the metal-free sulfonated phthalocyanines (H2PcSn, where n is the number of sulfonate groups per molecule) possess a considerable photoactivity. The relative phototoxicities of H2PcS1.5, H2PcS2.4, H2PcS3.1 and H2PcS3.8 on HEp2 human epidermoid carcinoma cells were 3.3, 20, 3.3 and 1, respectively, thus demonstrating dependence of the activity on the sulfonation degree, known for metallo-PcSn. A significant delay in tumor growth and a decrease in tumor regrowth rate were observed in mice after PDT with H2PcS2.4. The antitumor effect declined in the order H2PcS2.4 > H2PcS3.1 > H2PcS1.5 and vanished for H2PcS3.8. We demonstrate here that the high photodynamic activity of H2PcS2.4 can be explained by its physicochemical properties in living cells and tissues. Thus, H2PcSn (n is about 2) can be considered as a new alternative in PDT of light-accessible neoplasms and further clinic-oriented studies are warranted.     

Emodin-Based Nanoarchitectonics with Giant Two-Photon Absorption for Enhanced Photodynamic Therapy

Two-photon-excited photodynamic therapy (TPE-PDT) has significant advantages over conventional photodynamic therapy (PDT). However, obtaining easily accessible TPE photosensitizers (PSs) with high efficiency remains a challenge. Herein, we demonstrate that emodin (Emo), a natural anthraquinone (NA) derivative, is a promising TPE PS with a large two-photon absorption cross-section (TPAC: 380.9 GM) and high singlet oxygen (¹O₂) quantum yield (31.9 %). When co-assembled with human serum albumin (HSA), the formed Emo/HSA nanoparticles (E/H NPs) possess a giant TPAC (4.02×10⁷ GM) and desirable ¹O₂ generation capability, thus showing outstanding TPE-PDT properties against cancer cells. In vivo experiments reveal that E/H NPs exhibit improved retention time in tumors and can ablate tumors at an ultra-low dosage (0.2 mg/kg) under an 800 nm femtosecond pulsed laser irradiation. This work is beneficial for the use of natural extracts NAs for high-efficiency TPE-PDT.     

Tumour-localizing and -photosensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP)

A water-soluble meso-substituted porphyrin (H(2)TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield=0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H(2)TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 microM concentrations with no significant cytotoxic effect in the dark. Moreover, the H(2)TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H(2)TCP per mg of cell proteins after 24h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H(2)TCP toward B16F1 cells was studied for different irradiation (1-15 min) and incubation (1-24 h) times. Nearly complete (>95%) cell mortality was obtained upon incubation with 20 microM H(2)TCP and 10 min irradiation with red light (600-700 nm, 20 mW/cm(2)). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the tumour was achieved by administration of H(2)TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)-30% polyethyleneglycol (PEG 400)-50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy.