Embelin (2,5-dihydroxy-3-undecyl-p-benzoquinone): A bioactive molecule isolated from Embelia ribes as an effective photodynamic therapeutic candidate against tumor in vivo

The present study was carried out to assess the photosensitizing potential of embelin, the biologically active natural product isolated from Embelia ribes in photodynamic therapy (PDT) experiments in vivo. In vitro PDT clearly indicated that embelin recorded significant cytotoxicity in Ehrlich's Ascites Carcinoma (EAC) cells, which is superior to 5-aminolevulinic acid, a known photodynamic compound. For in vivo experiments solid tumor was induced using EAC cells in the male Swiss albino mice of groups I, II, III and IV. Group I served as the control (without solid tumor), group II served as tumor bearing mice without treatment and groups III and IV served as treatments. At the completion of 4 weeks of induction, the tumor bearing mice from group III and IV were given an intraperitoneal injection with embelin (12.5 mg/kg body weight). After 24 h, tumor area in the Group III and IV animals was exposed to visible light from a 1000 W halogen lamp. The mice from groups I to III were sacrificed 2 weeks after the PDT treatment and the marker enzymes (myeloperoxidase [MPO], β-d-glucuronidase, and rhodanese) were assayed and expression of Bcl-2 and Bax were analyzed in normal and tumor tissues. Animals from group IV were sacrificed after 90 days of PDT treatment and the above mentioned parameters were recorded. Reduction in tumor volume and reversal of biochemical markers to near normal levels were observed in the treated groups. This is the first report on PDT using a natural compound for solid tumor control in vivo. The uniqueness of the mode of treatment lies in the selective uptake of the nontoxic natural compound, embelin from the medicinal plant E. ribes used in Indian system of medicine, by the solid tumor cells and their selective destruction using PDT without affecting the neighboring normal cells, which is much advantageous over radiation therapy now frequently used.     

Photodynamic inactivation of multiresistant bacteria (KPC) using zinc(II)phthalocyanines

The worldwide increase in antibiotic resistance has led to search of alternatives anti-microbial therapies such as photodynamic inactivation. The aim of this paper was to evaluate the photodynamic activity in vitro of a neutral and two cationic Zn phthalocyanines. Their photokilling activity was tested on Escherichia coli ATCC 25922 and Klebsiella pneumoniae Carbapenemase (KPC)-producing. After treating bacteria with phthalocyanines, the cultures were irradiated with white light. As a result, the bacteria were inactivated in presence of cationic phthalocyanines. The photoinactivation was dependent of the irradiation time and phthalocyanine concentration. The most effective photosensitizer on KPC-producing was Zinc(II)tetramethyltetrapyridino[2,3-b:2′,3′-g:2″,3″-l:2?,3?-q]porphyrazinium methylsulfate (ZnTM2,3PyPz). After irradiation using the water soluble ZnTM2,3PyPz (3 μM) the viability of KPC (30 min of irradiation) and E. coli (10 min of irradiation) decreased ≈99.995%.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death

In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix^TM. Samples were prepared from bovine patellae and stored in Dulbecco’s Modified Eagle’s Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix?, stress-relaxation tests were conducted for the samples immersed in Hexabrix? and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix? were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8–10 MPa contact stresses, the samples immersed in full strength Hexabrix? showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.     

A naphthalocyanine based near-infrared photosensitizer: Synthesis and in vitro photodynamic activities

A hydrophilic near-infrared (NIR) photosensitizer featuring a naphthalocyanine core and peripheral carboxylate acid groups was synthesized and characterized. Its photophysical and photochemical properties were studied and compared with phthalocyanine. Due to the extended π-conjugation, both the Q band and fluorescence emit of this naphthalocyanine bathochromically shift to NIR region. It also exhibits superior NIR photodynamic efficiency to phthalocyanine as evidenced by high efficiency in generating singlet oxygen (Φ_Δ = 0.66) and in vitro phototoxicity toward Hela human cervical cancer cells. Therefore, this novel naphthalocyanine could potentially be a NIR photosensitizer for photodynamic therapy.     

Extended rhodamine photosensitizers for photodynamic therapy of cancer cells

Extended thio- and selenorhodamines with a linear or angular fused benzo group were prepared. The absorption maxima for these compounds fell between 640 and 700 nm. The extended rhodamines were evaluated for their potential as photosensitizers for photodynamic therapy in Colo-26 cells. These compounds were examined for their photophysical properties (absorption, fluorescence, and ability to generate singlet oxygen), for their dark and phototoxicity toward Colo-26 cells, and for their co-localization with mitochondrial-specific agents in Colo-26 and HUT-78 cells. The angular extended rhodamines were effective photosensitizers toward Colo-26 cells with 1.0 J cm⁻² laser light delivered at λ_max ± 2 nm with values of EC₅₀ of (2.8 ± 0.4) × 10⁻⁷ M for sulfur-containing analogue 6-S and (6.4 ± 0.4) × 10⁻⁸ M for selenium-containing analogue 6-Se. The linear extended rhodamines were effective photosensitizers toward Colo-26 cells with 5 and 10 J cm⁻² of broad-band light (EC₅₀’s ⩽ 2.4 × 10⁻⁷ M).     

First results from conservation studies of chlorophyllous spores of the Royal fern (Osmunda regalis, Osmundaceae)

Pteridophytes spore banks are a promising ex situ conservation tool used to increase the chances of survival of ferns, in fact that large quantities of germplasm with high genetic variation can be conserved in a small space with low economic and technical costs. However, methods to maintain the viability of chlorophyllous spores during storage are less understood.The aim of this study was to investigate the influence of long term storage on the viability of Royal Fern spores, which were stored under different conditions derived from various combinations of temperature and degrees of hydration. Survival and germination tests were performed after 1 and 28 months of storage. Our results showed the highest survival percentages for spores stored under Normal humidity at subzero temperatures (T = ? ?20 °C). These spores received no pre-treatment, dehydration, or cryoprotectants, which resulted in fast germination and gametophyte development which seemed to be stimulated by low temperatures.     

Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to generate reactive species that kill tumor and tumor vasculature cells. Nitric oxide produced by these cells could be procarcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. The purpose of this study was to determine whether tumor cells upregulate NO as a cytoprotective measure during PDT. Breast tumor COH-BR1 cells sensitized in their mitochondria with 5-aminolevulinic acid (ALA)-derived protoporphyrin IX died apoptotically after irradiation, ALA- and light-only controls showing no effect. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated > 3-fold within 4 h after ALA/light treatment, whereas other NOS isoforms were unaffected. Exposing cells to a NOS inhibitor (L-NAME or 1400W) during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2- to 3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by > 2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy and suggest NOS-based pharmacologic interventions for preventing this.     

Proteomics analysis of proteins in green alga Haematococcus lacustris (Chlorophyceae) expressed under combined stress of nitrogen starvation and high irradiance

This study is aimed at identifying the proteins that are up-regulated during astaxanthin accumulation in Haematococcus lacustris. For this H. lacustris cells were cultivated in photobioreactors under normal light irradiance of 40 μE m^?2 s^?1 for 6 days and then induced to accumulate astaxanthin for 3 days further by exposure to continuous high irradiance of 200 μE m^?2 s^?1 with fluorescent lamps as light source after the cells reached the stationary phase in a nitrogen-depleted condition. Under this condition, the average astaxanthin content per cell increased from 91 mg/l up to 406 mg/l after 3 days of induction. The proteomics data from a two-dimensional electrophoretic comparison demonstrated that a combination of nitrogen source depletion and 1 h high light have significantly changed the pattern of protein expression in H. lacustris. A total of 49 protein spots were picked after 1 h of stress induction. They consisted of 13 down-regulated proteins and 36 up-regulated proteins. Fifteen proteins which had highly up-regulated expression were further analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results will point toward interesting proteins that can be pursued for further analysis of astaxanthin biosynthesis pathway.     

Germination response and viability of an endangered tropical conifer Widdringtonia whytei seeds to temperature and light

The tropical conifer Widdringtonia whytei Rendle is an endangered species endemic to Mulanje Mountain in Malawi. A study was conducted for the first time under controlled conditions in order to assess the effects of temperature and light on germination and viability of W. whytei seeds. Seeds incubated at a constant temperature of 20 °C attained the highest cumulative germination percentage (100%) followed by 87% germination under fluctuating temperatures of 15 °C night/25 °C day. No seed germination occurred at temperatures below 15 °C. Seeds that failed to germinate at temperatures below 15 °C showed the highest (> 90%) viability compared to the seeds incubated at 25 °C (60%). Across temperature regimes, germination was significantly higher under light (44.7%) than dark (35.6%) conditions. It is concluded that temperature is one of the critical factors for germination of W. whytei seed. The ability of W. whytei seeds to germinate both in light and darkness implies that the species would unlikely form a persistent soil seed bank, an attribute which is common in species that survive in habitats frequently disturbed by fires.     

Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins

Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photodynamic action but not considered the main cause of cell death. The in vivo effect of the photodynamic process on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein.Two cationic porphyrins (Tetra-Py⁺-Me and Tri-Py⁺-Me-PF) were used to photoinactivate E. coli (5.0 μM; 10⁸ cells mL^?1) and S. warneri (0.5 μM; 10⁸ cells mL^?1) upon white light irradiation at 4.0 mW cm^?2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensitized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double-stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry.E. coli was completely photoinactivated with both porphyrins (5.0 μM), whereas S. warneri was only completely inactivated by Tri-Py⁺-Me-PF (0.5 μM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py⁺-Me-PF but almost unchanged with Tetra-Py⁺-Me after 40 min of irradiation. The amount of Tri-Py⁺-Me-PF bound to E. coli after washing the cells is higher than Tetra-Py⁺-Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells.     

Evaluation of Tris–citric acid,skim milk and sodium citrate extenders for liquid storage of Punjab Urial (Ovis vignei punjabiensis) spermatozoa

The Punjab Urial (Ovis vignei punjabiensis) is an endangered subspecie of ovidae, distributed as small scattered populations in the forest belt of the Himalayan foothills of Pakistan and in the areas enclosed by the Indus and the Jhelum rivers. The present study was conducted to evaluate the liquid storage of Punjab Urial spermatozoa in different extenders for use in future in situ conservation activities. Semen was collected by electro-ejaculation from three captive Punjab Urial rams. Suitable ejaculates of individual animals were pooled and divided into three aliquots for dilution with the experimental extenders (Tris–citric acid, skim milk and sodium citrate) at 37 °C. Extended semen was cooled from 37 °C to 5 °C in 2 h, and stored for three days at 5 °C. Sperm motility (%), viability (%; live/dead), acrosome integrity (%) and plasma membrane integrity (%) were assessed on days 1, 2 and 3 of storage. On day 1, sperm motility, viability as well as acrosome and plasma membrane integrity were similar (p > 0.05) in all three experimental extenders. On day 2, sperm motility, viability, acrosome and plasma membrane integrity were higher (p < 0.05) in Tris–citric acid extender compared to sodium citrate based extender. On day 3 of storage, the values of motility, viability and acrosome integrity were higher (p < 0.05) in Tris–citric acid extender than in skim milk and sodium citrate based extenders. In conclusion, Tris–citric acid extender appears to be a better option compared with skim milk and sodium citrate extenders for liquid storage of Punjab Urial semen.     

Optimized methodology for extraction of (1 → 3)(1 → 6)-β-d-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative

The cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with immunostimulant properties. The standard methodologies described for its extraction involve acid and alkaline washings, which degrade part of its glucose chains and reduce the final yield. In the present study, an optimized methodology for extraction of β-d-glucan from S. cerevisiae cells, involving sonication and enzyme treatment, with a yield of 11.08 ± 0.19%, was developed. The high-purity (1 → 3)(1 → 6)-β-d-glucan was derivatized to carboxymethyl-glucan (CM-G). In vitro tests with CM-G in Chinese hamster epithelial cells (CHO-k1) did not reveal any cytotoxic or genotoxic effects or influences of this molecule on cell viability. The method described here is a convenient alternative for the extraction of (1 → 3)(1 → 6)-β-d-glucan under mild conditions without the generation of wastes that could be potentially harmful to the environment.     

Photodynamic inactivation of oropharyngeal Candida strains

Oropharyngeal candidiasis (OPC) is an infection frequent in immunocompromised patients. Photodynamic therapy is an alternative to conventional treatments, based on the utilization of compounds that inhibit or kill microorganisms only under the effect of light, process known as Photodynamic Inactivation (PDI). In the present study, PDI of Candida spp. by the natural product α-terthienyl (α-T) was investigated following the guidelines of CLSI M27-A3, under UV-A light irradiation.The optimal values of two variables, exposure irradiation time (ET) and distance to the irradiation source (DIS) were established by employing Design Expert Software (DES). For this purpose, a panel of Candida strains isolated from OPC (C. albicans, C. tropicalis, C. parapsilosis and C. krusei) was employed and optimal values were 5 min (ET) and between 6.06 and 6.43 cm (DIS) with a desirability factor of 0.989. α-T plus UV-A light in the optimal conditions caused a complete reduction in viable cells in 5 min which was demonstrated by viable cells reduction assays and confocal microscopy after vital staining (propidium iodide/fluorescein diacetate). The germ tube formation of C. albicans was inhibited by α-T at sub-inhibitory concentrations. Results showed that α-T plus UV-A light could constitute an alternative for OPC treatments at the optimal conditions determined here.     

Novel 3,6-bis(imidazolidine)acridines as effective photosensitizers for photodynamic therapy

The photoeffect of new proflavine derivatives with DNA-binding and antitumour activities, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was studied to evaluate them as potential photosensitizers for photodynamic antitumor therapy. EPR measurements showed that superoxide radical anion and singlet oxygen were produced upon irradiation of AcrDIMs with UV-A light (>300 nm) in the presence of molecular oxygen. This indicates that AcrDIMs may act as photosensitizers. The most active pentyl-AcrDIM and hexyl-AcrDIM displayed photocytotoxic effect toward the mouse lymphocytic leukemia cell line L1210 and human ovarian cancer cells A2780. Antitumor activity of pentyl-AcrDIM increased as high as about 12 times (72 h incubation) after irradiation of A2780 cells (365 nm, 1.05 J/cm²). The photocytotoxicity seems to be associated with oxidative stress. Concerning the cell cycle, flow cytometry showed an arrest in the S-phase already 4 h after irradiation. In a comet assay, no genotoxicity of AcrDIMs was found. Typical morphologic changes and formation of DNA-ladders indicated induction of apoptotic cell death, though no activation of caspase-3 was observed. Investigation of intracellular localization of pentyl-AcrDIM confirmed its partial accumulation in mitochondria and lysosomes. After irradiation of the A2780 cells, colocalization of pentyl-AcrDIM with monodansylcadaverine, a lysosomal dye, was proven, suggesting that lysosomes in the irradiated cells may be involved in the cell death.     

Predation of Salmonella enterica serovar Typhimurium by the rumen protozoon Entodinium caudatum studied in vitro by fluorescence emission

Predation of bacteria by protozoa has important implications on rumen metabolism and bacterial populations. Protozoa can also restrict the passage of pathogenic bacteria to the host’s lower gastrointestinal tract. This work aimed to evaluate the predation by Entodinium caudatum (EC) and the intraprotozoal survival of Salmonella enterica serovar Typhimurium. EC cells from a monofaunated sheep were incubated for up to 105 min with a S. enterica strain producing a green fluorescent protein. Rumen fluid from a defaunated sheep (DEF) was used as a control. Fluorescence, as an index of predation, measured in the residual (protozoal) fraction was higher in EC than in DEF. 105 min after the beginning of the incubation it was higher than 30 min after. Intracellular survival of Salmonella within EC was assessed by means of a selective medium. Amounts of Salmonella in the residual fraction were higher in EC than in DEF only after 30 min. After 105 min, each protozoa engulfed 100 Salmonella cell per min. Intraprotozoal survival of ingested Salmonella was 0.0017. Predation of S. enterica by E. caudatum occurred and increased in proportion to time, but bacterial viability inside the protozoa was lower at 105 min. This study demonstrates that fluorescence emission combined with bacterial and protozoal cultures could be a reliable method for quantifying bacterial predation and viability in vitro.     

Magnetic susceptibility of iron in malaria-infected red blood cells

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0 ± 0.9) × 10^? 3 mL mol^? 1. The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88 ± 0.60) × 10^? 6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.     

In situ positron emission tomography monitoring of endothelial cells embedded in perfused fibrin gels

In tissue engineering, the continuous monitoring of cell and tissue cultures in vitro is crucial to assess their functional status over time. However, these constructs can be large, thick and non-transparent. Medical imaging techniques can allow real-time in situ monitoring of cell and tissue cultures in thick solid scaffolds. Here, human endothelial cells were embedded in fibrin gels that were continuously perfused by a culture medium. Positron emission tomography (PET) imaging was used to assess cell viability non-destructively over periods extending up to a few weeks. PET imaging protocols were adapted and validated to measure culture perfusion and cell metabolism using [¹⁸F]-fluorodeoxyglucose (¹⁸FDG). Cell densities down to 100,000 cells/mL were detectable after 12 h of culture and cell structures were localized within the fibrin gels after 1–2 weeks of culture. PET is a promising tool to investigate a wide range of cellular properties and reveal information on tissue development.     

Bladder cancer cell viability inhibition and apoptosis induction by baicalein through targeting the expression of anti-apoptotic genes

The study was aimed to investigate the effect of baicalein, a flavonoid molecule isolated from the plant Oroxylum indicum on bladder cancer cell viability. The results revealed that baicalein treatment of T24 and 253J bladder cancer cells targeted the expression of mRNA and proteins corresponding to the anti-apoptotic genes. RT-PCR assay showed that anti-apoptotic genes were markedly over-expressed in the bladder cancer cells. Exposure of the bladder cancer cells to baicalein at 5 mg/mL doses for 72 h led to reduction in the expression of mRNA levels of antiapoptotic genes. In T24 cells, the levels of BCL2, Bcl-xL, XIAP and surviving was reduced by 65, 69, 58 and 72%, respectively. In T24 and 253J cells exposure to baicalein for 72 h resulted respectively in 39 and 46% reduction in cell viability. Baicalein treatment also induced apoptosis in the bladder cancer cells. In T24 and 253J cells baicalein treatment at 5 mg/mL for 72 h induced apoptosis in 79 and 86% cells respectively. Thus, baicalein mediated reduction in antiapoptotic gene expression inhibits viability and induces apoptosis in bladder cancer cells. Therefore, baicalein is of therapeutic importance for the development of bladder cancer treatment strategy.     

Novel boronated chlorin e6-based photosensitizers: Synthesis,binding to albumin and antitumour efficacy

Chlorins, a class of plant porphyrins, are perspective as photosensitizing agents due to light absorption in the long wavelength spectral region and deeper photodamage of tissues. Aiming at optimization of antitumour properties of chlorins, we synthesized a series of boronated derivatives of chlorin e₆ and their complexes containing Zn(II), Pd(II) or Sn(IV). The compounds were synthesized by alkylation of amino or hydroxy derivatives of chlorin e₆ with 1-trifluoromethanesulfonylmethyl-o-carborane. Chlorin e₆ 13(1)-N-{2-[N-(o-carboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester (compound 5) formed complexes with serum albumin, a major porphyrin carrier. The binding constant of these complexes was ～4 times bigger than the respective value for the complexes of albumin with boron-free aminochlorin e₆. Compound 5 potently sensitized rat fibroblasts to illumination with monochromatic red light: >98% of cells were necrotic by 24 h post-illumination with 1 μM of 5. This compound demonstrated high efficacy in photodynamic therapy of rat M-1 sarcoma. After PDT with 25 mg/kg of 5 the residual tumours were significantly smaller than in animals subjected to PDT with equal concentration of boron-free aminochlorin e₆. No signs of general toxicity were detectable after PDT with 5. Thus, boronation can enhance the potency of chlorins in PDT, in particular, due to an increased binding to albumin. Our data expand the therapeutic applicability of boronated chlorins beyond boron neutron capture therapy; these agents emerge as dual efficacy photoradiosensitizers.