Modelling the Inactivation and Possible Regrowth of Salmonella enterica Treated with Chlorophyllin-Chitosan Complex and Visible Light.

The study focuses on predictive modelling of inactivation of Salmonella enterica after treatment with chlorophyllin-chitosan complex and visible light. Salmonella cells were incubated with chlorophyllin-chitosan complex (0.001% chlorophyllin and 0.1% chitosan) for different times (5-60 min) and then illuminated with visible light (λ=405 nm, H e=38 J/cm2). Inactivation curves and post-treatment regrowth curves were built based on microbiological viability tests and data were fitted to ten inactivation and two regrowth models. The photoactivated complex reduced Salmonella population, which were unable to regrow. Weibull and Baranyi models were the best to describe the inactivation and regrowth kinetics respectively. In conclusion, data from the kinetic analysis and predictive modelling confirmed that photoactivated chlorophyllin-chitosan complex is a promising non-thermal approach for inactivation of Gram-negative pathogens, since no bacterial regrowth after treatment has been predicted.

Toward better microbial safety of wheat sprouts: chlorophyllin-based photosensitization of seeds.

Sprouted seeds are gaining popularity worldwide due to their high nutritional value. At the same time, they are among the most highly contaminated fresh produce and have been recognized as the primary source of food-borne pathogens, such as E. coli O157 and harmful microfungi. The antifungal and antibacterial properties of chlorophyllin-based photosensitization in vitro together with successful application of this treatment for microbial control in wheat sprouts have been investigated. First, we examined the antimicrobial efficiency of chlorophyllin (Chl, 1.5 × 10-5-5 × 10-3 M) activated in vitro by visible light (405 nm, radiant exposure: 18 J cm-2) against the food-borne pathogen Escherichia coli and plant pathogen Fusarium oxysporum. Results revealed that this treatment (1.5 × 10-5 M Chl, incubation time 1 h, 405 nm, radiant exposure: 18 J cm-2) can reduce the E. coli population by 95%. Moreover, at higher chlorophyllin concentrations (5 × 10-4-5 × 10-3 M Chl), it is possible to delay the growth of F. oxysporum by 51-74%. The decontamination of wheat seeds by chlorophyllin-based photosensitization (5 × 10-4 M Chl, 405 nm, radiant exposure: 18 J cm-2) remarkably reduced the viability of surface-attached mesophilic bacteria (∼2.5log CFU g-1), E. coli (∼1.5log CFU g-1) and yeasts/fungi (∼1.5log CFU g-1). Moreover, SEM images confirmed that this treatment did not damage the grain surface microstructure. Most importantly, Chl-based photosensitization did not reduce the seed germination rate or seedling growth and had no impact on the visual qualities of sprouts. In conclusion, the chlorophyllin-based photosensitization treatment, being nonthermal, environmentally friendly and cost-effective, has huge potential for microbial control of highly contaminated germinated wheat sprouts and seeds used to produce sprouts, especially in organic farming.

Innovative Nonthermal Technologies: Chlorophyllin and Visible Light Significantly Reduce Microbial Load on Basil.

Due to the high amount of biologically active compounds, basil is one of the most popular herbs. However, several outbreaks have been reported in the world due to the consumption of basil contaminated with different food pathogens. The aim of this study is to apply nonthermal and ecologically friendly approach based on photosensitization for microbial control of basil which was naturally contaminated with mesophils and inoculated with thermoresistant food pathogen Listeria monocytogenes 56Ly. The obtained data indicate that soaking the basil in 1.5·10-4 M chlorophyllin (Chl) for 15 min and illumination with light for 15 min at 405 nm significantly reduced total aerobic microorganisms on basil by 1.3 log CFU/g, and thermoresistant L. monocytogenes 56Ly from 6.1 log CFU/g in control to 4.5 log CFU/g in the treated samples. It is important to note that this treatment had no impact on enzymatic activity of polyphenol oxidase and pectinesterase. Results obtained in this study support the idea that photosensitization technique with its high selectivity, antimicrobial efficiency and nonthermal nature can serve in the future for the development of safe nonthermal and environmentally friendly preservation technology for different fruits and vegetables.

Inactivation of bacterial biofilms using visible-light-activated unmodified ZnO nanorods.

Various zinc oxide (ZnO) nanostructures are widely used for photocatalytic antibacterial applications. Since ZnO possesses a wide bandgap, it is believed that only UV light may efficiently assist bacterial inactivation, and diverse crystal lattice modifications should be applied in order to narrow the bandgap for efficient visible-light absorption. In this work we show that even unmodified ZnO nanorods grown by an aqueous chemical growth technique are found to possess intrinsic defects that can be activated by visible light (λ = 405 nm) and successfully applied for total inactivation of various highly resistant bacterial biofilms rather than more sensitive planktonic bacteria. Time-resolved fluorescence analysis has revealed that visible-light excitation creates long-lived charge carriers (τ > 1 µs), which might be crucial for destructive biochemical reactions achieving significant bacterial biofilm inactivation. ZnO nanorods covered with bacterial biofilms of Enterococcus faecalis MSCL 302 after illumination by visible light (λ = 405 nm) were inactivated by 2 log, and Listeria monocytogenes ATCL3C 7644 and Escherichia coli O157:H7 biofilms by 4 log. Heterogenic waste-water microbial biofilms, consisting of a mixed population of mesophilic bacteria after illumination with visible light were also completely destroyed.

Effective photosensitization-based inactivation of Gram (-) food pathogens and molds using the chlorophyllin-chitosan complex: towards photoactive edible coatings to preserve strawberries.

This study is focused on the novel approaches to enhance the inactivation of the Gram (-) food pathogen Salmonella enterica and harmful molds in vitro and on the surface of strawberries using the chlorophyllin-chitosan complex. Salmonella enterica (∼1 × 10(7) CFU mL(-1)) was incubated with chlorophyllin 1.5 × 10(-5) M (Chl, food additive), chitosan 0.1% (CHS, food supplement) or the chlorophyllin-chitosan complex (1.5 × 10(-5) M Chl-0.1% CHS) and illuminated with visible light (λ = 405 nm, light dose 38 J cm(-2)) in vitro. Chlorophyllin (Chl)-based photosensitization inactivated Salmonella just by 1.8 log. Chitosan (CHS) alone incubated for 2 h with Salmonella reduced viability 2.15 log, whereas photoactivated Chl-CHS diminished bacterial viability by 7 log. SEM images indicate that the Chl-CHS complex under these experimental conditions covered the entire bacterial surface. Significant cell membrane disintegration was the main lethal injury induced in Gram (-) bacteria by this treatment. Analysis of strawberry decontamination from surface-inoculated Salmonella indicated that photoactivated Chl-CHS (1.5 × 10(-5) M Chl-0.1% CHS, 30 min incubation, light dose 38 J cm(-2)) coatings diminished the pathogen population on the surface of strawberries by 2.2 log. Decontamination of strawberries from naturally distributed yeasts/molds revealed that chitosan alone reduced the population of yeasts/molds just by 0.4 log, Chl-based photosensitization just by 0.9 log, whereas photoactivated Chl-CHS coatings reduced yeasts/molds on the surface of strawberries by 1.4 log. Electron paramagnetic resonance spectroscopy confirmed that no additional photosensitization-induced free radicals have been found in the strawberry matrix. Visual quality (color, texture) of the treated strawberries was not affected either. In conclusion, photoactive Chl-CHS exhibited strong antimicrobial action against more resistant to photosensitization Gram (-) Salmonella enterica in comparison with Gram (+) bacteria in vitro. It reduced significantly the viability of strawberry surface-attached yeasts/molds and inoculated Salmonella without any negative impact on the visual quality of berries. Experimental data support the idea that photoactivated Chl-CHS can be a useful tool for the future development of edible photoactive antimicrobial coatings which can preserve strawberries and prolong their shelf-life according to requirements of "clean green technology".

Visible light-activated ZnO nanoparticles for microbial control of wheat crop.

Every year 15-50% of cereals all around the world are lost due to fungal contamination and deterioration. In addition, 25% of crops, which are used for human and animal consumption, are contaminated with mycotoxins. It is obvious, that more effective and sustainable technologies for better microbial control of crops are required. For this purpose we evaluated antibacterial and antifungal activity of ZnO nanoparticles (NPs) (10-3-5 × 10-3M) activated with visible light (405 nm, 18-30 J/cm2). Obtained data indicated that this treatment can inactivate human pathogen E. coli B by 6 log CFU without any possibility to regrowth after treatment. Wheat pathogen Fusarium oxysporum was inactivated by 51.7%. Results indicated that reactive oxygen species took place in the mechanisms of inactivation. Moreover, visible light activated ZnO NPs reduced the population of mesophiles on the surface of grains by 2.5 log CFU/g, inoculated E. coli- by 2.0 log CFU/g and naturally distributed fungi-by 2.1 log CFU/g. This treatment had no impact on visual quality of grains, did not inhibit grain germination rate and slightly promoted grain seedling growth. Concluding, the use of visible light driven photocatalysis in ZnO nanoparticles has huge potential to control plant pathogens, reduce food-borne diseases and subsequently enhance the sustainability in agriculture, meeting the increasing demands of a growing world population.

Inactivation of molds on the surface of wheat sprouts by chlorophyllin-chitosan coating in the presence of visible LED-based light.

The present study clearly demonstrated the significant antifungal activity of chlorophyllin-chitosan complex (Chl-CHS) after activation with visible light. This phenomenon afterwards was successfully applied for better microbial control of highly popular food- germinated wheat sprouts. Obtained results indicated that photoactivated Chl-CHS complex (0.001% Chl-0.1% CHS and 0.005% Chl-0.5% CHS, 405 nm, 76 J/cm2) considerably inhibited (83%) the growth of dominating sprout pathogenic microfungus Fusarium graminearum in vitro. Moreover, obvious delay of fungus growth by 4 days after treatment was observed. The efficiency of antifungal treatment strongly depended on used Chl-CHS complex concentration. The coating of wheat grains with Chl-CHS (0.005% Chl-0.5% CHS) and illumination with visible light (405 nm; 76 J/cm2) inactivated the molds on the surface of grains by 79%. It is important to note, that no grain surface microstructure damage observed by SEM imaging have been found. No inhibiting effects on seed germination process, viability, average weight of grains, length of seedlings and content of chlorophyll a and chlorophyll b in the seedlings or eventually visual quality after Chl-CHS coating of grains and illumination with visible light have been observed. In conclusion, chlorophyllin-chitosan coating in the concert with visible light has great potential as cost-effective, environmentally friendly and sustainable strategy for better microbial control of highly contaminated sprouts.

Understanding Escherichia coli damages after chlorophyllin-based photosensitization.

Pathogenic strains of bacteria are causing various illnesses all around the world and have a major socio-economic impact. Thus, fast- and low-cost methods for the microbial control of foods are needed. One of them might be photosensitization. This study looks deeper into the mechanism of Escherichia coli damage by chlorophyllin-based photosensitization. Fluorimetric data indicate that after 15 minute incubation with chlorophyllin (Chl) (1.5 × 10-5 M Chl) 0.73 ± 0.03 µM of this compound was associated with E. coli cell surface. After photoactivation (405 nm, 6-30 J/cm2 ) significant reduction (88.2%) of bacterial viability was observed. Higher concentration of Chl (5 × 10-4 M Chl) reduced viability of bacteria more than by 98%. Results indicated that reactive oxygen species (ROS) took place in this inactivation. Colloidal surface enhanced Raman scattering (SERS) spectroscopy was employed to detect the molecular changes in the treated bacteria. It was found that Chl-based based photosensitization triggers multiple surface structure changes in E. coli what induce lethal unrepairable damages and inactivation of pathogen.

Innovative approach to sunlight activated biofungicides for strawberry crop protection: ZnO nanoparticles.

Strawberries are one of the most common and important fruits in the world, widely investigated for their nutritional and nutraceutical properties. However, after the emergence of several outbreaks of foodborne diseases some concerns regarding the microbiological safety of fresh strawberries have increased in recent years. In this paper new insights, based on application of ZnO nanoparticles (NPs) as alternative to chemical fungicides in the fields for preharvest preservation of strawberries are presented. Antifungal activity of ZnO NPs was tested on main strawberry plant pathogen Botrytis cinerea. Obtained data indicated that used ZnO NPs (5 × 10-3 M) in the dark just insignificantly (12%) inhibited the radial growth of B. cinerea. But photoactivated ZnO NPs (5 × 10-3 M, 405 nm, 34 J/cm2) inhibited the growth of B. cinera by 80%. Real-time field experiments revelead, that spraying of ZnO NPs in the strawberry field in sunny day reduced Botrytis incidences by 43%, enhanced the crop production by 28.5% and stoped the spoilage of harvested fruits during storage by 8 days, if compare with control. No harm to crowns and leaves of strawberry plant have been found, however this treatment increased the growth of inflorescence (37.5%) and reduced the growth of runners (32.8%). For comparison, spraying of conventionqal chemical fungicide fenhexamid (FEN) reduced Botrytis incidences in the same level as ZnO NPs, increased the harvest by 21.9% and delayed the spoilage of fruits by 8 days. The presented results look highly promising, since ZnO NPs in the presence of sunlight, activated by UV and visible light can protect strawberry fruits from Botrytis infection more effectively than conventional fungicide fenhexamid. This treatment significantly increased crop production and reduced spoilage of strawberries. It looks like ZnO NPs have great potential in the future to replace chemical fungicides.

Towards better microbial safety of fresh produce: Chlorophyllin-based photosensitization for microbial control of foodborne pathogens on cherry tomatoes.

The aim of this study is to evaluate the antimicrobial efficiency of Chlorophyllin-based photosensitization for microbial control of cherry tomatoes. Chlorophyllin-based photosensitization (1.5 × 10-4 M, 3 J/cm2) significantly (2.4 log) reduced the population of naturally distributed surface attached various mesophilic bacteria (microbiota) on tomatoes. Moreover, the population of thermoresistant strains of food pathogens Bacillus cereus and Listeria monocytogenes inoculated on tomatoes was reduced by 1.5 log and 1.6 log respectively after this treatment. Conventional washing with water reduced the population of Listeria on tomato by 0.6 log and Bacillus by 0.8 log. In comparison, hypochlorite treatment reduced Listeria on tomatoes by 1.4 log and Bacillus by 1.6 log. The regrowth of mesophilic bacteria and thermoresistant Listeria on the surface of tomatoes after photosensitization was delayed for 28 days and 14 days respectively. Moreover, photosensitization did not induce harmful effects on main parameter of nutritional quality of tomatoes, i.e. antioxidant activity of tomatoes remained unchanged (27.5 mM Fe2+/kg). Eventually, this treatment did not induce visible thermal effects in fruit matrix and prolonged the shelf-life of tomatoes by 4 days. In our opinion, chlorophyllin-based photosensitization has a huge potential as alternative to not-chemical food preservation technology, saving water and energy. In addition, fast development of light emitting diodes (LED's) and light sources based on LED technologies make this treatment low cost, environmentally friendly and easy to maintain.

New approach to the fungal decontamination of wheat used for wheat sprouts: effects of aminolevulinic acid.

Nowadays, there is a growing interest in natural, minimally processed, nutritional and healthy foods. Sprouted seeds can be offered as natural nutritive products. Regrettably, existing seed decontamination technologies are limited and have specific disadvantages. 5-aminolevulinic acid (5-ALA) as a novel and effective tool for wheat decontamination from microfungi is proposed in this work. Inhibition of wheat with 5-ALA revealed a drastically suppressed development of microfungi. Studies of wheat germination characteristics showed that 5-ALA stimulates the growth of wheat seedlings and roots without impairing the vigor of germination and the viability of seeds. 5-ALA also induces either marginal or significant activities of antioxidant enzymes which can be associated with enhanced cellular capacity to detoxify reactive oxygen species. The results indicate that 5-ALA application may be an effective, environmentally friendly and inexpensive technology to be used in producing sprouts for human consumption.

Towards environmentally and human friendly insect pest control technologies: photosensitization of leafminer flies Liriomyza bryoniae.

Development of new, ecologically safe technologies to control insect pest populations is of great importance. Photoactive compounds usually used for photosensitization might be effective as pesticide agents, with low impact on the environment, being non-toxic and not mutagenic. Phosensitizer accumulates within the insect body and, following exposure to visible light, induces lethal photochemical reactions and death. The aim of this study is to evaluate the possible usage of several photosensitizers (acridine orange, aminolevulinic acid, hematoporphyrin dimethyl ether, methylene blue) as photopesticides to control population of polyphagous plant pest Liriomyza bryoniae (Kaltenbach, 1858) (Diptera, Agromyzidae). Fluorescence measurements of intact cooled insects indicate that insect feeding with bait containing HPde and sugar induces remarkable accumulation of this compound in the body of insect. This accumulation is strongly dependent on sex and feeding duration. The highest HPde amount in the body of insect was detected 16 h after feeding, whereas no significant photosensitizer amount was detected in the same insect following 48 h. Following irradiation with visible light results in fast death of L. bryoniae. Of importance to note that survival of insects after feeding and irradiation depends on sex: female insect died much faster than males.

Public health and bioterrorism: renewed threat of anthrax and smallpox.

Bioterrorism is one of the main public health categorical domains. According to sociological analytics, in postmodern society terrorism is one of the real threats of the 21st century. While rare, the use of biological weapons has a long history. Recently, anthrax has been evaluated as one of the most dangerous biological weapons. Naturally occurring anthrax in humans is a disease acquired from contact with anthrax-infected animals or anthrax-contaminated animal products. Usually anthrax infection occurs in humans by three major routes: inhalational, cutaneous, and gastrointestinal. Inhalational anthrax is expected to account for most serious morbidity and most mortality. The clinical presentation of inhalation anthrax has been described as a two-stage illness. Many factors contribute to the pathogenesis of Bacillus anthracis. Antibiotics, anthrax globulin, corticosteroids, mechanical ventilation, vaccine are possible tools of therapy. Smallpox existed in two forms: variola major, which accounted for most morbidity and mortality, and a milder form, variola minor. Smallpox spreads from person to person primarily by droplet nuclei or aerosols expelled from the oropharynx of infected persons and by direct contact. In the event of limited outbreak with few cases, patients should be admitted to the hospital and confined to rooms that are under negative pressure and equipped with high-efficiency particulate air filtration. In larger outbreaks, home isolation and care should be the objective for most patients. Progress in detection, suitable vaccines, postexposure prophylaxis, infection control, and decontamination might be serious tools in fight against the most powerful biological weapon. To assure that the public health and healthcare system can respond to emergencies, the government should direct resources to strengthen the emergency-response system, create medication stockpiles, and improve the public health infrastructure.

Targeting PBR by hexaminolevulinate-mediated photodynamic therapy induces apoptosis through translocation of apoptosis-inducing factor in human leukemia cells.

Photodynamic therapy (PDT) with endogenous protoporphyrin IX derived from 5-aminolevulinic acid or its derivatives has been established for treatments of several premalignancies and malignancies; however, the mechanism of the modality is not fully elucidated. The mitochondrial permeability transition pore consists mainly of the mitochondrial outer membrane voltage-dependent anion channel and the peripheral benzodiazepine receptor (PBR) and the mitochondrial inner membrane adenine nucleotide translocator (ANT). These mitochondrial proteins are responsible for the permeability transition that leads to apoptosis. In the present study, the human leukemia cell line, Reh, was treated with PDT using hexaminolevulinate (HAL). More than 80% of apoptotic Reh cells were found after HAL-mediated PDT (HAL-PDT) with high-molecular-weight (50 kbp) DNA fragmentation. Addition of PK11195 or Ro5-4864, two ligands of PBR, during HAL-PDT significantly inhibited the apoptotic effect. Bongkrekic acid, a ligand for ANT, also reduced the PDT effect. Although the mitochondrial transmembrane potential collapsed, neither cytosolic translocation of mitochondrial cytochrome c nor activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase were found. However, nuclear translocation of mitochondrial apoptosis-inducing factor (AIF) was shown by both immunoblotting and immunocytochemistry. Because AIF is the sole one among all proapoptotic factors involved in caspase-dependent and caspase-independent pathways that induces the high-molecular-weight DNA fragmentation, we conclude that HAL-PDT specifically targets PBR, leading to apoptosis of the Reh cells through nuclear translocation of mitochondrial AIF. This study suggests PBR as a possible novel therapeutic target for HAL-based PDT of cancer.

Inactivation of Gram (-) bacteria Salmonella enterica by chlorophyllin-based photosensitization: Mechanism of action and new strategies to enhance the inactivation efficiency.

This study is focused on the enhancement of susceptibility of Gram (-) bacteria S. enterica to chlorophyllin-based (Chl) photosensitization combining it with other antimicrobial tools. In order to find best combinations, the mechanism by which Chl-based photosensitization inactivates bacteria must be identified. Data confirmed that photosensitization (Chl 1.5×10-5M, for 1-120min, 405nm, 0-46.1J/cm2) reduced S. enterica population, just by 2.05 log (CFU/ml). Fluorimetric measurements indicated that just minor part of Chl was bound to Salmonella in suspension. Addition of sodium azide (NaN3) (10mM) protected bacteria from killing, what means that 1O2 took place in photochemical reactions. Gene expression data confirmed that Chl-based photosensitization induced oxidative stress in bacteria cells, since mostly genes responsible for detoxification of ROS (OxyR, AhpC, GrxA) have been expressed in Salmonella. Moreover, the expression of genes, responsible for the inhibition of oxidative respiration (AtpC), cell division and down-regulation of metabolism (SulA) have been detected. In addition, Chl-based photosensitization induced significant release of intracellular components (absorbing at λ260 nm and λ280 nm) in bacteria that indicated increased membrane permeability. Thus, the combination of two antimicrobials (Chl-based photosensitization and chitosan (CHS)) with the same target (cellular membrane) in the presence of light drastically reduced viable Salmonella population (by 7.28 log). Combined treatment of photosensitization and high power pulsed UV light (HPPL) was also very effective, since reduced viable Salmonella by 7.5 log. Bacterial regrowth experiments clearly indicated that after both combined treatments Salmonella lost its ability to proliferate, and SEM images confirmed that after both treatments no viable bacteria have been found at all.

Radiosensitization of tumours by porphyrins.

Our previous data indicate, that hematoporphyrin dimethyl ether (HPde) can totally inhibit the growth of aggressive Ehrlich ascite tumour, when combined with low doses (2Gy) of ionizing radiation. Taking into account these findings, it appears of particular interest to evaluate the dependence of radiosensitizing efficiency of porphyrins on tumour aggressiveness. For this purpose two experimental tumour models (aggressive murine Ehrlich ascite carcinoma, (EAT), and not-aggressive hepatoma MH-22A) were used. Moreover, radiosensitizing properties of three porphyrin-type compounds of different chemical heterogeneity were evaluated (hematoporphyrin dimethyl ether (HPde), photofrin II (PII) and hematoporphyrin derivative (HPD)). Data obtained indicate, that HPde is the most effective one in this context (HPde>PII>HPD). It is important to note, that only the aggressive EAT tumours were radiosensitized by these dyes. No signs of radiosensitization (inhibition of tumour growth, injury of tumour tissue, evaluated by histological analysis) were observed in not-aggressive MH-22A hepatoma. Moreover, it was shown, that ligands of peripheral benzodiazepine receptors (PBR) might diminish the cell growth in aggressive EAT, but not in not-aggressive MH-22A hepatoma. The mechanism of radiosensitization by porphyrins, proposed in our previous studies, was strongly confirmed by these data. Actually, dicarboxylic porphyrins, being ligands of PBR, which are highly expressed in just aggressive tumours, can inhibit tumour cell proliferation and act in concert with ionizing radiation. Thus, combination of porphyrin and ionising radiation reflects the action of two antiproliferative factors, what eventually increases the response of aggressive tumours to the low doses of ionising radiation.

Mechanism of radiosensitization by porphyrins.

According to our previous data, hematoporphyrin dimethyl ether (HPde) at concentrations useful for photodynamic therapy can radiosensitize aggressive Ehrlich ascite carcinoma (EAT) to 2Gy irradiation inducing total tumour growth inhibition. The aim of this study was to further investigate the possible mechanism of radiosensitization of EAT by dicarboxylic porphyrin-HPde. Our results reveal that HPde is inducing several rearrangements in the EAT cells: 1.2 x 10-6 M of the photosensitizer diminishes the number of cells in mitosis by a factor of 3, increases the number of cells in the S phase of the cell cycle, modifies the activities of antioxidant enzymes glutation S-transferase (GST) and DT-diaphorase (DTD), and eventually induces slight apoptosis. Moreover, it was shown that HPde is a ligand of peripheral benzodiazepine receptor (PBR). Named "house keeper," PBR is usually responsible for all these perturbations, which, in our case, act in concert with the following ionizing radiation, producing the interaction of two antiproliferative/destructive factors.

Effective combination of LED-based visible light, photosensitizer and photocatalyst to combat Gram (-) bacteria.

The aim of this study was to investigate antimicrobial efficiency of combined treatments of visible light (405nm), chlorophyllin (Chl) and ZnO nanoparticles (NPs). Results obtained indicate that incubation of the Gram (-) Escherichia coli with Chl (1.5×10(-5)M) for 60min as well as illumination alone (405nm; 380kJ/m(2)) has no impact on the viability of E. coli. Just photosensitization (60min incubation with 1.5×10(-5)M Chl and following illumination, 405nm; 380kJ/m(2)) slightly reduces their population (∼1.5 log). ZnO NPs (5×10(-4)M) in the dark have negligible effect on E. coli viability. However, photocatalytic treatment (10min incubation with ZnO NPs (5×10(-4)M) and following illumination with light (λ=405nm; 380kJ/m(2)) resulted in significant reduction of viable cell population (4.6 log). Scanning electron microscopy (SEM) images indicate that cell surface-adhered NPs as well as chlorophyllin after illumination trigger cell shrinkage and eventually death. Simultaneous (at the same time) incubation 30min. with Chl and ZnO NPs and following illumination (380kJ/m(2)) was less effective than every treatment separately. Interaction of bacteria with photosensitizer and photocatalyst in the sequence (photosensitizer→photocatalyst→light) reduced the viability of bacteria by 3 log. But applying interaction of bacteria with photosensitizer and photocatalyst in opposite order (photocatalyst→photosensitizer→light) looks more promising as this reduced the Gram (-) E. coli microbial population by 4.5 log using 5 times lower photocatalyst concentration in comparison with efficiency of photoactivated ZnO NPs (4.6 log). In this case ZnO NPs interact electrostatically with negatively-charged cell surface and afterwards bind negatively charged Chl, thus act as mediators between negatively-charged cell surface and negatively-charged photosensitizer.

Inactivation of fungi in vitro by photosensitization: preliminary results.

Photosensitization is based on the interaction of 2 completely non-toxic agents--a photosensitizer, accumulated in microorganisms, and visible light. This interaction induces radical-based cytotoxic reactions in the presence of oxygen. The photosensitization phenomenon is widely involved in the treatment of tumors in oncology, in curing arthritis and atherosclerosis. In this work, the possibility to inactivate pathogenic and harmful fungi by photosensitization is shown. A new treatment methodology is proposed on the basis of effective inactivation of the several micromycetes, such as Aspergillus flavus, Trichothecium roseum, Fusarium avenaceum, Rhizopus oryzae, by photosensitization.

Experimental evidence on possibility to radiosensitize aggressive tumors by porphyrins.

Two murine experimental tumor models of different aggressiveness (murine Ehrlich ascitic carcinoma and MH-22A hepatoma) were used to investigate the radiosensitization by porphyrins. Data obtained clearly indicate that hematoporphyrin dimethyl ether, photofrin and hematoporphyrin derivative exert some radiosensitizing properties which are in clear correlation with purity of the compound. Of interest to note, that just aggressive Ehrlich ascitic carcinoma was radiosensitized to gamma-radiation, whereas no signs of radiosensitization were observed in MH-22A hepatoma tumor. Data obtained support the idea, that dicarboxylic porphyrins, being ligands of peripheral benzodiazepine receptors (responsible for proliferation and highly expressed in aggressive tumors) might induce several sublethal injuries in the cell which further work in concert with ionizing radiation producing synergistic interaction of two antiproliferative factors.