Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Inactivation of Foot-and-Mouth Disease Virus by Interaction of Dye and Visible Light

The inactivation of foot-and-mouth disease virus was studied by means of the interaction of neutral red, Toluidine Blue, and methylene blue with visible light. The virus, Type A, strain 1, CANEFA of Argentine origin, was grown in tissue culture and tested in the crude and clarified state. Virus and dye were mixed and incubated together at 4 C for 45 min in the dark, or were mixed and immediately exposed to the visible light source without prior incubation together. Mixtures of crude virus and dye, under any of the experimental conditions used, did not inactivate more than 1 to 2 logs of viral infectivity when held in the dark or when exposed to light during a period of 45 min. Complete inactivation of virus was achieved when clarified virus and dye were mixed and immediately exposed to the visible light source for 15 min. Prior incubation of clarified virus and dye permitted inactivation by methylene blue only, whereas no incubation prior to exposure resulted in three of the dyes contributing to inactivation. A concentration of 6 μg of neutral red, Toluidine Blue, methylene blue, and crystal violet was used per milliliter of virus suspension. Crystal violet was not a good viral inactivator under the conditions of the experimentation. Inactive virus induced the formation of neutralizing antibodies in adult chickens and mice. The antibody titer stimulated by the antigen treated with methylene blue and visible light was probably significant.     

Simulation of Na channel inactivation by thiazine dyes

Some dyes of the methylene blue family serve as artificial inactivators of the sodium channels when present inside squid axons at a concentration of approximately 0.1 mM. The dyes restore a semblance of inactivation after normal inactivation has been destroyed by pronase. In fibers that inactivate normally, the dyes hasten the decay of sodium current. Many dye-blocked channels conduct transiently on exit of the dye molecule after repolarization to the holding potential. In contrast, normally inactivated channels do not conduct during recovery from inactivation. Kinetic evidence shows that inactivation of a dye-blocked channel is unlikely or impossible, which suggests that dye molecules compete with inactivation "particles" for the same site. In the absence of tetrodotoxin, the dyes do not affect the ON gating current unless the interpulse interval is very short. If sufficient equilibration time is allowed during a pulse, the initial amplitude of the OFF gating current is reduced to near zero. This suggests that a dye molecule is a Na channel completely blocks that channel's gating current, even the fraction that is resistant to normal inactivation. Dyes block INa and Ig with the same time course. This provides the strongest evidence to date that virtually all of recorded "gating current" is associated with Na channels. Tetrodotoxin greatly slows dissociation of dye molecules from Na channels and reduced gating current during both opening and closing of the channels.     

Topo-optical reactions on the membrane of the human red cell ghost (author's transl)]

Previous studies have proved that the thiazin dyes toluidine blue, azure A, azure B, 1.9-dimethyl methylene blue and the quinolin dyes N,N'-diethylpseudoisocyanine chloride, N,N'-6,6'-dichlorpseudoisocyanine chloride are suitable for topo-optical reaction on the membrane of the red blood cells. In the present study the applicability of the thiazin and quinolin dyes on the membrane of the human red cell ghost was examined. Optical analysis revealed that the thiazin dyes are bound in radial position to the membrane, while the quinolin dyes are bound parallel to the membrane's plane.     

Photodynamic inactivation of Bacillus spores, mediated by phenothiazinium dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

Methylene blue directly oxidizes glutathione without the intermediate formation of hydrogen peroxide

Methylene blue stimulates the oxidation of glutathione in red blood cells in vitro and in vivo. This oxidation has been attributed to hydrogen peroxide that is generated from the autooxidation of leucomethylene blue arising from the reduction of methylene blue by NADPH. In this report we present evidence that methylene blue directly oxidizes glutathione and that oxidation of glutathione by hydrogen peroxide is a secondary reaction. Moreover, superoxide dismutase has no effect on the oxidation. Under aerobic conditions, methylene blue oxidizes glutathione 30 times faster than the spontaneous autooxidation of glutathione. Under anaerobic conditions the stoichiometry of the reaction of methylene blue with glutathione supports a direct chemical reaction. The reaction rates between glutathione and methylene blue suggest a second order reaction over the conditions tested. That neither oxygen radical formation nor significant amounts of hydrogen peroxide are produced by methylene blue, even in the presence of added glucose, is further confirmed by the failure to detect significant amounts of lipid peroxidation products, or hemolysis, in red blood cells incubated with the dye.     

Studies on the mechanism of the photosensitized inactivation of E. coli and reactivation phenomenon

In order to find a more satisfactory interpretation of the phenomenon of photosensitized inactivation of bacteria, studies were performed under various experimental conditions on methylene blue and E. coli. In summary the findings are as follow:— 1. The dye is absorbed by the bacteria according to the Langmuir isotherm and can be removed by ionic substitutions; the dye binding to the bacteria is predominantly ionic; the dye-bacteria complex produces a new absorption peak in the 610 mµ wave length region, and the action spectrum corresponds to the spectral absorption of the dye-bacteria complex. 2. There is an optimum dye concentration range for the photosensitized inactivation. 3. Photosensitized inactivation of bacteria can take place both in the frozen and liquid states and the presence of oxygen is essential to the inactivation process. 4. Hydrogen peroxide, formed by reoxidation of the reduced methylene blue, does not inactivate bacteria. 5. Following the photosensitized inactivation, E. coli lose their ability to reduce the methylene blue in the presence of various hydrogen donors, suggesting that enzymes are involved in the inactivation process. 6. Bacteria inactivated by photosensitization can be reactivated by prolonged storage after irradiation; the recovery rate increases with increasing temperature (maximum 37°), and is also influenced by the presence of various hydrogen donors. In view of collected experimental data, the basic reaction mechanisms are analyzed in photosensitized inactivation. The first step of the reaction seems to be excitation of the dye-bacteria, or dye-bacteria oxygen complex, by a photon which produces an activated complex. In such a state, molecular oxygen is capable of producing an oxidizing reaction, which results in the inactivation of the bacteria. Some aspects of the detailed reactions taking place at the cell surface are discussed.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

FURTHER STUDIES ON THE INHIBITION OF CYPRIDINA LUMINESCENCE BY LIGHT,WITH SOME OBSERVATIONS ON METHYLENE BLUE

1. Eosin, erythrosin, rose bengale, cyanosin, acridine, and methylene blue act photodynamically on the luminescence of a Cypridina luciferin-luciferase solution. In presence of these dyes inhibition of luminescence, which without the dye occurs only in blue-violet light, takes place in green, yellow, orange, or red light, depending on the position of the absorption bands of the dye. 2. Inhibition of Cypridina luminescence without photosensitive dye in blue-violet light, or with photosensitive dye in longer wave-lengths, does not occur in absence of oxygen. Light acts by accelerating the oxidation of luciferin without luminescence. Eosin or methylene blue act by making longer wave-lengths effective, but there is no evidence that these dyes become reduced in the process. 3. The luciferin-oxyluciferin system is similar to the methylene white-methylene blue system in many ways but not exactly similar in respect to photochemical change. Oxidation of the dye is favored in acid solution, reduction in alkaline solution. However, oxidation of luciferin is favored in all pH ranges from 4 to 10 but is much more rapid in alkaline solution, either in light or darkness. There is no evidence that reduction of oxyluciferin is favored in alkaline solution. Clark''s observation that oxidation (blueing) of methylene white occurs in complete absence of oxygen has been confirmed for acid solutions. I observed no blueing in light in alkaline solution.     

Additional Schiff-Type Reagents for Use in Cytochemistry

Twenty-four new Schiff-type reagents were discovered in a survey of 140 different dyes. These dyes include acid fuchsin, acridine yellow, acriflavine hydrochloride, azure C., Bismarck brown R, Bismarck brown Y, celestine blue B, chrysoidine 3R, chrysoidine Y extra, cresyl violet, crystal violet, gentian violet, methylene blue, neutral violet, phenosafranin, phosphine GN, proflavine, toluidine blue O, and toluylene blue. Positive results obtained with crystal violet and a few samples of methylene blue are considered due to impurities. Various chemical extractions, aldehyde blocking reagents, and enzymatic treatments were used to verify the aldehyde specificity of the above dye-SO₂, reagents as well as azure A, brilliant cresyl blue, neutral red, safranin O, and thionin which have been mentioned by other workers. These reagents were tested in the Feulgen reaction for DNA and the PAS reaction for polysaccharides. Absorption curves were obtained from individual nuclei stained for DNA. The absorption peaks ranged from 450 mμ, to 630 mμ. depending on the dye studied. The Feulgen reaction could be followed by the PAS reaction or vice versa in mouse intestine using reactive dyes of complementary colors. The evidence indicates that a potential Schiff-type reagent must have at least one free NH₂ group on the dye molecule.     

Chalcogenoxanthylium photosensitizers for the photodynamic purging of blood-borne viral and bacterial pathogens

Thio- and selenoxanthylium dyes were prepared by the addition of 2-lithiothiophene, 4-N,N-dimethylaminophenylmagnesium bromide, and 1-naphthylmagnesium bromide to the appropriate 2,7-bis-N,N-dimethylaminochalcogenoxanthen-9-one, followed by dehydration and ion exchange to the chloride salts. The corresponding chalcogenoxanthylium dyes were evaluated as photosensitizers for the inactivation of intracellular and extracellular virus in red blood cell suspensions and for the inactivation of selected strains of gram (+) and gram (-) bacteria in red blood cell suspensions. Selected combinations of photosensitizer and light gave >6 log10 inactivation of intracellular and extracellular virus, and >4 log10 inactivation of extracellular bacteria with varying levels of hemolyis, following a 42-day storage of red blood cell suspensions. Photocleavage experiments with plasmid DNA and the chalcogenoxanthylium dyes suggested the genomic material contained in the virus and in the bacteria as one possible target for the photodynamic action of some of these dyes.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

Induction of metachromasia and circular dichroism in the dye 1,9-dimethyl methylene blue by ATP

The theoretical prediction of induction of metachromasia [V Czikkely, H D Foersterling & H Kuhn (1970), Chem Phys Lett, 6,207] in a dye by a polyanion having only four to six anionic sites is proved experimentally, for the first time, in ATP--1.9-dimethyl methylene blue system. The findings show that ATP induces metachromasia in the dye at neutral pH, when ATP molecule remains fully charged providing four anionic sites to the dye cations. Conductometric titration shows that the dye molecules bind stoichiometrically to ATP (four dyes/ATP). However ATP at acidic pH and ADP and AMP at any pH fail to induce metachromasia. This is also the first report of induction of circular dichroism in bound dyes by ATP. Though the chiral moiety of ribose sugar in ATP may induce dichroism in the bound achiral dyes, the observed high molar ellipticity values indicate aggregation of bound dyes with twist in one sense initiated by the twisted conformation of the triphosphate chain in ATP. This inference on the state of conformation of ATP in its native environment is in agreement with that derived from PMR and spin lattice relaxation technique. It is thus interesting that the conformation of crystalline disodium ATP, as concluded from X-ray crystallography, is maintained by tetrasodium ATP in dilute aqueous solution--the native environment of ATP.     

Uptake of cationic dyes from aqueous solution by biosorption onto granular kohlrabi peel

A new, low cost, locally available biomaterial was tested for its ability to remove cationic dyes from aqueous solution. Granules prepared from kohlrabi peel had been utilized as a sorbent for uptake of three cationic dyes, methylene blue (MB), neutral red (NR) and acridine orange (AO). The effects of various experimental parameters (e.g., dye concentration, particle size, initial pH, contact time and other factors) were investigated and optimal experimental conditions were ascertained. Above the value of initial pH 4, three dyes studied could be removed effectively. The isothermal data fitted the Langmuir model in the case of NR sorption and the Freundlich model for all three dyes sorption. The biosorption processes followed the pseudo-first-order rate kinetics. The results in this study indicated that kohlrabi peel was an attractive candidate for removing cationic dyes from the dye wastewater.     

Studies on the toxin of Aspergillus fumigatus. XVIII. Photooxidation of asp-hemolysin in the presence of various dyes and its relation to the site of hemolytic activity

The site of hemolytic activity of a toxin isolated from Aspergillus fumigatus designated Asp-hemolysin was determined by photooxidation techniques. The hemolytic activity of this toxin was strongly inhibited by photooxidation with methylene blue, rose bengal, riboflavin, or eosin G as a sensitizer, whereas crystal violet, hematoxylin, naphthol yellow S, bromothymol blue, methyl orange, and cresol red had no effect. pH dependence of the inactivation with methylene blue was observed in the narrow range of pH values from 7.0 to 8.0, like that of the inactivation with rose bengal or riboflavin. The histidine, cysteine, methionine, tryptophan, and tyrosine content of methylene blue-photooxidized Asp-hemolysin was significantly decreased, while other amino acids were not affected. The hemolytic activity of the toxin was lost more slowly than the histidine residue, being maintained at about 50% even at the time when the histidine residue was completely lost after 30 min. Photooxidation of Asp-hemolysin in the presence of rose bengal also caused a decrease in histidine, methionine, and threonine content. These findings suggest that residues of cysteine, methionine, threonine, tryptophan, and/or tyrosine but not histidine may play an important role through stereostructure in the manifestation of the hemolytic activity of Asp-hemolysin.     

Mechanisms of Giemsa banding

Summary We investigated the capability of individual thiazins in Giemsa mixtures (methylene blue and azures A, B, and C) and of two related dyes (toluidine blue and thionin) to produce G-banding. We further tested the effects of variations of buffer composition and concentration, dye concentration, and staining time.G-banding was produced by all of the dyes at low concentrations, although differences were noted. Overall, methylene blue and azure B produced the best banding, azures A, C, and toluidine blue produced moderately good banding, and thionin produced poor banding. This order did not appear to be altered essentially by different treatments. The optimal conditions for G-banding for all dyes and treatments included the use of (1) 0.025–0.05M phosphate buffer, (2) dye concentrations of 0.002%–0.005%, and (3) staining times of 6–15 min.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.