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The photodecontamination of cellular blood components: Mechanisms and use of photosensitization in transfusion medicine
... Photodynamic action (PDA) is widely used for the therapy of neoplasms [1,2], infected wounds and other pathological foci of various etiologies [3][4][5], disinfection of biological fluids, blood components, and water [6]. The photodynamic effect is the photooxidative destruction of molecular structures that ensure the functioning of tumor cells and pathogens (bacteria, viruses) by reactive oxygen species (ROS). ...
... In this work, such an assessment was carried out for aqueous solutions of cationic PS, which are distinguished by high values of extinction and quantum yield of ROS generation: methylene blue, C16H18ClN3S (ε664 = 0.61 × 10 5 M −1 cm −1 ); zinc octakis(cholinyl)phthalocyanine, ZnPcChol8 (ε682 = 1.7 × 10 5 M −1 cm −1 ); meso-tetrakis[1-(2'bromoethyl)-3-pyridyl]-bacteriochlorin tetra-bromide, (3-PyBrE)4BCBr4 (ε762 = 1 × 10 5 M −1 cm −1 ). These PS do not aggregate in a wide range of concentrations and are promising both for photodynamic inactivation of pathogens [6,8,9,13] and for antitumor photodynamic therapy [10,14,15]. Fig. 1 shows the normalized spectral contours A(λ) of the absorption bands of these PSs (the absorption spectrum at low concentration divided by its maximum value) and the normalized emission spectra Inorm(λ) of LEDs (mLED-664, mLED-684, mLED-763, Biospec, Russia), which can be used to excite these PSs (light intensity spectral density divided by the integral intensity). ...
... The loss of a part of the light energy reduces the efficiency of the PDT as a whole. This loss must be taken into account when predicting PDA results and optimizing its tactics, particularly when choosing light sources and parameters of irradiation for photodynamic disinfection of surfaces from viral and bacterial infections [2,5], or clinical photodynamic treatment to local virus-infected foci [5,6]. The obtained results are also important for in vitro studies and comparing the photodynamic efficiency of PSs [10,14] since the values of RAPD are very different for PSs with different extinction, especially when using broadband light sources. ...
... Cationic and amphiphilic derivatives of phthalocyanines readily inactivated enveloped herpes viruses [78]. Phthalocyanines with positively charged residues on the central silicon atom were successfully used for disinfection of blood components from human immunodeficiency virus [79]. Cationic substituted phthalocyanines with high quantum yield of singlet oxygen and photostability [80,81] were effective against enveloped viruses [21,82,83]. ...
The COVID-19 pandemic has updated research on inactivation of coronaviruses with physical, chemical, and physical-chemical treatments. The review focuses on the inactivation of coronaviruses including the recent SARS-CoV-2 and viruses of other groups with analogous structure using optical radiation. The antiviral effects of different optical ranges from vacuum ultraviolet to infrared radiation are described in terms of the mechanisms of virus photoinactivation, sensitive molecular targets and efficacy. Direct and photosensitized damaging effects of light on the viral molecular structures are considered. Information on the applied pathogen photoinactivation technologies and the advantages of light sources for future applications is provided.
... Les photo-sensibilisateurs sont utiles dans des domaines variés, en particulier car il est aisé de contrôler le déclenchement des réactions qu'elles provoquent, en contrôlant l'irradiation lumineuse. Ces molécules peuvent être naturelles, comme la chlorophylle, l'hématoporphyrine ou la protoporphyrine IX ( fig. 25 Dans un cadre industriel, les photo-sensibilisateurs sont aussi utilisés pour la décontamination de produits sanguins [Ben-Hur et al., 1996], au cours de synthèses chimiques nécessitant une oxydation forte, ou même pour le retraitement des eaux usées [Esser et al., 1994 ;Jiménez-Hernandez et al., 2005]. ...
La mitochondrie est à la fois le lieu de la production d’énergie de la cellule et le centre régulateur de la mort cellulaire. L’efficacité de ces 2 fonctions est liée au niveau d’oxydation de la membrane mitochondriales interne (MMI). Le but de ce travail est de comprendre l’influence des lipides de la membrane sur sa résistance au stress oxydant. Les expériences sont menées sur des liposomes mimant la MMI. Leur oxydation est déclenchée par l’irradiation de la Ce6, une molécule photo-sensibilisatrice ayant une forte affinité pour les bicouches lipidiques. D’abord, l’association entre membranes et Ce6 est quantifiée par fluorimétrie. Ces mesures permettent de garder constant le ratio Ce6/lipides d’une composition membranaire à l’autre. Nous étudions ensuite, par microscopie à contraste de phase, l’influence de la nature des têtes des lipides et de la position, du nombre et de la conformation des insaturations des chaines lipidiques sur la perméabilisation du liposome déclenchée par son oxydation. Cette perméabilisation est due à deux phénomènes : la formation de défauts dans la bicouche et l’ouverture de pores micrométriques dans la membrane. Les seconds ne permettent une fuite significative que pour des membranes de composition proche des MMI. La présence du cardiolipide, spécifique aux mitochondries, retarde et réduit la perméabilité induite. La perméabilisation est d’autant plus efficace que les lipides comportent des insaturations et qu’elles sont proches de la tête polaire. La spectrométrie Raman donne en outre accès à la chimie du phénomène. Notamment, l’oxydation de la membrane n’est possible que si les électrons engagés dans la double liaison peuvent être délocalisés.
... The LABAs are readily soluble in water, ethanol, and acetone, and they generate reactive oxygen species (ROS) when exposed to a visible light source 156,159,190,191 . The ROS produces bacteria cellular injuries including DNA damage, a disruption of membrane integrity, and this induces bacteria death 192,193 . ...
This thesis details the anti-biofouling property of superhydrophobic surface and white light-activated bactericidal polymers, and self-cleaning and bactericidal paints for preventing hospital associated infection. To investigate the anti-biofouling property of superhydrophobic surface over a long period of time, superhydrophobic surfaces were made using 1H, 1H, 2H, 2Hperfluorooctyltriethoxysilane, P25 TiO2 nanoparticles, ethanol, and double sided tape. The bacteria adhesion of the superhydrophobic surface was tested through full immersion of four different bacteria suspensions for 1, 4, 8, 16, and 24 h and then the result was compared with other surfaces containing glass, polystyrene, and polyurethane. Changes of the tested surfaces were investigated by water contact angle meter, SEM, AFM, and confocal microscope. Through a simple swell-encapsulation shrink process, white light-activated bactericidal polyurethane was produced. Toluidine blue O and silver nanoparticles were encapsulated into a polyurethane and characterised by water contact angle meter, UV/Vis spectrometer, fluorescence microscope, and material testing and inspection device. Crystal violet and acrylic latex, which is a widely used paint material for home decoration, were mixed together to produce photobactericidal paints for the first time. At various mixing ratio, crystal violet and acrylic latex were combined together. The paint coated slide was characterised using water contact angle meter, and UV/Vis spectrometer, and its stability was investigated through liquid leaching test Crystal violet, toluidine blue O, P25 TiO2 nanoparticles, and 1H, 1H, 2H, 2Hperfluorooctyltriethoxysilane were used to produce dual functional paints with superhydrophobic and bactericidal behaviour. TiO2, TBO, and CV paints were fabricated via physical and chemical reaction. The dual functional paint coated slides were investigated in terms of water repellence, self-cleaning, and anti-biofouling properties, and was also characterised by SEM, AFM, and UV/Vis spectrometer. Bactericidal properties of the treated polyurethane and paint, dual functional paints were assessed with Escherichia coli and Staphylococcus aureus. The tested samples demonstrated not only potent photobactericidal activity in white light (typical 5 hospital lamp) but also bactericidal activity in dark. It is expected that bactericidal materials detailed in this thesis will be useful for use in healthcare facilities in order to reduce hospital associated infections.
... 15 These pathogen reduction devices include: the use of a combination of a psoralen and ultraviolet A (UVA) light; riboflavin and visible light; ultraviolet B irradiation; and the addition of methylene blue or phthalocyanines with visible light. [57][58][59][60] With regard to the bacterial inactivation of contaminated platelet concentrates, Lin et al reported that the novel psoralen amotosalen (S-59), combined with UVA light exposure has effective bacterial inactivation capability. 61 Thus, a combination of 150 µmol/L of the psoralen S-59 and 3 joules/cm UVA light result in significant bacterial reduction of contaminated apheresis platelets, as well as whole blood-derived platelets. ...
Bacterial contamination of transfusion products, especially platelets, is a longstanding problem that has been partially controlled through modern phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage. Bacterial contamination of platelet products has been acknowledged as the most frequent infectious risk from transfusion occurring in approximately 1 of 2,000–3,000 whole-blood derived, random donor platelets, and apheresis-derived, single donor platelets. In the US, bacterial contamination is considered the second most common cause of death overall from transfusion (after clerical errors) with mortality rates ranging from 1:20,000 to 1:85,000 donor exposures. Estimates of severe morbidity and mortality range from 100 to 150 transfused individuals each year.
Concern over the magnitude and clinical relevance of this issue culminated in an open letter calling for the “blood collection community to immediately initiate a program for detecting the presence of bacteria in units of platelets.” Thereafter, the American Association of Blood Banks (AABB) proposed new standards to help mitigate transfusion of units that were contaminated with bacteria. Adopted with a final implementation date of March 1, 2004, the AABB Standard reads “The blood bank or transfusion service shall have methods to limit and detect bacterial contamination in all platelet components.”
This Joint ASH and AABB Educational Session reviews the risks, testing strategies, and regulatory approaches regarding bacterial contamination of blood components to aid in preparing practitioners of hematology and transfusion medicine in understanding the background and clinical relevance of this clinically important issue and in considering the approaches currently available for its mitigation, as well as their implementation.
In this chapter, Drs. Hillyer and Josephson review the background and significance of bacterial contamination, as well as address the definitions, conceptions and limitations of the terms risk, safe and safety. They then describe current transfusion risks including non-infectious serious hazards of transfusion, and current and emerging viral risks. In the body of the text, Dr. Blajchman reviews the prevalence of bacterial contamination in cellular blood components in detail with current references to a variety of important studies. He then describes the signs and symptoms of transfusion-associated sepsis and the sources of the bacterial contamination for cellular blood products including donor bacteremia, and contamination during whole blood collection and of the collection pack. This is followed by strategies to decrease the transfusion-associated morbidity/mortality risk of contaminated cellular blood products including improving donor skin disinfection, removal of first aliquot of donor blood, pre-transfusion detection of bacteria, reducing recipient exposure, and pathogen reduction/inactivation. In the final sections, Drs. Vostal, Epstein and Goodman describe the regulations and regulatory approaches critical to the appropriate implementation of a bacterial contamination screening and limitation program including their and/or the FDA’s input on prevention of bacterial contamination, bacterial proliferation, and detection of bacteria in transfusion products. This is followed by a discussion of sampling strategy for detection of bacteria in a transfusion product, as well as the current approval process for bacterial detection devices, trials recommended under “actual clinical use” conditions, pathogen reduction technologies, and bacterial detection and the extension of platelet storage.
... These pathogen reduction approaches include the combined use of a psoralen and ultraviolet (UVA) light; riboflavin and visible light; ultraviolet B irradiation; and the addition of methylene blue and pthalocyanines. [20][21][22][23] Lin et al. reported that the novel psoralen amotosalen (S-59) combined with UVA light exposure has effective bacterial inactivation capability in platelet concentrates. 24 A combination of 150 mm/L of S-59 and 3 joules per cm 2 of UVA light resulted in significant bacterial reduction of contaminated apheresis platelets as well as whole-blood-derived platelets. ...
Transfusion reactions associated with bacteria and/or their products, during or following a blood transfusion, were one of the earliest recognized complications of allogeneic blood transfusions. Bacterial contamination of blood products has, therefore, been a problem for many decades and, at the present time, is likely the most common microbiological cause of transfusion-associated mortality and morbidity. Septic reactions associated with platelet concentrates appear to be much more common than those associated with contaminated red blood cells. The overall prevalence of contaminated cellular blood products has been estimated to be approximately 1 in 3000; however, the transfusion of contaminated platelet or red blood cell units may not necessarily be associated with clinically apparent morbidity in recipients because the majority of contaminated blood product units contain relatively few organisms. Unfortunately, though, in a minority of instances, contaminated blood product units contain clinically significant numbers of bacteria as well as endotoxins that may be associated with significant mortality and/or morbidity. The incidence of clinically evident severe septic episodes has not yet been clearly established, but is probably in the order of 1 per 50,000 platelet units transfused and 1 per 500,000 red blood cell units transfused. In recent years, a variety of interventions have been proposed, and in some instances implemented, in an effort to reduce this significant and potentially preventable transfusion risk.
... Currently PLTs are stored for up to 5 days to prevent bacterial contamination [108]. Over the past 10 years, a number of pathogen reduction methods have been reported, which enable the reduction of viruses, bacteria, and protozoa that are present in cellular blood products by means of photodynamic or photochemical methods [109][110][111][112][113][114][115]. Even in absence of contaminating agents, storage itself causes a variety of changes in PLT function and morphology, the so-called PLT storage lesions [116,117]. ...
Since the genomic era has not fully kept its promises, studies addressing the protein complement to the genome have been recently gaining momentum. Proteomics investigations could be potentially used from bench to bedside, in order to test the quality of collected blood components prior to or during storage. In parallel, proteomics could be used to verify the effects of the production and pathogen reduction processes of plasma derivatives and blood components on the protein fractions, or to reduce the effects of storage lesions. Another area of interest is represented by the discovery of peculiar biomarkers readily adoptable for targeted evaluation of blood-component integrity or functionality, as well as to assess the proliferative capacity of hematopoietic stem/progenitor cells. These accumulating basic research evidences will hopefully be accompanied by actual applications in routine clinical practice. Whether the costs of the needed facilities (instruments and trained personnel) will meet the current demand of the clinical market, proteomic-expert transfusionists will no longer only inform, but also perform a role in clinical routine.
... Com o aparecimento do HIV na década de 80 e o descobrimento de novos agentes virais transmitidos por via transfusional (HCV, HTLV I/II, etc.), a constatação de que bactérias também criam sérios problemas ao receptor, e o recente descobrimento de agentes emergentes ou re-emergentes na população mundial, o processo de inativação universal de patógenos presentes no sangue doado passou a ser uma meta prioritária na pesquisa hemoterápica. Desta forma, ao longo dos últimos 10 anos, vários compostos foram pesquisados e desenvolvidos 4 10 13 48 . ...
... Photodynamic therapy (PDT)? has been widely studied as a tumor therapy and also for other applications such as the sterilization of blood products (1). The efficacy of the treatment depends on the type of sensitizer and its concentration, the oxygen tension in the tumor, the total light dose and the sensitivity of the tumor for the treatment. ...
It has been shown previously that the efficiency of photodynamic therapy (PDT) both in vivo and in vitro is dependent on fluence rate. In this study, different in vitro experiments showed that tetrasulfonated aluminum phthalocyanine (AIPcS4) is more efficient in photosensitization if the light is delivered at low fluence rate. Erythrocyte damage, virus inactivation and photooxidation of reduced glutathione (GSH) and histidine were all enhanced if light was delivered at 100 W/m2 as compared to 500 W/m2. Bleaching did not occur under these conditions. Oxygen depletion, shown to be important in fluence rate effects observed in vivo, does not seem to be involved. On theoretical grounds saturation of the triplet state is not likely under these conditions. A possible explantation for the observed fluence rate effects might be found in different reaction pathways, that are favored under high or low fluence rate illuminations. These reactions might involve uni- or bimolecular reactions of intermediate products, resulting in less efficiency at higher fluence rate. It proves to be important, under all circumstances, to monitor fluence rate, because a change in fluence rate, even with similar total fluences, might influence photobiological results in an unexpected way.
... Again, enveloped viruses such as HIV, VSV and HSV are generally amenable to photoinactivation, [60][61][62] whereas the non-enveloped virus, cephalomyocarditis virus, is not, indicating that the viral envelope is a target for phthalocyanine photosensitization. 63 The use of phthalocyanines against various forms of HIV infection has been reported, and once again paralleling the development of these compounds in cancer PDT, aluminium and silicon phthalocyanines appear to show considerable promise, both groups of compounds exhibiting light absorption in the near infrared and efficient sensitization of singlet oxygen. In terms of chemical make-up, the silicon phthalocyanines used as photodynamic agents are functionalized axially through the silicon atom, rather than in the periphery of the aromatic system and this appears to produce highly active compounds. ...
Whereas the photodynamic therapy (PDT) of cancer has recently shown rapid clinical acceptance, photodynamic antimicrobial chemotherapy (PACT)--which predates the related cancer regimen--is not widely appreciated. Like PDT, PACT utilizes photosensitizers and visible or ultraviolet light in order to give a phototoxic response, normally via oxidative damage. Currently, the major use of PACT is in the disinfection of blood products, particularly for viral inactivation, although more clinically-based protocols are being developed, e.g. in the treatment of oral infection. The technique has been shown to be effective in vitro against bacteria (including drug-resistant strains), yeasts, viruses and parasites. A wide range of photosensitizers, both natural and synthetic, is available with differing physicochemical make-up and light-absorption properties. PACT is proposed as a potential, low-cost approach to the treatment of locally occurring infection.
The problem of blood-borne infections remains relevant in transfusion medicine. Pathogen reduction technologies (PRT) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. To date, PRT widely used for a number of blood components, however, the use of these technologies for the treatment of erythrocyte-containing components has not been studied. Objective: to conduct a comparative analysis of the clinical efficacy of transfusions of pathogen-reduced and gamma-irradiated erythrocyte suspension in pediatric patients with various oncological and hematological diseases. Seventy transfusions of red blood cell suspensions (RBC-S) (35 transfusions of pathogen-reduced RBC-S and 35 transfusions of gammairradiated RBC-S) in pediatric patients with oncological and hematological diseases were analized. Clinical efficacy parameters such as the hemoglobin and the hematocrit increment after transfusion, the interval between transfusions, the frequency and severity of transfusion reactions were estimated. We also evaluated the correlation between the hemoglobin and the hematocrit increment with age, patient’s body weight, the hemoglobin concentration and patient's hematocrit before transfusion, the volume of transfusion, the hemoglobin dose and the adjusted hemoglobin dose received for transfusion. We found that the clinical efficacy and safety of RBC-Ss of the compared groups did not differ: the hematocrit and the hemoglobin increment, the frequency and severity of transfusion reactions, and the interval between transfusions were comparable between groups. There was no evidence of immune elimination and allo-sensibilization caused by pathogen-reduced RBC-S. In the group of patients receiving pathogen-reduced RBC-S, a correlation was found between the increase in the hemoglobin and hematocrit values with some of the EV indices. According to our data, the spectrum of efficiency and safety indicators of pathogen-reduced RBC-S is no worse than that of gamma-irradiated RBC-S, provided that RBC-S is used for 14 days of storage.
Carbon (identified by a symbol C in the periodic table of elements, Fig. 1.3) is an interesting element; you cannot see (C, pronounced as see) the C all the time, but the existence of the element (C) is almost everywhere. It is in your clothes, in your backpack, in your food, and in your body. It provides the framework for all tissues of plants and animals (rational and irrational). These tissues are built of elements grouped around chains or rings made of carbon atoms. Indeed, most of the structures that make up animals, plants, and microbes are made from three basic classes of molecules: amino acids, carbohydrates, and lipids (often called fatty acids), and all of these materials contain carbon as their backbone [1, 2]. As these molecules are vital for life, metabolic reactions either focus on making these molecules during the construction of cells and tissues, or breaking them down and using them as a source of energy during digestion [3]. As a matter of fact, carbon (C) occurs in nature as the sixth most abundant element in the universe and the 19th element in order of mass in the Earth’s crust.
Toluidine blue O (TBO) dye together with either silver (Ag) nanoparticles (NPs), gold (Au) NPs, or a mixture of Ag and Au NPs (Mix Ag-Au NPs) were incorporated into polyurethane to make antimicrobial surfaces using a swell-encapsulation-shrink process. Antimicrobial testing against Escherichia coli showed that inclusion of the NPs significantly enhanced the antimicrobial activities of the TBO polyurethane samples. In particular, samples containing Ag NPs exhibited potent antimicrobial activity under white light and surprisingly, also in the dark. The numbers of viable bacteria decreased below the detection limit on the TBO/Ag NPs incorporated samples within 3 h and 24 h under white light and dark conditions. A mechanistic study using fufuryl alcohol indicated that the enhanced photobactericidal activity was most likely due to a type I photochemical reaction. To the best of our knowledge, this is the first report of an antimicrobial surface comprised of a combination of Ag NPs and a light activated agent to provide a dual kill mechanism. These surfaces are promising candidates for use in healthcare environments to reduce the incidence of hospital-acquired infections.
Great improvements have been made in the past to ensure the safety of blood supplied during emergencies by donor screening, selection and deferral techniques. Despite these improvements, the risk of transmitting infections through blood transfusion remains. This concern coupled with the fears that new pathogens will be identified in future that escape detection has led to the development of methods for the inactivation of pathogens in the blood products to ensure complete safety. Photochemical sterilization of blood components is considered to be the most promising technology that may reduce the risk of transmitted diseases while sparing the structure and functions of the blood components. Various photochemical approaches depending upon the target have been adopted for sterilization of blood and its components. Advances in photochemical approaches for blood sterilization has been reviewed in this article.
IntroductionDescription and Brief History of PDTPDT MechanismsBlood and PDTProperties of Blood, Blood Cells, and Photosensitizers: Before Photodynamic ReactionPhotodynamic Reactions in Blood and Blood Cells, Blood Components, and CellsTypes of Photodynamic Reactions in Blood: In vitro versus In vivoBlood Sample In vitro as a Model Studying Photodynamic ReactionMonitoring of Oxygen Consumption and Photobleaching in Blood during PDT In vivoPhotodynamic Disinfection of BloodPhotodynamic Therapy of Blood Cell CancerSummaryAcknowledgmentsGlossaryReferences
There is considerable current interest in photodynamic inactivation (PDI) as potential antimicrobial therapy. This study reports successful implementation of PDI of Staphylococcus
epidermidis using methylene blue (MB) in combination with biogenic gold nanoparticles (GNP). Monodispersed colloidal GNP were synthesized by reduction of Au+3 in the presence of cell-free filtrate of Trichoderma koningii and were characterized by a number of techniques including UV–Vis and fluorescence spectroscopy, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) to be 12 ± 3 nm spherical gold particles coated with proteins. Studies on the role of the cell-free filtrate proteins in the synthesis of the GNP indicate that the process is nonenzymatic but involves interactions of various amino acids with gold ions. A Xe lamp (550–780 nm) or a He–Ne laser (632 nm) was used as light sources to study the effect of MB alone, the GNP alone, and the MB–GNP mixture on the viability of bacterial cells. Lethal photosensitization of S. epidermidis with the MB–GNP mixture was achieved after 5 and 10 min exposure to laser or Xe lamp, respectively. It has been found that the MB–GNP mixture exhibits a significant antibacterial activity already in the absence of any light source and gives an enhanced antimicrobial response when using either a laser or a Xe lamp source for photosensitization.
Polyurethane polymer sheets were embedded with methylene blue (MB), toluidine blue (TBO) and 2nm gold nanoparticles by a swell–shrink encapsulation process using an acetone water mixture. The polymers were characterised by IR, UV–vis, SEM/WDX and mechanical testing. The dye impregnated polymers were potent at killing suspensions of Staphylococcus aureus under white illumination with a hospital light source (2000lux) showing a 2.8log10 kill (MB) and a 4.3log10 kill (TBO) compared to controls when exposed to 24h of illumination. Notably the incorporation of gold nanoparticles with the dyes enhanced the observed kill to 3.8log10 (MB) and 4.8 log10 (TBO). This is the first time that a derivatised polyurethane polymer used in catheters has been shown to be effective in killing bacteria using white light illumination.
— Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.
During the past decades major improvements in blood safety have been achieved, both in developed and developing countries. The introduction of donor counseling and screening for different pathogens has made blood a very safe product, especially in developed countries. However, even in these countries, there is still a residual risk for the transmission of several pathogens. For viruses such as the human immunodeficiency virus (HIV), and the hepatitis viruses B and C, this is due mainly to window-period donations. Furthermore, the threat of newly emerging pathogens which can affect blood safety is always present. For example, the implications of the agent causing new variant Creutzfeld-Jakob disease for transfusion practice are not yet clear. Finally, there are several pathogens, e.g. CMV and parvo B19, which are common in the general donor population, and might pose a serious threat in selected groups of immunosuppressed patients. In the future, further improvements in blood safety are expected from the introduction of polymerase chain reaction for testing and from the implementation of photochemical decontamination for cellular blood products. The situation in transfusion medicine in the developing world is much less favorable, due mainly to a higher incidence and prevalence of infectious diseases.
The photochemical reactions of dipyridamole (DIP), a healing agent, was studied to predict the photobiological implications of its use which will be the subject of a future article. Its photolysis product under UV-A and aerobic conditions was isolated and identified. DIP was shown to be photostable under inert atmosphere (argon). On the other hand, under the same conditions a process of electron transfer was detected in the presence of nitro blue tetrazolium (NBT) which can be inhibited by oxygen. The photodegradation of DIP occurs probably via a type II mechanism involving irreversible trapping of self-photogenerated reactive oxygen species (ROS). This fact could be indicative of its antioxidant activity. The formation of singlet oxygen and superoxide during the DIP photodegradation makes one suspicious of a possibility that DIP could also be involved in oxidative stress in biological systems. Further studies on biological systems will contribute to elucidate this probable dual behavior.
BACKGROUND: All studied photosensitizers for virus inactivation impair RBCs. To reduce damage to the RBCs without affecting virucidal activity, selective protection of the RBCs is necessary. The ability of the band 3 ligand, dipyridamole, to react with singlet oxygen and to increase the selectivity of photosterilization was investigated.
STUDY DESIGN AND METHODS: Solutions of dipyridamole were illuminated in the presence of tetrasulfonated aluminum phthalocyanine (AlPcS4) and dimethylmethylene blue (DMMB). Solutions of amino acids, RBCs, and vesicular stomatitis virus (VSV) in RBC suspensions were photodynamically treated in the presence or absence of dipyridamole.
RESULTS: Illumination of a solution of dipyridamole in the presence of AlPcS4 or DMMB resulted in changes in the optical spectrum of dipyridamole. The photooxidation of dipyridamole was inhibited by azide and augmented by D2O, which suggests the involvement of singlet oxygen. Photooxidation of amino acids and photodamage to RBCs was strongly reduced in the presence of dipyridamole. In contrast, photoinactivation of VSV in RBC suspensions was only slightly affected by dipyridamole.
CONCLUSION: Dipyridamole can improve the specificity of photodynamic sterilization of RBC concentrates, thereby increasing the practical applicability of this photodecontamination method.
Blood components have replaced whole-blood transfusion because they allow better maintenance of quality, more effective use of the donor's blood and more appropriate dosage to the patients. The techniques for the preparation and storage of components, both from whole blood and via apheresis, have developed gradually. The oxygen-releasing capacity of the erythrocytes is still influenced negatively even after short storage with currently used systems, but better methods have been described. Leukocyte removal from red cell and platelet preparations results in different degrees of purity. Filtration as well as some apheresis techniques have improved in recent years so that the aim, <5 × 106 leukocytes per transfusion, can be achieved confidently. Transfusion-associated graft-versushost disease requires attention. Collection of haematopoietic stem cells from peripheral blood for transplantation has become an important new task. Bacterial contamination, being a greater problem in platelet concentrates than earlier believed, can be detected by testing and probably soon counteracted by decontamination.
A number of potentially therapeutical drugs, which are membrane impermeant, are captured and degraded in the lysosomes after cellular uptake, and are thereby prevented from executing their biological function. However, in the present thesis a technology is presented that makes it possible to overcome this substantial barrier for the cellular delivery of functional macromolecules. The technology, photochemical internalisation (PCI), is a unique procedure for light-induced release of several types of membrane impermeant molecules from endocytic vesicles to the cytosol of the target cells. The PCI technology can be utilised as a new efficient and site-specific method for drug delivery in the therapy of cancer and other diseases, and it can also be applied as research tool for macromolecule delivery both in vitro and in vivo.
The safety of blood transfusion can be increased by introducing methods that eliminate blood-borne pathogens such as viruses and bacteria. In this thesis, the use of photodynamic treatment (PDT) to inactivate pathogens in cellular blood products is described. Various photosensitizers, from phenothiazines to porphyrins, were tested for their efficacy to inactivate a wide range of pathogens with minimal induction of blood cell damage. From this series of photosensitizers, meso-substituted mono-phenyl-tri (N-methyl-4-pyridyl) porphyrin [Tri-P(4)] stood out as the most efficacious. Besides the virucidal and bactericidal efficacy of PDT, we also studied the effect of the treatment on the quality and functionality of red blood cells and of haematopoietic stem cells from cord blood stem cell products. In addition, we evaluated the potential of Tri-P(4)-mediated PDT to induce immunomodulation
The silicon phthalocyanine, HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc 4), is a new photosensitizer that can inactivate lipid-enveloped viruses in red blood cell concentrates (RBCC) upon exposure to red light. Because Pc 4 is insoluble in water, it was delivered either as an emulsion in saline and cremophor EL (CRM) or as a solution in dimethyl sulfoxide (DMSO). In RBCC, Pc 4 added in either vehicle distributed between the plasma and red blood cells (RBC) in a ratio of 4:6, similar to the ratio of these components in RBCC 3:7 (i.e. a hematocrit of 70%). Light exposure did not affect this distribution and caused only marginal degradation of Pc 4 at a light dose that inactivates > 5 log10 vesicular stomatitis virus (VSV). Among human plasma proteins, Pc 4 bound mainly (about 70%) to lipoproteins and to a lesser extent to albumin and lower molecular weight proteins when delivered in DMSO. When delivered in CRM, distribution between lipoproteins and albumin became more even. Among the lipoproteins Pc 4 bound almost exclusively to very low-density lipoproteins (VLDL) when delivered in DMSO and to both VLDL and low-density lipoproteins when added in CRM. The rate of VSV inactivation was independent of the delivery vehicle but there was less RBC damage, as measured by hemolysis during storage, when Pc 4 was added in CRM. These results indicate that using CRM as emulsifier can enhance the specificity of Pc 4-induced photochemical decontamination of RBCC for transfusion.
The use of phthalocyanines in conjunction with red light has been shown to inactivate model lipid-enveloped viruses in red cell concentrates. The ability of this treatment to inactivate multiple forms of human immunodeficiency virus (HIV) was evaluated in this study.
The phthalocyanines used were aluminum phthalocyanine tetrasulfonate (AIPcS4) and the silicon phthalocyanines HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), and HOSiPcOSi(CH3)2 (CH2)3N+(CH3)3I-(Pc 5). HIV was studied in a cell-free form, in an actively replicating form, in latently infected cells, and in blood from HIV-positive patients.
All three phthalocyanines inactivate > or = 10(5) infectious doses of cell-free HIV. However, only Pc 4 effectively inactivated actively replicating HIV and latently infected cells. The latter was about four times as sensitive to inactivation as was actively replicating HIV. Increasing the hematocrit of red cells during treatment decreased the rate of inactivation, especially at lower light doses. Under treatment conditions that completely inactivated the laboratory isolates of HIV, cell-associated HIV in blood from HIV-positive patients was also completely inactivated. The polymerase chain reaction signal from the gag gene of HIV was not affected on treatment of cell-free virus, but it was reduced after treatment of cell-associated HIV, particularly in some latently infected cell lines.
Pc 4 and red light are effective in eliminating the infectivity of HIV in red cell concentrates. The usefulness of this approach for blood banking depends on future demonstration of the preservation of red cell circulatory survival and function in vivo.
Phthalocyanines are useful sensitizers for the photodynamic sterilization of red blood cell concentrates. The mechanism of photoinactivation of lipid-enveloped viruses is not completely understood. Vesicular stomatitis virus (VSV) was used as a model virus to study the primary targets of photoinactivation by aluminum phthalocyanine tetrasulfonate (AIPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Inactivation conditions for VSV in buffer were determined using an end point dilution assay, and viral RNA synthesis in host cells was measured to determine the loss of infectivity in a direct way. The very rapid decrease in the viral RNA synthesis after photodynamic treatment was correlated with respect to different potential primary targets that are involved in different steps of the viral replication cycle. Damage to the viral proteins, induced by treatment with AIPcS4 or Pc4 and analyzed by gel electrophoresis, could not account for the observed loss of infectivity. Binding of VSV to host cells was only slightly impaired after photodynamic treatment with both sensitizers and could therefore not be responsible for the rapid decrease in viral RNA synthesis in cells. A very strong inhibition of viral RNA polymerase activity after treatment with AIPcS4 and red light was detectable using an in vitro assay. This decrease correlated well with the loss of infectivity, indicating that either the RNA or the viral RNA polymerase is the primary target for photoinactivation of VSV with AIPcS4. Treatment with Pc4 did not cause inhibition of viral RNA polymerase activity to an extent that could account for the observed very rapid loss of infectivity. It was therefore concluded that neither the viral proteins nor the binding to the host cells nor the RNA or RNA polymerase are the primary targets for photoinactivation of VSV by Pc4.
Photochemical decontamination of red blood cell concentrates (RBCC) with the silicon phthalocyanine Pc 4 and red light is being studied to enhance the viral safety of blood transfusion. Recent reports indicate that treatments with radiation and various phototsensitizing agents can activate the promoter of human immunodeficiency virus (HIV). This raises the possibility that an inadequate, sublethal photochemical treatment of RBCC could induce HIV in latently infected cells. This question has been addressed using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV promoter. In control studies, 8-methoxypsoralen (8-MOP) excited by UVA light caused activation of the HIV promoter in a dose- and time-dependent manner. At 0.1 microgram/mL of 8-MOP, maximal activation occurred with 18 J/cm2, 30 h after light exposure, With Pc 4 at 20 nM, over 90% of HeLa cells were killed after 24 h when exposed to 1 J/cm2 of red light. During that time interval and over a wide range of light doses no activation of the HIV promoter occurred. It is concluded that RBCC sterilization with Pc 4 and red light is unlikely to induce HIV production in latently infected cells.
Phthalocyanines are useful sensitizers for the photodynamic sterilization of red cell concentrates. The use of the phthalocyanine Pc4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2) and red light is very efficient in killing various viruses. The addition of scavengers of Type I photodynamic reactions and the use of cremophor to deliver Pc4 give protection to the red cells.
Various red cell components, either white cell-enriched, buffy coat-removed, or white cell-reduced, have been used to study the effect of photodynamic treatment with Pc4 on hemoglobin and potassium leakage and on ATP and glucose levels after prolonged storage.
After treatment, storage interval-dependent damage to the red cells could be observed. In components with 26 x 10(9) white cells per L, virus inactivation was less efficient than that in components with no or 2 x 10(9) white cells per L. Similarly, red cells were less affected by the treatment in components with a large number of white cells. Pretreatment storage and use within 1 week after photodynamic treatment induce less damage to the red cells at the moment of transfusion.
Various improvements in the treatment protocol may ultimately lead to the implementation of photodynamic treatment in transfusion practice. In this respect, the white cell content of the red cell concentrates should be taken into account.
Treatment of platelet concentrates (PCs) with psoralens and broad-band ultraviolet A (UVA) radiation is being examined for the elimination of pathogens that might be present in donated blood. Previous studies have demonstrated the inactivation of cell-free viruses and the maintenance of platelet integrity with common in vitro assays.
Human immunodeficiency virus (HIV) in three forms-cell-free, activity replicating, and latently infected cell lines-was added to PCs and treated with 50-microgram per mL of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), 0.35 mM rutin, and broad- and narrow-band UVA light (320-400 nm and 360-370 nm [UVA1], respectively). The inactivation of added HIV was assessed in tissue culture; platelet hemostatic activity was assessed in thrombocytopenic rabbits.
Each form of HIV was inactivated completely (> or = 10(5) infectious units) on treatment with 30 J per cm2 of UVA1 light. Similar results were obtained on treatment of 2.5 mL of PCs in test tubes or intact PC units (50 mL) in blood bags. Latently infected cell lines were substantially more sensitive than cell-free HIV or HIV that was actively replicating. Human platelets treated with 40 J per cm2 of UVA1 light had a fully corrected bleeding time shortly after treatment or after 5 days' storage, as assessed in thrombocytopenic rabbits. Platelet hemostatic function began to decrease with 81 J per cm2 of UVA1 light and was abolished with 113 J per cm2. At similar fluences, broad-band UVA light was more injurious to platelets than was UVA1 light.
HIV transmission might be eliminated by PCs after treatment with AMT and UVA1 light and without a reduction in platelet hemostatic function.
Zinc phthalocyanine tetrasulfonate (ZnPcS4), a potential photosensitizer for photodynamic therapy (PDT), has been studied using femtosecond laser spectroscopy. The excited-state dynamics in water have been found to be fast (< 80 ps) and dominated by intermolecular aggregation. Since the proposed mechanism for PDT is energy transfer from the triplet excited state of the photosensitizer to triplet O2 creating singlet O2, the short lifetime is expected to be unfavorable for producing singlet O2. This leads to the suggestion that the presence of biological substrates may have an effect on the excited-state dynamics. To test this hypothesis, the lifetimes of the excited states of ZnPcS4 have been directly measured in the presence of a model membrane, n-hexadecyltrimethylammonium bromide (CTAB). The excited-state dynamics of ZnPcS4 in buffer solutions and with human serum albumin (HSA) have also been measured. The presence of HSA and CTAB increases the excited-state lifetime significantly relative to that observed in water. The longer lifetime of ZnPcS4 in CTAB (> 1 ns) indicates that the micellar surface favors monomer formation. By increasing the excited-state lifetime, the surface substantially increases the photosensitizing potential of ZnPcS4.
Illumination of erythrocytes in the presence of merocyanine 540 (MC540) resulted in changed binding characteristics of MC540, i.e. a red shift in the emission maximum of bound dye with an increase in the relative fluorescence quantum yield. Aluminum phthalocyanine tetrasulfonate-mediated photodynamic treatment, before addition of MC540, resulted in a comparable change in the MC540-binding characteristics with, in addition, an increase in the concentration of MC540 in the membrane. Both photodynamic treatments induce depolarization of the red cell membrane, with a dose dependency comparable to that of changed MC540 binding. Also depolarization, induced by incubation of the cells with A23187 in the presence of Ca2+ in high [K+] buffer, resulted in similar changes in the MC540 binding characteristics. These results indicate a relation between photodynamically induced membrane depolarization and changed MC540-binding characteristics. Hyperpolarization induced by incubation with A23187 in low [K+] buffer resulted in decreased binding of MC540. In accordance, the MC540-mediated photodamage to red cells decreased upon hyperpolarization of the cells. The results indicate that the binding of MC540 to erythrocytes is strongly dependent on the membrane potential and that hyperpolarization of the membrane could be a possible protection mechanism for erythrocytes against MC540-mediated photodynamic damage.
Background:
All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9-dimethylmethylene blue (dimethyl-methylene blue), had superior screening results and was chosen for further characterization.
Study design and methods:
White cell reduced RBC suspensions (30% hematocrit) were deliberately inoculated with extracellular virus or virus-infected VERO cells, incubated with 4 microM dimethyl-methylene blue and illuminated with cool-white fluorescent light. Control and treated samples were titered for virus inactivation. In parallel studies, RBC suspensions were exposed to dimethylmethylene blue and light under identical conditions and assayed for in vitro RBC storage properties.
Results:
Phototreatment of RBC suspensions inactivated > 4.4 log10 of extracellular vesicular stomatitis virus (VSV), > 3.0 log10 of intracellular VSV, > 5.0 log10 of extracellular pseudorabies virus (PRV), > 4.8 log10 of intracellular PRV, > 4.7 log10 of extra-cellular bovine virus diarrhea virus, 5.8 log10 of bacterio-phage phi 6 and > 7 log10 of bacteriophage R17. Encephalo-myocarditis virus, a nonenveloped picornavirus, was resistant to photoinactivation. Virucidal conditions resulted in no detectable IgG binding in 11 of 13 samples, unchanged RBC morphology, normal banding patterns of RBC membrane proteins on SDS PAGE, and unaltered characteristics of 12 of 13 RBC antigens during storage as measured by antibody titrations. In addition, minimal changes were observed in RBC osmotic fragility, lysis, potassium efflux, ATP and 2,3-DPG levels, and the strength of one RBC antigen during storage of phototreated samples compared with controls.
Conclusion:
Dimethylmethylene blue photo-treatment can inactivate several intracellular and extracellular model viruses under conditions which minimally alter RBC properties during 42 days storage at 1-6 degrees C.
Three of four recipients of transfusion in the United States are patients undergoing surgery, and despite promising advances in the development of alternatives to allogeneic blood transfusion, it is likely that for years to come this patient population will remain dependent on blood donated by volunteers. The safety of the blood supply has been questioned seriously since it became known that the human immunodeficiency virus could be transmitted by transfusion. In response to this threat, enforcement of strict donor eligibility criteria, removal of high risk donors from the donor pool, and testing of each donation with a panel of viral markers were instituted which have reduced the infectious risks of allogeneic blood transfusion dramatically during the last decade. The current safety of the blood supply is reviewed and the ongoing efforts to improve the safety of transfusions in the future are summarized briefly.
Vesicular stomatitis virus (VSV) was used as a model virus to study the processes involved in photoinactivation by aluminum phthalocyanine tetrasulfonate (AlPcS4) or silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc4) and red light. Previously a very rapid decrease in the intracellular viral RNA synthesis after photodynamic treatment was observed. This decrease was correlated to different steps in the replication cycle. Binding of VSV to host cells and internalization were only slightly impaired and could be visualized by electron microscopy. The capability of the virus to fuse with membranes in an acidic endosomal environment was studied using both pyrene-labeled liposomes and a hemolysis assay as a model. These tests indicate a rapid decrease of fusion capacity after AlPcS4 treatment, which correlated with the decrease in RNA synthesis. For Pc4 treatment no such correlation was found. The fusion process is the first step in the replication cycle, affected by AlPcS4 treatment, but also in vitro RNA polymerase activity was previously shown to be inhibited. Inactivation of VSV by Pc4 treatment is apparently caused by damage to a variety of viral components. Photodynamic treatment of virus suspensions with both sensitizers causes formation of 8-oxo-7,8-dihydroguanosine in viral RNA as measured by HPLC with electrochemical detection. This damage might be partly responsible for inhibition of the in vitro viral RNA polymerase activity by photodynamic treatment.
Phthalocyanines are useful sensitizers for photodynamic sterilization of red cell concentrates. Various lipid-enveloped viruses can be inactivated with only limited red cell damage. Because white cells are involved in the immunomodulatory effects of blood transfusions, the study of the effect of photodynamic treatment on these cells is imperative.
White cell-enriched red cell suspensions were photodynamically treated with either the hydrophobic Pc4 (HOSiPcOSi-(CH3)2(CH2)3N(CH3)2) or water-soluble aluminum phthalocyanine tetrasulfonate (AIPCS4) under virucidal conditions. Viability of white cell subpopulations on Days 0, 1, and 4 after treatment was determined by fluorescence-activated cell sorting by flow cytometric analysis of propidium iodide uptake. Apoptosis induction was studied by DNA ladder formation and staining for an early marker of apoptosis (annexin V).
Treatment with Pc4 causes a significant decrease in cell viability of all white cells, as shown by prodidium iodide uptake. Monocytes and granulocytes are the most sensitive, and lymphocytes are relatively more resistant. Some of the cells die by apoptosis, which is induced within 30 minutes after treatment. Treatment with AIPCS4 damages only monocytes; other cell populations are not affected.
Physicochemical properties of the photosensitizers partly determine their effect on white cells. Differences in intracellular localization are likely to be responsible for the effects observed.
Recombinant adenovirus vectors are popular tools for gene transfer and gene therapy. However biosafety constraints require that all handling of the vectors and vector-containing samples is restricted to dedicated containment laboratories, unless they had undergone a validated virus-inactivation procedure, which decontaminates the samples from any active virus. In this study we evaluated the feasibility of photodynamic treatment (PDT) with visible light to inactivate recombinant adenovirus vectors in biological samples, with minimum associated effects on other biological activities. Several photosensitizers were tested for their capacity to inactivate a model human adenovirus vector, AdCMVLuc, upon illumination. Four photosensitizers (methylene blue (MB), rose bengal (RB), uroporphyrin (UP) and aluminum phthalocynine tetrasulphonate (AIPcS4)) could inactivate the adenovirus, as measured by expression of the luciferase reporter gene and by plaque assay. Of these, MB demonstrated to be the most effective sensitizer in phosphate-buffered saline (PBS), giving > 7 log10 inactivation of the adenovirus. DNA isolated from MB- and light-treated virions was inefficient as a template for transcription. Furthermore, Southern blot analysis revealed fragmentation of the viral DNA. Based on its preference for DNA, MB is suited for adenovirus inactivation in blood plasma. Spiking experiments in which AdCMVLuc was added to plasma samples demonstrated a reduction (> 4 log10-fold) of reporter gene expression to almost background levels. In contrast to MB, photodynamic treatment with RB, UP or AIPcS4 did not lead to DNA damage. Although alterations of the viral capsid could not be detected, the binding pattern of the particles to target cells was significantly changed. Taken together, our data demonstrate that PDT is an efficient, convenient and useful method for the inactivation of adenovirus vectors in biological samples.
Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted. Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC). This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification. The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin- and a ruthenium (ORIGEN)-labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin-coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium-labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal. Results using PC and RBC samples, spiked with clinically relevant gram-negative and -positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL(-1). Signals were generated in the range of 1400-80000 and 3500-500000 ECL units for unwashed and washed samples, respectively. This is equivalent to 10(5)-10(8)CFU mL(-1). These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.
Merocyanine 540 (MC540)-mediated photodynamic damage to erythrocytes was strongly reduced when illumination was performed at pH 8.5 as compared to pH 7.4. This could be explained by high pH-mediated hyperpolarization of the erythrocyte membrane, resulting in decreased MC540 binding at pH 8.5. In accordance, the MC540-mediated photooxidation of open ghosts was not inhibited at pH 8.5. Photoinactivation of vesicular stomatitis virus (VSV) was not inhibited at pH 8.5. This suggests that illumination at increased pH could be an approach to protect red blood cells selectively against MC540-mediated virucidal phototreatment. With tetrasulfonated aluminum phthalocyanine (AIPcS4) as photosensitizer, damage to erythrocytes, open ghosts and VSV was decreased when illuminated at pH 8.5. A decreased singlet oxygen yield at high pH could be excluded. The AIPcS4-mediated photooxidation of fixed erythrocytes was strongly dependent on the cation concentration in the buffer, indicating that the surface potential may affect the efficacy of this photosensitizer. This study showed that altering the environment of the target could increase both the efficacy and the specificity of a photodynamic treatment.
Photodynamic therapy (PDT) is a cytotoxic treatment, which can induce cells to initiate a rescue response, or to undergo cell death, either apoptosis or necrosis. The many signaling pathways involved in these processes are the topic of this review. The subcellular localization of the photosensitizer has been shown to be a key factor in the outcome of PDT. Mitochondrial localized photosensitizers are able to induce apoptosis very rapidly. Lysosomal localized photosensitizers can elicit either a necrotic or an apoptotic response. In the plasma membrane, a target for various photosensitizers, rescue responses, apoptosis and necrosis is initiated. Several protein phosphorylation cascades are involved in the regulation of the response to PDT. Finally, a number of stress-induced proteins play a role in the rescue response after PDT. Notably, the induction of apoptosis by PDT might not be crucial for an optimal outcome. Recent studies indicate that abrogation of the apoptotic pathway does alter the clonogenic survival of the cells after PDT. Further studies, both in vitro and especially in vivo could lead to more efficient combination therapies in which signaling pathways, involved in cell death or rescue, are either up- or downregulated before PDT.
The in vitro antioxidant and photo-oxidant activity of dipyridamole was studied by its effect on superoxide- and singlet oxygen-mediated photohemolysis and viability of neutrophils. Dipyridamole was found to be phototoxic when examined by the photohemolysis on human erythrocytes and on linoleic acid as lipid peroxidation model at concentrations above 3.0 x 10(-5) M. On the contrary, when lower concentrations (1.0 x 10(-5) to 1.0 x 10(-6) M) were used, dipyridamole showed a protector action against singlet oxygen-mediated photohemolysis by other phototoxic compounds like triamterene. This antioxidant property is proposed to result from quenching of triamterene mediated by fluorescence energy transfer. Auto-oxidation and fluorescence-energy transfer is clearly an important mechanism for protection for this drug.
A series of amino- and hydroxytetraphenylporphyrin derivatives was found to have activity against the human immunodeficiency virus type 1 (HIV-1) in human peripheral blood mononuclear cells. Activity tends to be associated with increased hydrophilicity of the porphyrins for the porphyrins with one substituent on each phenyl ring but there is no clear pattern for the porphyrins with two substituents on each phenyl ring. The antiviral activity of certain porphyrins has been demonstrated in the absence of light, suggesting a non-photochemical process. Whereas only some of the porphyrins that inhibit HIV-1 in culture inhibit HIV-1 reverse transcriptase in a cell-free system, none of those tested inhibit DNA polymerase α.
A wide range of enveloped viruses, including human herpes simplex virus type 1, human cytomegalovirus, human T cell leukemia/lymphoma virus type I, human immunodeficiency virus type 1, Sindbis virus, and Friend erythroleukemia virus, are highly susceptible to merocyanine 540 (MC 540)-sensitized photoinactivation. By contrast, human pluripotent hematopoietic stem cells, red cells, factor VIII, and von Willebrand factor are much less sensitive. This suggests that MC 540 may be useful for the inactivation of enveloped viruses in blood and blood products. The dye has a low acute systemic toxicity, is rapidly eliminated from the blood stream, and has little or no mutagenic potential. The currently available data support the view that MC 540-sensitized photo-inactivation interferes with early events in the infectious process, notably the ability of the virus to adhere to and penetrate its host cell. The viral envelope is a major target of photodynamic damages which appear to be mediated at least in part by singlet molecular oxygen.
The well-documented viral safety of albumin arises from several factors operating in concert, including virus removal during preparation, immune neutralization, serendipitous inactivation, virus sterilization through pasteurization. Safety with respect to HBV transmission was achieved even prior to the development of sensitive screening tests for HBsAg, as can be predicted given the initial virus load and the influence of factors affecting removal and inactivation. Coagulation factor concentrates, as traditionally prepared, are known to have transmitted the viral agents of hepatitis and AIDS with high frequency. Application of virucidal procedures to these concentrates, in some cases, appears to have eliminated virus transmission, raising the question as to whether absolute safety has now been achieved. Clinical proof of absolute safety is made difficult by the small number of eligible patients who can be monitored, lengthy and expensive monitoring procedures, and opportunity for transmission of virus by product-independent routes. Based on viral load analysis, modern coagulation factor concentrates are predicted to have the same probability of freedom from HIV, HBV, and HCV transmission as that exhibited by albumin.
To explore the mechanisms involved in the pathogenesis of human multiple myeloma (MM), we investigated the potential role of interleukin-6 (IL-6), a B-cell differentiation factor in humans, and a growth factor for rat/mouse heterohybridomas and murine plasmacytomas. Using a heterohybridoma assay, we found that two well-documented human myeloma cell lines, RPMI 8226 and U266, did not secrete IL-6 and did not express RNA messengers for IL-6. Neutralizing antibodies to IL-6 did not inhibit their proliferation, and recombinant IL-6 did not stimulate it. Taken together, these data show that IL-6 is not the autocrine growth factor of these human myeloma cell lines. A high production of IL-6 was found in the bone marrows of patients with fulminating MM, compared with patients with inactive or slightly active MM, or to healthy donors. This IL-6 production was assigned to adherent cells of the bone-marrow environment but not to myeloma cells. A spontaneous proliferation of myeloma cells freshly isolated from patients was observed in short-term cultures. Recombinant IL-6 was able to amplify it two- to threefold. The spontaneous proliferation of the myeloma cells was inhibited by anti-IL-6 antibodies and reinduced by recombinant IL-6. After 2 to 3 weeks of culture, the myeloma-cell proliferation progressively declined and no IL-6-dependent myeloma cell lines could be obtained despite repeated additions of fresh IL-6 and costimulation with other cytokines such as tumor necrosis factor (TNF)beta, or IL-1 beta. These data demonstrated a paracrine but not autocrine regulation of the growth and differentiation of myeloma cells by IL-6.
Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.
It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.
In vitro incubation of leukemic bone marrow with the lipophilic fluorescent dye merocyanine 540 and simultaneous exposure to light reduced the concentration of L1210 leukemia cells by about 5 orders of magnitude but spared enough normal pluripotent hematopoietic stem cells to allow hematopoietic reconstitution of lethally irradiated syngeneic mice. This simple and rapid procedure may find an application in the purging of tumor cells from autologous bone marrow grafts.
Platelet concentrates (PC) may be infrequently contaminated with low levels of bacteria that can cause septicemia and death in patients receiving transfusion therapy. We evaluated the efficacy of a photochemical decontamination (PCD) technique using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA) to inactivate bacteria in standard therapeutic PC. Twelve phylogenetically distinct pathogenic bacteria, 5 gram-positive and 7 gram-negative organisms, were seeded into PC to a final challenge dose ranging from 10(5) to 10(7) colony- forming units (CFU)/mL. Contaminated PC were treated with 8-MOP (5 micrograms/mL) and 5 J/cm2 of UVA, a PCD treatment regimen found to adequately preserve in vitro platelet function. Greater than 10(5) CFU/mL of all 5 gram-positive (Staphylococcus aureus, Streptococcus epidermidis, Streptococcus pyogenes, Listeria monocytogenes, and Corynebacterium minutissimum) and 2 of the gram-negative (Escherichia coli and Yersinia enterocolitica) organisms were inactivated. The remaining 5 gram-negative organisms were more resistant, with less than 10(1) to 10(3.7) CFU/mL inactivated under these conditions. The inactivation efficiency for this resistant group of gram-negative organisms was improved when PC were resuspended in a synthetic storage medium with reduced plasma protein concentration (15%) and an increased 8-MOP concentration (23.4 micrograms/mL). Illumination with 3 J/cm2 of UVA in this system inactivated greater than 10(5) CFU/mL of 4 resistant gram-negative organisms (Salmonella choleraesuis, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae) and 10(4.1) CFU/mL of the most resistant gram-negative organism (Pseudomonas aeruginosa). This level of PCD treatment did not adversely affect in vitro platelet function. These results demonstrate that PCD using 8-MOP (5 to 23.4 micrograms/mL) effectively inactivated high levels of pathogenic bacteria in PC with adequate preservation of in vitro platelet properties.
Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.
The photochemical aminomethyltrimethyl psoralen (AMT), in conjunction with UV A light (UVA), has been shown to inactivate human immunodeficiency virus-1 and model viruses in platelet suspensions under conditions that have only a minimal effect on in vitro platelet properties. A rabbit ear bleeding time technique was used to assess the hemostatic effectiveness of human platelet suspensions treated with AMT/UVA. New Zealand White rabbits were made thrombocytopenic by a combination of irradiation and heterologous antirabbit platelet antiserum. Reticuloendothelial function in these rabbits was suppressed by the intravenous administration of ethyl palmitate. The hemostatic function of 1- and 5-day-old human platelet suspensions (14.5% plasma) that had been treated on day 1 with 40 micrograms/mL AMT and 24 kJ/m2 UVA (1 x UVA) was evaluated by measuring microvascular bleeding times after a standard incision. Comparable bleeding times were observed after infusion with both control and AMT/UVA-treated platelets stored for either 1 or 5 days. With the transfusion of AMT/1 x UVA-treated platelets stored for 5 days, the mean (+/- SD) bleeding time was 156.3 +/- 39.2 seconds (n = 10). With untreated platelets (no AMT/no UVA), stored for 5 days, the mean bleeding time was 189.2 +/- 36.4 seconds (n = 10). Neither AMT nor 1 x UVA treatment alone influenced the observed bleeding times. In contrast, the hemostatic effectiveness of human platelet suspensions was diminished if they were exposed to three times the standard UVA dose (72 kJ/m2) on day 1 and stored for 4 more days, regardless of whether AMT was present, with the mean bleeding time increasing to 442.2 +/- 122.6 seconds (n = 15, AMT present) or 396.0 +/- 45.9 seconds (n = 10, AMT absent). These results are consistent with data obtained from in vitro studies and indicate that virucidal AMT/1 x UVA treatment does not influence platelet hemostatic function. However, the final conditions to achieve these results must be carefully controlled.
We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.
Of the naturally occurring substances that are necessary to sustain human life, oxygen is unique in that it is taken up and used by organisms in its elemental form, i.e., as an O2 molecule.
This chapter describes the photochemistry and photobiology of furocoumarins and the applications of furocoumarins in biology and medicine. Furocoumarins and their congeners are found in many plants. The photoreactivity of psoralens regarding the cycloaddition to DNA is determined by three main factors: kinetic, steric, and electronic. RNA can also be photochemically modified with psoralens and near-UV radiation. When psoralens are intercalated in DNA, the singlet reaction can be a predominant path, because the singlet excited psoralen intercalated need not diffuse too far to encounter its substrate. The probability of the photocycloaddition of psoralen to thymine in solution is enhanced if the excited singlet state of the psoralen encounters thymine and forms an exciplex. In mammalian cells, PUVA-induced mutagenesis is linear with dose, following one-hit kinetics. PUVA treatment is a well-established clastogen. Caffeine enhances the yield of chromosome aberrations when added after PUVA treatment. PUVA treatment inactivates transforming DNA in Bacillus subtilis. DNA cross-links contribute to this effect by preventing entry of DNA into the bacterial cells.
Currently transfused cellular components of blood are not available in a sterile form and carry a small risk of transmitting viral and parasite diseases. Using phthalocyanines and red light, lipid enveloped viruses, e.g., HIV-1, can be inactivated in red blood cell concentrates (RBCC). Under conditions leading to virus sterilization the blood borne parasites Trypanosoma cruzi (Chagas disease) and Plasmodium falciparum (malaria) could be eliminated to undetectable levels (> 4 log10 kill). RBC damage during treatment could be avoided by increasing the light fluence rate to 80 mW/cm², and by including the free radical scavenger glutathione and the vitamin E derivative Trolox during light exposure. Similar sterilization of platelet concentrates was achieved with the psoralen derivative AMT and UVA light. Platelet damage due to PUVA treatment was avoided by including the plant flavonoid rutin during irradiation. It is concluded that elimination of the risk of transmitting pathogens during blood transfusion is feasible with photochemical treatments.
The photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) has been studied regarding its ability to destroy enveloped viruses in blood and blood products when activated by light. Its maximum wavelength of absorption (690 nm) has proven useful in terms of activation of the photosensitizer in materials containing red blood cells. Experiments conducted on whole blood of patients infected with the human immunodeficiency virus (HIV) demonstrated that BPD-MA and light could effectively eliminate the virus when treated materials were placed in culture and tested for viral p24, but did not appear to damage blood cells or blood components. Since HIV is largely intracellular in infected individuals, these results were investigated further. We have shown, using flow cytometry, that in HIV-infected blood, BPD-MA and light appear to selectively destroy white cells that bear the interleukin 2 receptor and the DR antigen. These markers are prevalent on activated lymphocytes, and since HIV replicates only in CD4+ T cells which are activated, this finding provides an explanation for the selective killing of HIV.
Thymine and cytosine homo‐ and heterodimers ate produced when E. coli DNA is irradiated at wavelength > 300 mμ in the presence of acetone as photosensitizer. Employing acetophenone as sensitizer only thymine containing dimers are obtained. In heat denatured DNA photosensitized dimerization of thymine occurs in a higher proportion of the TT dinucleotide sequences than in native DNA and the trans‐syn thymine dimer isomer is formed as a minor photoproduct.
Abstract— The photodynamic inactivation of HSV-1, a virus having a membranous envelope, with both a decaalkyl sapphyrin and its dicarboxy-substituted analog was studied. The decaalkyl sapphyrin was as efficient in the inactivation of HSV-1 on a per macrocycle basis as DHE, whereas the efficiency of the dicarboxy-substituted sapphyrin was approximately two orders of magnitude less. Fluorescence studies of sapphyrin's binding to liposomes and VSV suggested that the decaalkylsapphyrin bound monomerically to cholesterol-rich regions of the viral envelope, whereas its charged analog localized in a more polar environment.
Abstract— Furocoumarins, potent skin therapy and tanning agents, form covalent adducts in a photochemical reaction with unsaturated fatty acids. These adducts and the chemical kinetics of their formation have been characterized by chromatography, isotopic tracers, electronic absorbance and fluorescence spectroscopy and mass spectrometry. Adduct formation does not require oxygen. The quantum yield of adduct formation in ethanol or methanol-water solutions is comparable to the quantum yield for formation of furocoumarin-thymine adducts in DNA.
The newly synthesized psoralen derivatives, 4′ hydroxy-methyl 4,5′,8 trimethylpsoralen, 4′ methoxymethyl 4,5′,8 trimethylpsoralen,
and 4′ aminomethyl 4,5′,8 trimethylpsoralen hydrochloride photoreact with the single-stranded RNA animal virus, Vesicular
Stomatitis virus, VSV. This virus is inactivated 103 times more effectively by photoreaction with these compounds than when photoreacted with 4,5′,8 trimethylpsoralen. Under
these conditions the RNA virus remains more than 103 times less sensitive to inactivation by these new photoreagents than were two double-stranded DNA viruses, Herpes Simplex
type 2 (HSV-2) and Vaccinia. Preliminary evidence for the generality of this result is discussed.
Three instances of Serratia marcescens septicemia in two patients following infusion of platelet concentrates stored at 22 C, and the isolation of the organism from one unit of a platelet concentrate, led to a study to determine the possible sources of such contamination. Cultures of the available blood products, derived from the same blood donations used to prepare the suspect platelet concentrates, yielded Serratia marcescens from two units of cryoprecipitate and from one unit of red blood cells. All other available blood products were sterile. Serratia marcescens was isolated in considerable numbers from 82 per cent of the vacuum tubes from one manufacturer's lot in use in the transfusion center at the time of the septic episodes. Six other lots of vacuum tubes prepared by the same manufacturer in use at the same time were sterile. The organism was not found in samples from other equipment, materials or personnel involved in the preparation of the blood products. Simulation of the blood collection technique using vacuum tubes from the contaminated lot, when filled from an in-line needle as used following the blood collection procedure, gave contamination of the primary pack with Serratia marcescens in five of the six experiments attempted. The contaminated vacuum tubes were thus considered the most likely source of contamination of the platelet concentrates.
The relative importance of type I and type II mechanisms in the photodynamic treatment of red blood cell concentrations (RBCC) to inactivate viruses was studied using aluminum phthalocyanine tetrasulfonate (AlPcS4), visible light and quenching or enhancing agents of reactive forms of oxygen. Treatment of a human RBCC with 10-13 microM AlPcS4 and 25-26 mW/cm2 visible light resulted in the rapid and complete inactivation of added vesicular stomatitis virus (VSV). The addition of mannitol, glycerol, reduced glutathione (GSH), or superoxide dismutase (SOD), known quenching agents of type I mechanisms, had little to no effect on the rate of inactivation of VSV. Significant inhibition of VSV kill was observed on addition of tryptophan or sodium azide, known quenchers of type II mechanisms. Additionally, the rate of VSV kill was enhanced in the presence of D2O. Taken together, these results indicate a predominant role of singlet oxygen in the inactivation of VSV on photodynamic treatment of RBCC. The relative importance of type I and type II mechanisms on cellular toxicity was also evaluated. Little, if any hemoglobin release was observed on treatment of human or rabbit RBCC with 10 microM AlPcS4 and 44 J/cm2 of visible light in the presence or absence of the above mentioned quenchers. The effect of the addition of quenchers on the recovery and circulatory survival of treated, autologous rabbit RBCC, labeled with 51Cr, was also assessed.(ABSTRACT TRUNCATED AT 250 WORDS)
The virucidal and functional effect of the treatment of platelet concentrates (PCs) with long-wave ultraviolet light (UVA) and the psoralen derivative 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) was studied. Cell-free vesicular stomatitis virus (VSV) was completely inactivated (greater than or equal to 6.5 log10) on treatment of PCs with 25 micrograms per mL (85 microM) of AMT and with 20.7 J per cm2 (30 min) of UVA in the presence of air, or with 82.8 J per cm2 (2 hours) of UVA under conditions of reduced oxygen tension. When treatment was in air, the extent and rate of platelet aggregation in response to collagen measured after overnight storage were reduced to about 70 and 50 percent of control values, respectively; however, aggregation responses were similar to those of controls when PCs were treated under reduced oxygen tension. As a means of eliminating the necessity of oxygen depletion during AMT and UVA treatment, we examined the effects of the addition of quenchers of reactive oxygen species. The presence of 2 mM (2 mmol/L) mannitol during treatment of PCs with 25 micrograms per mL of AMT and 20.7 J per cm2 of UVA in air significantly improved the aggregation response and other in vitro indicators of platelet function and had little or no effect on VSV inactivation. Less benefit was observed with the other quenchers examined. Thus, the nucleic acid specificity of psoralen photoinactivation under reduced oxygen conditions may also be attainable when selected free radical scavengers such as mannitol are present during treatment in air.
The photodynamic inactivation of retroviruses was investigated using aluminium and zinc phthalocyanine (Pc) derivatives. The N2 retrovirus packaged in either of the two murine cell lines, Psi2 and PA317, was used as a model for enveloped viruses. AlPc derivatives were found to be more effective photodynamically for inactivation of the viruses than the corresponding ZnPc derivatives. Sulphonation of the Pc macrocycle reduced its photodynamic activity progressively for both AlPc and ZnPc. Fluoride at 5 mM during light exposure completely protected viruses against inactivation by AlPc. In the presence of F-, inactivation by the sulphonated derivatives AlPcS1 and AlPcS4 was reduced 2.5- and twofold respectively. In a biological membrane (erythrocyte ghosts), F- had no significant effect on AlPcS4-sensitized lipid peroxidation. Under similar conditions, cross-linking of spectrin monomers in ghosts is drastically inhibited (E. Ben-Hur and A. Orenstein, Int. J. Radiat. Biol., 60 (1991) 293-301). Since Pc derivatives do not inactivate non-enveloped viruses, it is hypothesized that inactivation occurs by photodynamic damage to envelope protein(s). Substitution of sulphonic acid residues reduces the binding of Pc derivatives to the envelope protein(s), thereby diminishing their photodynamic efficacy and the ability of F- to modify it.
The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern. Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus. A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood. Herpes virus (HSV-1) was used to screen compounds. Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias. Irradiation was achieved with a filtered xenon light source and/or tunable dye laser. Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins. No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment. Platelet condition is minimally modified with time. Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.
Aluminum phthalocyanine tetrasulfonates (AIPcS) are photoactive compounds with absorption maxima at 665-675 nm. The inactivation of viruses (vesicular stomatitis virus, VSV; human immunodeficiency virus, HIV) added to either whole blood or red blood cell concentrates (RBCC) and platelet concentrates (PC) on treatment with tetrasulfonated AIPc (AIPcS4) was evaluated. Treatment of RBCC with 10 microM AIPcS4 and 44 J/cm2 visible light resulted in the inactivation of greater than or equal to 10(5.5) infectious doses (TCID50) of cell-free VSV, greater than or equal to 10(5.6) TCID50 of cell-associated VSV, and greater than or equal to 10(4.7) TCID50 of cell-free sindbis virus. Both greater than or equal to 10(4.2) TCID50 of cell-free and greater than or equal to 10(3.6) TCID50 of cell-associated forms of HIV were also shown to be inactivated. Encephalomyocarditis virus, used as a model for nonenveloped viruses, was not inactivated. Equivalent virus kill with Photofrin II required a substantially higher concentration of dye and longer exposure to visible light. Following AIPcS4 treatment, red cell integrity was well maintained as judged by the low level (less than 2%) of hemoglobin release immediately following treatment and on subsequent storage, by measurements of erythrocyte osmotic fragility, and by the normal recovery and circulatory survival on infusion of treated, autologous red blood cells in baboons. Treatment of PC with 10 microM AIPcS4 and 44 J/cm2 visible light also resulted in effective virus kill (greater than or equal to 10(5.5) TCID50) of VSV; however, both the rate and extent of platelet aggregation in response to collagen addition declined by at least 50%. Based on these results, further characterization of AIPcS4-treated RBCC is justified.
Four key measures help keep the use of blood safe. First, all who prescribe blood should try to limit the frequency of homologous transfusion by responding only to patients' physiologic needs and by using alternatives such as autologous transfusion and intraoperative blood salvage. Second, the selection of safe donors provides the greatest safety. For example, the current detection rate for human immunodeficiency virus (HIV) infection among active blood donors in the United States is between 5 and 10 confirmed positive results per 100,000 — that is, 1/40 to 1/80 of the rate anticipated in a random sample of the population, . . .
To determine the clinical and economic effects of granulocyte macrophage colony-stimulating factor (GM-CSF) as adjunct therapy in relapsed or refractory Hodgkin disease.
A randomized, double-blind, phase III clinical trial.
A tertiary referral center.
Twenty-four patients (twelve of whom were controls) treated with high-dose chemotherapy and autologous bone marrow transplantation.
The 12 patients treated with GM-CSF, when compared with placebo recipients, had shorter periods of neutropenia (median duration of an absolute neutrophil count of less than 1000 cells/mm3, 16 days compared with 27 days; P = 0.02), shorter periods of platelet-transfusion dependency (median duration, 13.5 days compared with 21 days; P = 0.03), and shorter hospitalizations (median hospital stay, 32 days compared with 40.5 days; P = 0.004). Other clinical outcomes, such as frequency and severity of toxicities, development of pneumonia or infection, in-hospital death, and response rate were similar in the two groups. Actuarial long-term disease-free survival was 64% for patients treated with GM-CSF and 58% for patients who received placebo after 32 months of follow-up (P = 0.15). The group treated with GM-CSF had lower total charges after infusion of autologous marrow than the placebo group (median in-hospital charges, $39,800 compared with $62,500; P = 0.005) because of lower post-infusion charges for room and board, antibiotic therapy, transfusions, laboratory tests, and physical therapy visits.
Administration of GM-CSF was associated with acceleration of myeloid and platelet recovery and was cost effective in the treatment of patients with relapsed Hodgkin disease who received intensive chemotherapy.
The inactivation of viruses added to whole blood and a red cell concentrate with aluminum phthalocyanine and its sulfonated derivatives was studied. A cell-free form of vesicular stomatitis virus (VSV), used as a model, was completely inactivated (greater than 10(4) infectious units; TCID50) on treatment of whole blood with 10 microM (10 mumol/L) aluminum phthalocyanine chloride (AIPs) and visible light dosage of 88 to 176 J per cm2. At 44 J per cm2, complete VSV inactivation was achieved on raising the concentration of AIPc to 25 microM (25 mumol/L). Results at least as good were achieved on similar treatment of a red cell concentrate. Also inactivated were a cell-associated form of VSV and both cell-free and cell-associated forms of human immunodeficiency virus; encephalomyocarditis virus, used as a model for non-lipid-enveloped viruses, was not inactivated by this procedure. This inactivation of cell-free VSV suggests that a similar degree of inactivation could be achieved with a lower concentration of the sulfonated forms of aluminum phthalocyanine. Throughout the above studies, red cell integrity was well maintained, as judged by the absence of hemoglobin release (less than or equal to 2%) during the course of treatment or on subsequent storage. Red cell osmotic fragility was decreased on treatment of whole blood with AIPc. This study indicates that AIPc may be a promising method for the inactivation of viruses in cellular blood products.
The ability of two fundamentally different photochemical procedures to inactivate model viruses in platelet suspensions was compared. Merocyanine 540 (MC 540) with visible light was used as an example of an oxygen-dependent chemical-directed at the viral membrane, and aminomethyl trimethyl psoralen (AMT) with ultraviolet A light (UVA) was used as an example of a nucleic acid-directed system. Antiviral conditions in petri dishes were identified and the effects of these procedures on platelet suspensions in plastic storage containers were studied. Concentrations of photochemicals in the 10 to 150 mumol range with 30 to 60 minutes of visible light (MC 540) or 1 to 2 minutes of UVA (AMT) readily inactivated 5 to 6 log10 of vesicular stomatitis virus (VSV) and other model viruses in platelet suspensions, provided the plasma concentration was reduced to about 15 percent by the use of a synthetic platelet storage medium. Extracellular pH, morphology scores, and aggregation response dropped markedly when platelets were treated with MC 540 and visible light. However, treatment with 136 mumol per L of AMT and 1 to 3 minutes of UVA could inactivate 5 log10 of VSV in platelet suspensions with retention of platelet characteristics for 4 days, particularly if oxygen levels were reduced during treatment. These studies demonstrate that AMT-UVA treatment meets the initial requirements for virus inactivation in platelet suspensions.
To determine the medical and laboratory characteristics of bacteremia secondary to transfusion of microbiologically contaminated platelet concentrates.
Febrile transfusion reactions were prospectively monitored over 42 months. Units involved in reactions were evaluated with Gram's stain and culture tests.
Comprehensive cancer center.
Patients receiving platelet transfusions for thrombocytopenia secondary to bone marrow failure.
Seven cases of transfusion-associated sepsis were observed. Multidonor platelet products stored for 5 days resulted in an incidence of sepsis five times higher than those stored for 4 days or less (P less than .01). Investigation indicates that contamination most likely occurred at the time of blood collection. Clinically, septic reactions were associated with greater temperature elevations (average increase, 2.0 degrees C) than febrile reactions to sterile products.
Contamination of platelet concentrates remains a significant clinical problem. Septic episodes may be reduced by transfusion of platelets with shorter storage intervals.
A model has been developed to demonstrate the use of photodynamic treatment (PDT) to eradicate viral contaminants from donated blood and blood products. Whole blood, spiked with vesicular stomatitis virus (VSV), was treated with the photosensitizer benzoporphyrin derivative-monoacid ring A (BPD-MA). After light activation of BPD-MA, a neutral red dye uptake assay was carried out to determine virus inactivation. Various drug incubation times and light intensities were tested as well as red cell lysis and distribution of VSV in blood. At BPD-MA concentrations between 2 and 4 micrograms per mL in whole blood, up to 10(7) VSV were inactivated. Several photosensitizers were also tested with this model to determine their relative efficacy in viral inactivation.
Dry or wet heat, solvents, and detergents combined with ultraviolet irradiation provide effective means of sterilizing soluble blood products such as albumin or factor VIII. For obvious reasons, these procedures are not applicable to cellular blood components. We have recently shown that simultaneous exposure to the photosensitizer, merocyanine 540 (MC 540) and white light rapidly inactivates the Friend erythroleukemia virus complex and Friend virus-transformed cells, but causes relatively little damage to pluripotent hematopoietic stem cells. In this communication, we show that several lipid-enveloped human pathogenic viruses are also highly susceptible to MC 540-sensitized photoirradiation, and we report on an initial evaluation of the ability of MC 540-sensitized photoirradiation to sterilize blood products.
In 1986 the allowable platelet storage time was reduced from 7 to 5 days because of a recent increase in septic deaths associated with platelet transfusion. In this study, the growth curves of two gram-positive and two gram-negative organisms in platelets stored for 7 days in CLX and PL-732 bags were evaluated. Platelets in CLX bags were inoculated with 10(1), 10(2), and 10(3) organisms and 10(2) organisms were introduced into PL-732 bags. Test organisms were inoculated into trypticase soy broth as a control. All four bacteria grew rapidly in trypticase soy broth, reaching 10(9) organisms per mL within 48 hours. In both CLX and PL-732 bags, the growth pattern of gram-positive organisms was generally logarithmic during the first few days of storage. A concentration of 10(8) organisms per mL was present by Day 3 or 4, after which further proliferation was inhibited by the high density of bacteria in the platelets. In PL-732 bags, the proliferation of gram-negative organisms followed a pattern similar to that of the gram-positive bacteria. However, gram-negative organisms grew less well in CLX bags.(ABSTRACT TRUNCATED AT 250 WORDS)
A photodynamic flow system employing a dihematoporphyrin ether (DHE) was tested for its ability to inactivate the in vitro infectivity of simian immunodeficiency virus (SICMac) at 630 +/- 5 nm with a light fluence of 5 J/cm2. Cell-free SIVMac was inactivated by photoactivated hematoporphyrin derivative in a dose-dependent fashion. Since SIVMac is closely related to human immunodeficiency virus type 2 (HIV-2) and we have previously reported the successful photodynamic inactivation of HIV-1 in cell-free medium as well as in whole human blood, this technology has the potential for the eradication of transfusion-associated acquired immunodeficiency diseases caused by the above-mentioned retroviruses.
Transmission of viral diseases through blood products remains an unsolved problem in transfusion medicine. We have developed a psoralen photochemical system for decontamination of platelet concentrates in which platelets are treated with long wavelength ultraviolet radiation (UVA, 320-400 nm) in the presence of 8-methoxypsoralen (8-MOP). Bacteria, RNA viruses, and DNA viruses ranging in genome size from 1.2 x 10(6) daltons, encompassing the size range of human pathogens, were inoculated into platelet concentrates and subjected to treatment. This system inactivated 25 to 30 logs/h of bacteria Escherichia coli or Staphylococcus aureus, 6 logs/h of bacteriophage fd, 0.9 log/h of bacteriophage R17 and 1.1 logs/h of feline leukemia virus (FeLV) in platelet concentrates maintained in standard storage bags. Platelet integrity and in vitro function before, immediately following photochemical treatment, and during prolonged storage after treatment, were evaluated by measuring: (1) extracellular pH; (2) platelet yields; (3) extracellular lactate dehydrogenase (LDH) levels; (4) platelet morphology; (5) platelet aggregation responsiveness; (6) thromboxane beta-2 (TXB-2) production; (7) dense body secretion; and (8) alpha granule secretion. These assays demonstrated that this photochemical inactivation system inactivated bacteria and viruses in platelet concentrates with minimal adverse effects on the in vitro function of platelets in comparison to untreated control concentrates maintained under current, standard blood bank conditions.
A survey was conducted to determine the prevalence of infection with Trypanosoma cruzi, the protozoan etiologic agent of American trypanosomiasis (Chagas' disease), among Nicaraguan and Salvadoran immigrants living in the Washington, D.C., area. The serum samples of study subjects were tested for reactivity with T. cruzi antigens in an enzyme-linked immunosorbent assay, and also tested for antibody specific for the 72 and 90 kilodalton (kDa) surface glycoproteins of the parasite in an immunoprecipitation and electrophoresis procedure. Xenodiagnosis using reduviid bugs to detect parasites, and clinical evaluations for cardiac and gastrointestinal disease were performed in patients in whom results of both serologic tests were positive. Of 205 subjects studied, 4.9 percent were infected with T. cruzi, and parasites were isolated from 50 percent of those in whom xenodiagnosis was attempted. No significant cardiac or gastrointestinal abnormalities were detected in the six infected patients who were evaluated clinically. These findings suggest that a sizable proportion of persons in this immigrant group are infected with this organism. Thus, routine serologic testing for antibody to T. cruzi may be warranted in immigrants from these countries, especially in view of the potentially serious consequences of infection with this parasite, and also because of the risk of transmission of T. cruzi by blood transfusion.
The potential value of sulphonated aluminium phthalocyanine (AISPc) as a purging agent for bone marrow autografts in acute myeloblastic leukaemia (AML) has been studied using in vitro clonogenic assays for normal (GM-CFC) and leukaemic (AML-CFC) progenitor cells. In nine out of 13 cases, the leukaemic blasts were found to be highly sensitive to AISPc. In six of the sensitive cases clonogenic assays revealed that only 2 +/- 1% of AML progenitor cells survived AISPc treatment under conditions which permitted a GM-CFC recovery of 60 +/- 11%. AISPc photosensitization was also shown to selectively eliminate the leukaemic cell line K562 from an in vitro model of minimal residual disease. Thus photosensitization using AISPc may be an effective method of purging marrow autografts in some cases of AML. Evaluation of the sensitivity of AML clonogenic cells at diagnosis may identify those patients in whom AISPc photo-purging may be of benefit at the time of an autologous bone marrow transplant.
A modification of photodynamic inactivation of herpes simplex viruses (HSV) has been developed employing hematoporphyrin derivative (HPD) and visible light. Isolates of HSV Type 1 rapidly and completely lose their plaqueforming ability when a mixture of virus and HPD is exposed to light. This effect is dependent upon optimal concentrations of the tetrapyrrole, light intensity, and the presence of molecular oxygen. Photodynamically inactivated virions do not cause viral cytopathic effects following infection of susceptible cells, and fail to induce viral antigens. The treated particles are unable to adsorb to or penetrate cells, and therefore cannot initiate an infection. These observations are consistent with photooxidative damage to the viral envelope occurring through the interaction of HPD plus light.
Photodynamic therapy (PDT) is a cancer treatment based on the accumulation in malignant tissue of a photosensitiser with low systemic toxicity. Subsequent illumination induces a type II photochemical reaction with singlet oxygen production that results in destruction of biomolecules and subcellular organelles.
The first full clinical report of PDT dates from 1976. Haematoporphyrin derivative, a complex mixture of porphyrins, was initially used as a photosensitiser. An enriched fraction (porfimer sodium) is now the most commonly used clinical agent. After systemic administration porphyrins bind to albumin and lipoproteins. Accumulation occurs mainly in tumours and organs of the reticuloendothelial system. The light of an argon-dye laser can be tuned to the appropriate wavelength and delivered either superficially, interstitially or intraluminally. Light distribution can be assessed by using a radiation transport model and tissue optical properties, or direct measurement with light detectors.
The effects of PDT depend in a complex way on: characteristics, tissue concentration and localisation of the photosensitiser; the target tissue optical properties and oxygenation; activation wavelength, power density and treatment regimen. Future research is directed towards: better photosensitisers (i.e. phthalocyanines, chlorins or protoporphyrin IX endogenously produced from 5-aminolevulinic acid); improved light generation and delivery; and combination with hyperthermia, chemotherapy, radiotherapy or surgery. Adjuvant intraoperative PDT is a promising approach to destroying residual tumour after surgery.
For virus sterilization of platelet concentrates (PCs), treatment with aminomethyltrimethyl psoralen (AMT) and long-wavelength ultraviolet A light (UVA) has shown efficacy. It has been found that treatment with 50 micrograms per mL of AMT and 38 J per cm2 of UVA in the presence of 0.35-mM rutin efficiently kills viruses while maintaining platelet integrity. There is, however, concern about the mutagenic potential of psoralens and UVA (PUVA)-treated PCs.
Adsorption of PUVA-treated PCs with a hydrophobic resin containing C18 as the ligand was used for AMT removal, which was quantitated by the use of radioactive AMT. PUVA-treated PCs, with and without C18 treatment, were examined for solution pH and platelet aggregation response to agonists. In addition, residual AMT activity was determined by AMT's virucidal activity or incorporation into cellular DNA upon a second UVA irradiation and by its mutagenic potential in the Ames test.
After PUVA treatment of PCs, residual AMT retained virucidal and adduct-forming ability upon re-exposure to UVA, but activities were less than those observed originally. As has been found previously, AMT had mutagenic potential following incubation in the dark with rat liver S9 microsomal enzymes. The PUVA treatment reduced this potential by 90 percent. C18 adsorption following PUVA treatment had no negative effect on platelet integrity and eliminated 50 percent of the added radioactive AMT. In addition, all detectable virucidal, nucleic acid-modifying, and mutagenic activities of AMT-treated PCs were removed by C18.
These results suggest that hydrophobic resin adsorption of PUVA-treated PCs will conveniently remove functional psoralens and eliminates their mutagenic potential.
The mechanism of the antiviral activity of hypericin was characterized and compared with that of rose bengal. Both compounds inactivate enveloped (but not unenveloped) viruses upon illumination by visible light. Human immunodeficiency and vesicular stomatitis viruses were photodynamically inactivated by both dyes at nanomolar concentrations. Photodynamic inactivation of fusion (hemolysis) by vesicular stomatitis, influenza, and Sendai viruses was induced by both dyes under similar conditions (e.g., I50 = 20-50 nM for vesicular stomatitis virus), suggesting that loss of infectivity resulted from inactivation of fusion. Syncytium formation, between cells activated to express human immunodeficiency virus gp120 on their surfaces and CD4+ cells, was inhibited by illumination in the presence of 1 microM hypericin. Hypericin and rose bengal thus exert similar virucidal effects. Both presumably act by the same mechanism--namely, the inactivation of the viral fusion function by singlet oxygen produced upon illumination. The implications of this photodynamic antiviral action for the potential therapeutic usefulness of both hypericin and rose bengal are discussed.
Irradiation with red light of red cells (RBCs) containing the photodynamically active phthalocyanine (Pc) dyes is being studied for inactivation of lipid-enveloped viruses. One of the outstanding problems with this treatment is the binding of IgG to RBCs. The effects of oxygen and type I or type II quenchers on this IgG uptake were evaluated.
The Pc compounds used were aluminum phthalocyanine tetrasulfonate (AIPcS4), HOSiPcOSi(CH3)2(CH2)3N(CH3)2 (Pc 4); HOSiPcOSi(CH3)2(CH2)3N+(CH3)3I- (Pc 5); and SiPcOSi[(CH3)2(CH2)3N+(CH3)3](2)2I- (Pc 6). RBCs were analyzed by flow cytometry for the presence of IgG.
Irradiation with red light for 30 minutes of RBCs containing either 2 microM Pc 4, 2 microM Pc 5, 2 microM Pc 6, or 6.5 microM AIPcS4 resulted in an uptake of IgG. These conditions completely inactivated the lipid-enveloped vesicular stomatitis virus (VSV) (> 5 log10 kill). IgG uptake was reduced when oxygen was depleted. The addition of reduced glutathione (GSH) or mercaptoethanol prevented the binding of IgG with RBCs treated with AIPcS4, Pc 4, Pc 5, and Pc 6. Specific binding of IgG2 but not of C3d was observed upon irradiation of RBCs with Pc 5 and Pc 6 in the absence of GSH. No gross changes were observed in RBC antigen strength after irradiation with the dyes in the presence of GSH. Inactivation of VSV by Pc plus light was not affected by GSH.
Sulfhydryl compounds are useful in preventing IgG binding to RBCs following Pc photosensitization. Since virus inactivation proceeds at the same rate in the presence and the absence of sulfhydryl compounds, their addition to treated RBCs should allow crossmatching for transfusion after treatment. The binding of IgG depends to a large extent on the generation of reactive oxygen species.
The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems.
Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent.
Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 x 10(-6), from a spontaneous frequency of 28 x 10(-6).
Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.
Photodynamic treatment of red cells (RBCs) with phthalocyanines and red light inactivates lipid-enveloped viruses, such as vesicular stomatitis virus (VSV) and human immunodeficiency virus. To protect RBCs from photodynamic damage, type I free radical quenchers, such as mannitol, which did not affect virus inactivation, were added.
Aluminum phthalocyanine tetrasulfonate (AIPcS4) was found to inactivate VSV at a rate one-fourth that of the silicon phthalocyanines (Pc 4 and Pc 5). However, the latter also caused more RBC damage. To protect RBCs against this photodynamic damage, Trolox, a water-soluble vitamin E analogue, was used. RBC damage was measured as potassium leakage or hemolysis during storage after treatment. In addition, reduction in negative surface charge on RBCs was measured immediately after treatment, and the effect of Trolox on VSV inactivation in RBCs was evaluated.
Trolox at a concentration of 5 mM was found to reduce potassium leakage during storage after Pc 4 and AIPcS4 photodynamic treatment of RBCs. Hemolysis during storage of RBC concentrates treated with Pc 4 or Pc 5 was drastically reduced by the addition of 5 mM Trolox prior to light exposure. At the same concentration, Trolox inhibited the reduction of negative surface charges on RBCs following Pc 4 and Pc 5 photodynamic treatment. Under these conditions, VSV inactivation by photodynamic treatment with all phthalocyanines was not affected by Trolox. In aqueous solution, Trolox formed a complex with AIPcS4, thus quenching the excited triplet state of AIPcS4 at a constant rate of 8.8 x 10(6) per M per second.
These findings indicate that Trolox protects RBCs from phthalocyanine-photosensitized damage without affecting virus kill. The addition of Trolox would be beneficial for improving the quality of RBCs subjected to photodynamic treatment.