Protoporphyrin IX-sensitized photoinactivation of 5-aminolevulinate-treated leukemia cells: effects of exogenous iron

Photodynamic therapy with 5-aminolevulinic acid (ALA) is based on metabolism of ALA to a photosensitizing agent, protoporphyrin IX (PpIX), in tumor cells. Photosensitivity of target cells may be influenced by mitochondrial iron levels because ferrochelatase-catalyzed insertion of Fe2+ into PpIX converts it to heme, a nonsensitizer. To investigate this prospect, we exposed L1210 cells (approximately 10(6)/mL in 1% serum-containing medium) to a lipophilic iron chelate, ferric-8-hydroxyquinoline (Fe[HQ]2, 0.5 microM), prior to treating with ALA (0.2 mM, 4 h) and irradiating with broadband visible light. When Fe(HQ)2 was added to cells immediately or 1 h before ALA, the initial rate of photokilling, as measured by thiazolyl blue (mitochondrial dehydrogenase) assay, was markedly less than that of non-iron controls. The HPLC analysis of cell extracts indicated that ALA-induced PpIX was at least 50% lower after this Fe(HQ)2 treatment, presumably explaining the drop in photolethality. By contrast, cells treated with ALA and light 20 h after being exposed to Fe(HQ)2 contained the same amount of PpIX as non-iron controls and were photoinactivated at nearly the same rate. The 20 h delayed cells contained approximately 12 times more immunodetectable ferritin heavy subunit than controls or 1 h counterparts, which could account for the disappearance of iron's antisensitization effects in the former. Consistent with this idea, the short-term effects of Fe(HQ)2 on ALA-induced sensitization were found to be blunted significantly in ferritin-enriched cells. The Fe(HQ)2 produced strikingly different results when cells were sensitized with exogenous PpIX, stimulating photokilling after short-term contact but inhibiting it after long-term contact while having no significant effect on the level of cell-associated PpIX in either case. Thus, iron can have diverse effects on PpIX-mediated photokilling, depending on contact time with cells and whether the porphyrin is metabolically derived or applied as such.     

MODULATION BY THIOLS OF THE MEROCYANINE 540-SENSITIZED PHOTOLYSIS OF LEUKEMIA CELLS, RED CELLS, AND Herpes simplex VIRUS TYPE 1

This paper reports on the role of endogenous and exogenous thiols in the merocyanine 540 (MC 540)-sensitized photoirradiation of L1210 leukemia cells, human erythrocytes, and human Herpes simplex virus type 1. Several measures taken to decrease the intracellular content of glutathione enhanced the cells' sensitivity to MC 540-sensitized photoirradiation while stimulation of glutathione biosynthesis or supplementation of the extracellular or extraviral thiol content decreased the photosensitivity of cells and viruses. Taken together, these data suggest that endogenous and exogenous thiols can modulate the sensitivity of cells and enveloped viruses to MC 540-sensitized photoirradiation. They also pose new questions as to the mechanism of MC 540-sensitized photolysis.     

POTENTIATION OF MEROCYANINE 540-MEDIATED PHOTODYNAMIC THERAPY BY SALICYLATE and RELATED DRUGS

Abstract— Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.     

LIMITED CELL-CYCLE DEPENDENCE OF THE MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF L1210 LEUKEMIA CELLS

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.     

Delayed Hyperresistance of Endothelial Cells to Photodynamic Inactivation After Contact with Hemin

Hemin (ferriprotoporphyrin IX), the oxidized prosthetic group of hemoglobin, is a potential source of prooxidant iron in heavily vascularized tumors. We have evaluated hemin's effects on photodynamic inactivation of bovine artery endothelial cells, using a partially purified oligo-meric fraction of hematoporphyrin derivative (HPD-A) as the sensitizing agent. Confluent cells in 5% serum/ RPMI medium showed a progressive loss of thiazolyl blue (MTT)-detectable viability when irradiated with broadband visible light in the presence of HPD-A. Cells pretreated with desferrioxamine (DFO) were substantially less sensitive to photokilling, implying that non-heme iron plays a role in cytotoxic activity. Hemin (10–20μM) had remarkably different effects on photokilling, depending on the time interval between adding it to cells and exposing them to photodynamic action. For example, cells were more sensitive when photostressed immediately after 1 h hemin treatment and washing but much more resistant when photostressed 23 h later. Similar responses were observed when cells were challenged with glucose oxidase. Immunoblot analysis following hemin treatment revealed a progressive induction of the heavy (H) subunit of ferritin that paralleled the development of hyperresistance. After incubation with saturating levels of the synthetic iron donor [⁵⁵Fe]ferric-8-hydroxyquino-line, hemin-stimulated cells contained about four times more immunoprecipitable ferritin ⁵⁵Fe than controls. This is consistent with the notion that sequestration of toxic iron as a result of induction of H-chain-enriched ferritin is a key factor in hyperresistance. Inflammatory injury in tumor vasculatures could expose endothelial and neoplastic cells to chronic hemoglobin-derived iron. Consequent upregulation of ferritin could impact negatively on the efficacy of photodynamic therapy and other oxidant-based cancer therapies.     

MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF LEUKEMIA CELLS: EFFECTS OF DOSE FRACTIONATION

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.     

CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.     

ANTIVIRAL ACTIVITY OF MEROCYANINE 540

Abstract Simultaneous exposure to the lipophilic dye merocyanine 540 (MC 540) and white light inactivates several enveloped viruses. The same treatment appears to have little or no effect on pluripotent hematopoietic stem cells, mature red cells, and mature leukocytes. At least some components of the clotting system are spared, too. The molecular basis of the virucidal effect of MC 540 and light is not yet completely understood. Based on what is known about the interactions of MC 540 with cells and artificial membranes, it seems likely that MC 540 binds to and damages the viral envelope. MC 540-mediated photosensitization may have implications for the sterilization of bone marrow and blood products, the preparation of vaccines, and selected areas of antiviral therapy.     

TUMOR CELL SPECIFIC DARK CYTOTOXICITY OF LIGHT-EXPOSED MEROCYANINE 540: IMPLICATIONS FOR SYSTEMIC THERAPY WITHOUT LIGHT

Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light-exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light-exposed MC540 resulted in 70-90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte-macrophage colony forming cells (CFU-GM) survived the treatment. The observed cytotoxicity of light-exposed MC540 to the tumor cells was significantly greater (P less than 0.05) than the native MC540. Results show that tumor cell specificity and cytotoxicity in the light activated dye are retained for at least 30 days. Addition of catalase and mannitol decreased the cell kill by light-exposed compound, indicating that the observed effects may be due to reactive oxygen species. The electron micrographs of treated cells show a progression towards apoptosis in a majority of the cells. The life span of L1210 leukemia-bearing mice treated with light-exposed MC540 was prolonged compared to the untreated and native MC540 treated mice. High pressure liquid chromatography (HPLC) analysis of light-exposed material shows a completely different elution profile compared to the native compound. Results presented here show that light-exposed photoactive compounds can be used without further illumination and may have significant clinical applications. Photoactive mechanisms dependent on events other than short-lived transient elevations in energy or singlet oxygen must be invoked to explain the reported cytotoxicity.     

A Comparative Study of 8‐Hydroxyquinoline and 8‐Hydroxyquinoline‐5‐sulfonic Acid for Antimony(III) Determination by AdSV. Substituent Effect on Sensitivity II

A sensitive and selective method for the determination of Sb³⁺ based on the formation of its complexes with 8‐hydroxyquinoline (HQ) and 8‐hydroxyquinoline‐5‐sulfonic acid (HQS) is proposed. The best analytical conditions are: pH 5.4 and 2.2 for HQ and HQS, respectively; C_HQ from 15.0 to 25.0 µmol L^?1 and C_HQS from 70.0 to 200.0 µmol L^?1. The detection limits are 100.0 and 14.0 ng L^?1 (t_acc=30 s) for Sb³⁺ with HQ and HQS, respectively. The method using HQS as ligand has a 2.2‐fold higher sensitivity than that with HQ and the former was chosen for Sb³⁺ determination.     

PHOTOBLEACHING OF MEROCYANINE 540: INVOLVEMENT OF SINGLET MOLECULAR OXYGEN

The purpose of this study was to assess the mechanism of merocyanine 540 (MC540) photobleaching in a liposomal system. Broad based visible irradiation of MC540 in unilamellar dilauroylphosphatidylcholine (DLPC) vesicles resulted in dye bleaching that was strictly O2 dependent. The rate of self-sensitized photobleaching was enhanced in D2O and inhibited by both azide and histidine, consistent with 1O2 intermediacy (Type II chemistry). Supportive evidence for this mechanism was obtained by using a Type II sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS lambda max = 678 nm). Irradiation of AlPcS and MC540 in DLPC with lambda greater than 630 nm (absorbed only by AlPcS) light resulted in rapid bleaching of MC540, which was stimulated by D2O and inhibited by azide. A rate constant of 10(7) M-1 s-1 was determined for the chemical quenching of 1O2 by MC540. The rate constant for physical quenching of 1O2 by MC540 was estimated to be ca 10(9) M-1 s-1.     

Preferential inactivation of paediatric solid tumour cells by sequential exposure to Merocyanine 540-mediated photodynamic therapy and Edelfosine: implications for the ex vivo purging of autologous haematopoietic stem cell grafts

Paediatric solid tumours exhibit steep dose-response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms' tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.     

ACTION SPECTRA OF THE ANTILEUKEMIC and ANTIVIRAL ACTIVITIES OF MEROCYANINE 540

Action spectra of the antileukemic and antiviral activities of merocyanine 540 (MC540) were determined using L1210 leukemia cells and human Herpes simplex virus type 1. The major peak of both action spectra aligned closely with the absorption spectrum of membrane-bound dye monomer, and by implication, the action spectrum of 1O2 generation. These results are compatible with the notion that the antileukemic and antiviral activities of MC540 are primarily attributable to membrane-bound monomer and at least in part mediated by 1O2.     

Subcellular localization of merocyanine 540 (MC540) and induction of apoptosis in murine myeloid leukemia cells

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.     

Genetic variability in the response of normal murine hematopoietic progenitor cells to extracorporeal photochemotherapy

Normal hematopoietic progenitor cells from 129S6/SvEv mice are substantially less sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than hematopoietic progenitors from sex- and age-matched C57BL/6 mice. When exposed to a combination of MC540 and light commonly used for the extracorporeal purging of hematopoietic stem cells, granulocyte/macrophage progenitors (CFU-GM) from C57BL/6 mice are depleted 7.9-fold whereas CFU-GM from 129S6/SvEv and (C57BL/6 x 129S6/SvEv) F1 mice are depleted 1.4- and 2-fold, respectively. The same rank order of sensitivity is also found with regard to unipotent progenitors of granulocytes and macrophages and with regard to early and late erythroid progenitors. The resistance of hematopoietic progenitors from 129S6/SvEv mice to MC540-PDT appears to be the result of reduced dye binding rather than the result of high levels of intracellular glutathione. These findings have practical implications for the design of preclinical tests of PDT in animal models. They may also provide a useful tool for future investigations into the molecular determinants of sensitivity to MC540-PDT.     

Potentiation of the antitumor effect of Merocyanine 540-mediated photodynamic therapy by amifostine and amphotericin B

Leukemia and lymphoma cells are much more sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than normal pluripotent hematopoietic stem cells and normal colony forming unit-granulocyte/macrophage progenitors (CFU-GM). By contrast, most solid tumor cells are only moderately sensitive to MC540-PDT. The limited activity against solid tumor cells has detracted from MC540's appeal as a broad-spectrum purging agent. We report here that noncytotoxic concentrations of amifostine (Ethyol, Ethiofos, WR-2721) and amphotericin B used either alone or in combination potentiate the MC540-sensitized photoinactivation of leukemia cells, wild-type small cell lung cancer cells and cisplatin-resistant small cell lung cancer cells. Amphotericin B also enhances the MC540-sensitized photoinactivation of normal CFU-GM, whereas amifostine protects CFU-GM against the cytotoxic action of MC540-PDT. The yield of CD34-positive normal hematopoietic stem and progenitor cells is only minimally diminished by pretreatment with amifostine, amphotericin B or combinations of amifostine plus amphotericin B. Purging protocols that combine MC540-PDT with amifostine or with amifostine plus amphotericin B could offer a simple and effective approach to the purging of autologous stem cell grafts that are contaminated with solid tumor cells or the purging of stem cell grafts from heavily pretreated leukemia patients that contain reduced numbers of normal stem and progenitor cells and, therefore, can ill afford additional losses caused by purging.     

Postirradiation hyperthermia selectively potentiates the merocyanine 540-sensitized photoinactivation of small cell lung cancer cells

Lung cancer has long been considered a disease that might benefit from the dose escalation of radio/chemotherapy afforded by a stem cell transplant. However, the clinical experience with high-dose chemotherapy and autologous bone marrow transplantation in lung cancer has been disappointing, with most trials showing little or no improvement in long-term survival. Unfortunately, lung cancer has a tendency to metastasize to the bone marrow, and lung cancer cells are known to circulate in the peripheral blood. Therefore, there is concern that autologous stem cell grafts from lung cancer patients may reinoculate recipients with live tumor cells. Photochemical purging of stem cell grafts with Merocyanine 540 (MC540) is highly effective against a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Most solid tumor cells (including lung cancer cells), however, are only moderately sensitive or refractory to MC540-mediated photodynamic therapy (PDT). We report here that postirradiation hyperthermia (< or = 42 degrees C, 3 h) potentiates the MC540-mediated photoinactivation of both wild-type (H69) and cisplatin-resistant mutant (H69/CDDP) small cell lung cancer cells by several orders of magnitude, while only minimally enhancing the depletion of normal human granulocyte/macrophage progenitor cells. Our data suggest that postirradiation hyperthermia provides a simple and effective means of extending the utility of MC540-PDT to the purging of stem cell grafts contaminated with lung cancer and possibly other solid tumor cells.     

Effect of Membrane Potential on the Binding of Merocyanine 540 to Human Erythrocytes

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.     

Sub-lethal Photodynamic Damage to ARPE-19 Cells Transiently Inhibits Their Phagocytic Activity†

Efficient phagocytosis of photoreceptor outer segments (POS) membranes by retinal pigment epithelium (RPE) plays a key role in biological renewal of these highly peroxidizable structures. Here, we tested whether photodynamic treatment, mediated by merocyanine 540 (MC 540), rose Bengal or a zinc-substituted chlorophyllide inhibited phagocytic activity of ARPE-19 cells in vitro. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS isolated from cow retinas and nonspecific phagocytosis of fluorescent polystyrene beads were measured by flow cytometry. Photodynamic treatment, mediated by all three photosensitizers with sub-threshold doses, induced significant inhibition of the cell-specific phagocytosis. The nonspecific phagocytosis was inhibited by photodynamic treatment mediated only by MC 540. The inhibition of phagocytosis was a reversible phenomenon and after 24 h, the photodynamically treated cells exhibited phagocytic activity that was comparable with that of untreated cells. This study provides proof of principle that sub-threshold photodynamic treatment of ARPE-19 cells with appropriate photosensitizers is a convenient experimental approach for in vitro study of the effects of oxidative stress on specific phagocytic activity of RPE cells. We postulate that oxidative damage to key components of the cell phagocytic machinery may be responsible for severe impairment of its activity, which can lead to retinal degeneration.     

DIFFERENTIAL INACTIVATION OF SURROGATE VIRUSES WITH MEROCYANINE 540

Bacteriophages may be useful as surrogates for animal viruses when the virucidal properties of different photosensitizing compounds are initially investigated. We studied photoinactivation of four bacteriophages, phi X174, T7, PRD1, and phi 6, by the dye merocyanine 540 (MC540) (15 micrograms/mL). Merocyanine 540 (MC540) should be most effective with lipid-containing viruses, since it is primarily lipophilic (but also binds to proteins). Two of the phages, PRD1 and phi 6 contain lipid, with only phi 6 having an external lipoprotein envelope. Filtered radiation (450-600 nm) from a 750 W projector was used at 16-100 W/m2. The survival curves of the different viruses clearly demonstrated different levels of sensitivity to photoinactivation by MC540, with phi 6 (Do = 1.5 kJ/m2) being the most sensitive, followed by T7 (21-fold less sensitive). While both PRD1 and phi 6 have lipid components, only phi 6 was photoinactivated by MC540. Thus the internal lipid components of PRD1 were not sufficient to allow photoinactivation by this dye, at fluences up to 300 kJ/m2. For comparison, we also photoinactivated Herpes simplex virus (Do = 0.053 kJ/m2) and found it to be 28-fold more sensitive than phi 6 to photoinactivation by the same concentration of MC540. Thus phi 6 may be used as a surrogate for enveloped human viruses for photoinactivation by lipophilic dyes, but the results may only be useful qualitatively.