Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies.

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

The use of dimethylmethylene blue for virus photoinactivation of red cell suspensions.

Phenothiazine dyes and light have been known to have virucidal properties for over seventy years. This review will describe recent progress in the use of one phenothiazine dye, dimethyl-methylene blue, for photo-inactivation of a number of RNA and DNA viruses in red cell suspensions under conditions that minimally affect red cell in vitro properties during 42-day 1-6 degrees C storage. Dimethylmethylene blue has a higher affinity for nucleic acid than the closely related phenothiazine, methylene blue. Virus photoinactivation appears to be mediated by singlet oxygen. The kinetics of photoinactivation depends on the virus studied, but for a given virus, is similar for both intracellular and extracellular forms. The similarity for inactivation of intracellular and extracellular virus suggests that a common target, such as nucleic acid, is involved. Finally, lymphocytes, which can harbour transfusion-associated viruses and can mediate transfusion-associated-graft-versus host disease, are sensitive to dimethylmethylene blue photoinactivation under virucidal conditions.

Role of the immune system in mediating the antitumor effect of benzophenothiazine photodynamic therapy.

The role of the host immune system in contributing to tumor regression following benzophenothiazine photodynamic therapy (PDT) was examined. Photodynamic therapy with 2-iodo-5-ethylamino-9-diethylaminobenzo[a]-phenothiazinium chloride (2I-EtNBS) eradicated EMT-6 mammary fibrosarcomas in 75-100% of treated mice. In contrast, PDT failed to inhibit tumor growth in T-cell-deficient nude mice. Furthermore, T-cell depletion studies with anti-CD8 antibody revealed that the CD8+ T-cell population was critical for an effective PDT response (tumor volume 14 days post-PDT: 262 mm3 vs 59 mm3 in controls; P < 0.01). Because anti-CD4 antibody inhibited tumor growth in the absence of PDT, the role of CD4+ T cells remains unclear. Depletion of natural killer (NK) cells in vivo with anti-asialo-GM1 antibody significantly reduced a suboptimal PDT effect relative to vehicle controls (tumor volume 13 days post-PDT: 513 mm3 vs 85 mm3, respectively; P < 0.001). However, splenic NK cells obtained from PDT-treated tumor-bearing mice were not cytotoxic in vitro against EMT-6 cells, suggesting that NK cells contribute to the PDT effect in vivo by an indirect mechanism. In addition, when mice with complete tumor regression following PDT were rechallenged 28 days later with 5 x 10(5) EMT-6 cells, tumor growth was significantly inhibited as compared to controls (tumor volume 40 days postrechallenge: 137 mm3 vs 833 mm3 in controls; P < 0.03; percent animals without tumor in five experiments: 67% vs 8% in controls). Collectively, these results demonstrate that CD8+ T cells are required to prevent tumor regrowth after 2I-EtNBS-PDT, NK cells contribute to this response and such PDT can elicit protective antitumor immunity.

Photodynamic therapy of naturally occurring tumors in animals using a novel benzophenothiazine photosensitizer.

5-Ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride (EtNBS) is a novel photodynamic therapy (PDT) photosensitizer with efficacy against experimental murine tumors. In this preliminary study, dogs and cats with naturally occurring tumors were treated with EtNBS-PDT to determine safety and efficacy. Fifteen treatments were performed on 13 animals (9 treatments in 8 cats and 6 treatments in 5 dogs), generally using 400 J of 652 nm light. Two feline sublingual squamous cell carcinomas (SCCs) responded briefly (minor response). Six feline facial SCCs were treated, resulting in two partial responses and four long-term complete responses (CR). Two canine intraoral SCCs were treated; one responded minimally for 2 weeks (minor response), and one achieved long-term CR. One canine cutaneous mast cell tumor achieved CR, and one canine ocular mast cell tumor responded briefly. One canine ocular melanoma did not respond to treatment. Systemic reactions included nausea associated with photosensitizer injection in two cats and two dogs, elevated body temperatures during treatment in two dogs, elevated body temperature 2 days after PDT in one cat, and inappetance for 2 weeks in one cat. A peripheral neuropathy of undetermined cause occurred in one cat 2 weeks after PDT and resolved without treatment. Local reaction was well tolerated in 13 of 15 treatments. All animals were exposed to normal daylight after less than 5 days (mean, 3.5 days) without residual photosensitization. EtNBS-PDT is safe for dogs and cats and has activity against selected naturally occurring tumors, with an overall objective response rate (partial response + CR) of 61.5%.

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue.

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.

Factors affecting virus photoinactivation by a series of phenothiazine dyes.

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1,9-dimethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dibutylaminophenothiazine [compound 4-140], 1,9-dimethyl-3-dimethylamino-7-diethylaminophenothiazine [compound 6-136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (> 0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140. The dyes had the following affinities for DNA: 1,9-dimethylmethylene blue > compound 6-136 > MB approximately compound 4-140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue. Compound 4-140 had a high affinity for protein and a low affinity for DNA. Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9-dimethylmethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anucleate red cells.

Benzophenothiazine and benzoporphyrin derivative combination phototherapy effectively eradicates large murine sarcomas.

The tumoricidal effects of photochemotherapy with two photosensitizers, 5-ethylamino-9-diethylaminobenzo[a] phenothiazinium chloride (EtNBS) and benzoporphyrin derivative monoacid ring A (BPD-MA), were evaluated separately and in combination against the EMT-6 fibrosarcoma implanted subcutaneously in BALB/c mice. Animals carrying tumors 8-10 mm in diameter were divided into eight different groups (approximately 20/group) and subjected to various photoirradiation and drug conditions. The tumor response to photodynamic therapy (PDT) was measured as the mean tumor wet weight 2 weeks post-PDT. The combination treatment with 5.25 mg/kg EtNBS and 2.5 mg/kg BPD-MA followed by photoirradiation with 100 J/cm2 at 652 nm and then by 100 J/cm2 at 690 nm resulted in a 95% reduction in the average tumor weights compared to controls (no light, no drugs) with 76% of the mice being tumor free 2 weeks post-PDT. Because treatment with EtNBS or BPD-MA at twice the light dose and drug concentration resulted in either no significant reduction in tumor weights or increased the lethality of treatment, respectively, the data suggest that the enhanced PDT effect observed with the combination of drugs is synergistic rather than additive. Histology of tumors 24 h post-PDT with the combination of drugs showed nearly complete destruction of the tumor mass with little or no damage to the vasculature and no extravasation of red blood cells. There was no damage to the normal skin adjacent to the tumor. Fluorescence microscopy of EMT-6 cells incubated in vitro with the two photosensitizers revealed that they were localized to different intracellular compartments. The fluorescence pattern from frozen tumor tissue slices following the in vivo administration of the photosensitizers indicated a greater intracellular localization for EtNBS vs BPD-MA.

Novel photodynamic effects of a benzophenothiazine on two different murine sarcomas.

The photochemotherapeutic properties of a novel benzophenothiazine, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride, were assessed in vitro and in vivo against two murine mammary sarcoma models (EMT-6 and RIF). Photodynamic therapy (PDT) of EMT-6 and RIF cells following a 30-min incubation with dye (0.4 microgram/ml) and a light dose of 3.3 J/cm2 killed 87.0 and 99.6% of the cells, respectively. Over this same time period, RIF cells accumulate more than twice the amount of dye than the EMT-6 cell line [7.54 +/- 0.17 (SD) versus 3.11 +/- 0.15 nmol/10(6) cells] which probably accounts for their increased sensitivity to PDT. Conversely, in vivo, the EMT-6 tumor accumulates 3 times more dye (34.66 +/- 2.16 micrograms/g dry weight) than the RIF tumor (12.28 +/- 1.27 micrograms of dye/g) 3 h post-s.c. injection of dye (15 mg/kg). A study of the concentration dependent uptake of dye (following s.c. injection) in the tumor and plasma of mice bearing the EMT-6 tumor indicated a nonlinear relationship for both compartments. Maximum tissue uptake of dye and discrimination between tumor and skin or muscle occur 3-8 h following s.c. injection of dye. The ratios of dye in the tumor to the dye in surrounding skin and gastrocnemius muscle 8 h following dye injection were 4:1 and 8:1, respectively. At 24 h after dye injection, the dye was not detectable by absorption spectroscopy in the tumor, skin, or muscle. Decreasing the fluence rate from 200 to 50 mW/cm2 at a total light dose of 100 J/cm2 optimized the PDT effect. At 3 h following s.c. administration of dye, PDT of EMT-6 (7.5 mg of dye/kg; 50 mW/cm2; 100 J/cm2) and RIF tumors (15 mg dye/kg; 50 mW/cm2; 150 J/cm2) resulted in 100 and 70% cures, respectively. Histology at 24 and 72 h post-PDT showed minimal or no damage to the surrounding tissue (skin) while 70-90% of the tumor cells were destroyed or damaged. Moreover, 50-60% of the tumor cells isolated and cultured immediately following PDT were found to be nonviable. Similarly, the administration of 60 mg 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride/kg also resulted in no damage to the skin 24 h following PDT. It is suggested that the redox properties of the dye coupled with the differing metabolic states of the tumor and skin, which increase the amount of photoactive, oxidized dye present in the tumor and decrease it in the skin, are responsible for this unique differential PDT effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Photodynamic destruction of lysosomes mediated by Nile blue photosensitizers.

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS-6I and sat-NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal. This is indicated by the light-and drug-dose-dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosome-containing subcellular fraction, and impairment of the lysosomes to take up and sequester acridine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat-NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat-NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Phototoxicity, redox behavior, and pharmacokinetics of benzophenoxazine analogues in EMT-6 murine sarcoma cells.

Structural modifications to the photoinactive benzophenoxazine Nile blue A have led to three novel derivatives which include 5-ethylamino-9-diethylaminobenzo[a]phenoxazinium (EtNBA), 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium (EtNBS), and 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium (EtNBSe) chlorides. The incorporation of sulfur and selenium into the benzophenoxazine moiety results in lipophilic, red-absorbing (650-660 nm) chromophores which possess significantly increased singlet oxygen yields (0.025 and 0.65, respectively, compared to 0.005 for EtNBA). This study examines the photosensitizing efficacies and pharmacokinetics in vitro in the EMT-6 murine mammary sarcoma cell line as well as the physicochemical, photochemical, and redox properties of these new analogues. Comparisons with Photofrin II, the only photosensitizer available clinically, were made in an attempt to high-light their different pharmacological characteristics. The photodynamic activity of the benzophenoxazine dyes correlates with their ability to generate the phototoxin singlet oxygen and increases in the following order: EtNBA < EtNBS << EtNBSe. At an extracellular dye concentration of 0.5 microM, the light dose required to kill approximately 50% of the cells was 2.0 and < 0.5 J/cm2 for the sulfur and selenium dyes, respectively. The light dose required to kill approximately 50% of the cells for both EtNBA and Photofrin II could not be determined because of their weak phototoxic effect under these conditions. At a light dose of 3.3 J/cm2, EtNBSe is approximately 1000 times more phototoxic than Photofrin II. All three benzophenoxazine derivatives are characterized by a similar uptake/efflux pattern in vitro consisting of a rapid and extensive cellular accumulation followed by a slow efflux rate. Contrary to their rapid uptake, 50% of the accumulated EtNBS and EtNBSe is retained intracellularly after a 6-h period in dye-free medium. Video-enhanced fluorescence microscopy corroborates the rapid uptake measurements as well as indicating the intracellular localization of the dyes in both living and thermally inactivated cells. Low extracellular dye concentrations (0.05 microM) result in a punctate fluorescence pattern in the perinuclear region, while higher dye concentrations (> 0.1 microM) lead to additional fluorescence in the cytoplasm, cytomembranes, and other organelles but apparently not the nucleus. Absorption spectrometry revealed that living cells rapidly reduce the dyes to their colorless leuko form (photoinactive) if oxygen is not readily available in the environment. It is shown that the cellular reduction is an enzymatic process and that an oxygen-free and cell-free medium containing both the coenzyme NADH and the hydride transfer enzyme diaphorase is capable of reducing the dyes to the colorless leuko form.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells.

Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells.

Photosensitization, uptake, and retention of phenoxazine Nile blue derivatives in human bladder carcinoma cells.

The overall goal of our research is to develop effective new photosensitizers for tumor-selective photodynamic therapy. Phenoxazine dyes, including several Nile blue analogues, are known to localize selectively in animal tumors. Structural modifications yielded several series of analogues with substantially higher 1O2 yields and different photochemical and physicochemical properties. This study examined the photosensitization potency, cellular uptake, and retention of these derivatives in human bladder carcinoma cells (MGH-U1) in culture. Nile blue derivatives containing halogens and/or sulfur substitutes were selected to exhibit different 1O2 yields, pKa values, and hydrophobicities. The effectiveness of these derivatives in mediating photokilling of tumor cells in vitro corresponded well with the 1O2 yields of these compounds, indicating that structural modifications which resulted in increased 1O2 yields enhanced potency in mediating photocytotoxicity in vitro. Using derivatives (sat-NBS and sat-NBS-61) with the highest 1O2 quantum yield (0.35 and 0.821), over 90% cell kill was achieved at a sensitizer concentration of 5 x 10(-8) M, about 3 orders of magnitude more effective than hematoporphyrin derivative, the only sensitizer currently available clinically. This result suggests that some of the oxazine derivatives could potentially be effective photosensitizers. The correspondence between 1O2 yield and photosensitizing potency, together with results showing enhanced photocytotoxicity in the presence of D2O and reduced photocytotoxicity under hypoxic conditions, strongly suggests that the generation of 1O2 is a major mechanism mediating the photocytotoxic effect. The uptake of Nile blue derivatives by cells in culture exhibited a pattern of rapid initial uptake followed by a gradual increase in cellular dye contents. The uptake does not correlate directly with the individual pKa values or hydrophobicities of the derivatives, indicating that the structural modifications that increased 1O2 yields did not significantly alter the uptake and retention of Nile blue derivatives. The highly concentrative uptake by and slow efflux from dye-loaded cells were consistent with an active mechanism for the cellular accumulation of these dyes. On the other hand, the retention of the compounds was directly proportional to dye concentration in the medium over a 1000-fold range of concentrations, and the uptake could proceed at temperatures below 2 degrees C; these observations excluded endocytosis or a carrier-mediated mechanism for the uptake. The uptake was also unaffected by the presence of serum in the medium. Based on these results, we hypothesize that Nile blue derivatives transport across the cell membrane possibly as deprotonated forms and, upon entering the cell, either partition into lipophilic areas of the cell membranes and/or become sequestered in certain intracellular organelles.

Mechanisms of mitochondrial photosensitization by the cationic dye, N,N-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC): preferential inactivation of complex I in the electron transport chain.

We investigated mechanisms of mitochondrial phototoxicity caused by the cationic cyanine dye N,N'-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC), examining the role of the mitochondrial membrane potential on the dye uptake by carcinoma cells in vitro, and both the dark and photosensitizing effects of the dye on the function of isolated mouse liver mitochondria. When human bladder carcinoma cells (EJ) were pretreated with 2,4-dinitrophenol or nigericin, cellular uptake of EDKC decreased or increased, respectively, consistent with dye uptake that is dependent on membrane potentials. In isolated liver mitochondria, during NADH linked substrate oxidation (using glutamate plus malate or beta-hydroxybutyrate as substrates), low concentrations of the dye (0.25-0.5 microM) sensitized mitochondria to illumination with long wavelength light and inhibited both basal and ADP-stimulated respiration. Similar effects were observed during succinate oxidation, but only at higher concentrations of EDKC (greater than 5 microM) and at 10-fold greater light doses. NADH coenzyme Q reductase (Complex I) activity was inhibited by dye with or without light to an extent comparable to the inhibition of glutamate plus malate oxidation. Activity of cytochrome c oxidase, the terminal enzyme in the electron transport chain, was photosensitized with high dye doses (greater than 5 microM) and light, but the extent of inhibition was much less than the inhibition of respiration with succinate as substrate. ATP synthetase (F0F1 ATPase) activity was minimally affected by 4.0 microM EDKC with or without 24 J/cm2 light. We conclude that at low concentrations of dye, respiratory Complex I is a primary target for EDKC dark and light-induced toxicities. If Complex I is bypassed by using succinate as a respiratory substrate, the mitochondria can tolerate much higher dye concentrations and light doses.

Strategies for selective cancer photochemotherapy: antibody-targeted and selective carcinoma cell photolysis.

A principle objective in chemotherapy is the development of modalities capable of selectively destroying malignant cells while sparing normal tissues. One new approach to selective photochemotherapy, antibody-targeted photolysis (ATPL) uses photosensitizers (PS) coupled to monoclonal antibodies (MAbs) which bind to cell surface antigens on malignant cells. Selective destruction of human T leukemia cells (HBP-ALL) was accomplished by coupling the efficient PS chlorin e(6) to an anti-T cell MAb using dextran carriers. Conjugates with chlorin: MAb ratios of 30:1 retained > 85% MA b binding activity, and had a quantum yield for singlet oxygen production of 0.7 +/- 0.1, the same as that of free chlorin e(6). Cell killing was dependent on the doses of both MAb-PS and 630-670 nm light and occurred only in target cell populations which bound the MAb. On the order of 10(10) singlet oxygen molecules were necessary to kill a cell. A second approach to specific photochemotherapy, selective carcinoma cell photolysis (SCCP), relies on preferential accumulation of certain cationic PS by carcinoma cell mitochondria. We have evaluated several classes of cationic dyes, and in the case of N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC) and some of its analogs, have demonstrated highly selective killing of human squamous cell, bladder and colon carcinoma cells in vitro. In isolated mitochondria, EDKC uptake and fluorescence depended on membrane potential, and the dye specifically photosensitized damage to Complex I in the electron transport chain. N,N'-bis-(2-ethyl-1,3-dioxolane)-kryptocyanine and some of its analogs accumulated within subcutaneous xenografts of human tumors in nude mice with tumor:skin ratios > 8. Photoirradiation caused significant inhibition of tumor growth, without cutaneous phototoxicity.

Intramitochondrial dyes allow selective in vitro photolysis of carcinoma cells.

Carcinoma cell mitochondria preferentially accumulate and retain certain cationic dyes to a much greater extent than most normal cells. Thus, they can potentially serve as targets for highly selective photochemotherapy. We evaluated 10 rhodamine and cyanine dyes as carcinoma-specific mitochondrial photosensitizers in vitro. The most effective, N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine (EDKC), caused marked, light-dependent killing of human bladder, squamous, and colon carcinoma cell lines after 30-min incubations at 1-0.01 microM but was minimally toxic to human keratinocytes and to normal monkey kidney epithelial cells (CV-1). Carcinoma cell phototoxicity was proportional to the amount of dye incorporated by the different cell lines. Selective killing ratios were 70-1000 for 0.1 microM dye and light doses of 100-175 J/cm2 between 680 and 720 nm.