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,

Selective in vitro photodestruction of HPB-ALL human T-cell leukemia cells was accomplished using the photosensitizer chlorin e6 coupled through dextran molecules to an anti-T-cell monoclonal antibody (mAb), anti-Leu-1.Conjugates with mAb/chlorin molar ratios as high as 1:36 retained mAb binding activity, and the absorption spectrum and quantum efficiency for singlet oxygen production of bound chlorin (0.7 ± 0.2) were unchanged from that of the free photosensitizer. Phototoxicity, as measured by a clonogenic assay and by uptake of ethidium bromide, was dependent on the doses of both mAb-chlorin and 630-to 670-nm light, was enhanced by 2H20, and was observed only in target populations that bound the mAb. Similarly, free chlorin e6 in solution had no photodynamic effect in amounts 100 times more than that carried by the mAb. For this antibody-targeted system, approximately 1010 molecules of singlet oxygen were necessary to kill a cell.A principal objective in the development of chemotherapeutic strategies is to devise modalities that are capable of selectively acting on the offending cells while sparing normal tissues. Selectivity may be provided by the preferential action of a particular agent or by preferential localization of molecules that act nonspecifically. Monoclonal antibodies (mAbs) that bind to cell surface antigens have great promise for directing cytotoxic agents to appropriate sites; and a substantive body of literature has already accumulated on the use of monoclonal antibodies to enhance selectivity of cytotoxic drugs, radioisotopes, or toxins (1-4).The selectivity ofmAb-directed treatment may be reduced, however, by cross-reactivity due to shared antigens on normal tissues and by nonspecific tissue uptake (5, 6). These problems can be obviated by utilizing mAbs that carry light-activated molecules (i.e., photosensitizers, PSs), which are innocuous without illumination but which produce toxic or reactive species such as singlet oxygen (102) upon absorbing light. By confining illumination to areas containing specifically bound PSs, toxicity can be limited to target cells. Nontarget cells, which adventitiously bind the mAb-PS but which are outside the illuminated volumes, would be spared.The present report describes selective cytolysis of human T-cell leukemia cells in vitro using mAb conjugated to the PS chlorin e6, which has a molar extinction coefficient of about 59,000 M-1-cm-1 at 660 nm (7). Chlorin/mAb conjugation ratios of approximately 30:1 were achieved, and mAb and chlorin activities were retained. Selective destruction of target T cells was accomplished by irradiation of cells containing mAb-PS conjugates bound to their cell surface. MATERIALS AND METHODSChromophore Preparation. In brief, chlorin e6-monoethylenediamine monoamide (chlorin e6-A) was prepared as follows: A monoactivated species (mixed anhydride) was formed by sequential addition oftriethylamine and ethyl chloroformate to chlorin e6 dissolved in anhydrous N,N'-dimethylformamide. TLC analysis using C18-bonded silica pla...

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.

The effects of depleting glytolytic ATP on EDKC mediated cellular phototoxicity has been investigated using squamous carcinoma cells (FaDu) in vitro. Cells were pretreated with a non -metabolizable glucose analog 2-deoxy -D-glucose (5 mM) for 13 -15 hr that resulted in 40 -45 % decrease of cellular ATP. Subsequent exposure of the cells to EDKC resulted in twice as much dye uptake compared to control cells and upon radiation induced 4 -5 fold more killing. Pretreatment with 2-deoxy -D-glucose (2 -DG) alone did not have any effect on cell survival. We conclude that if cellular glycolytic ATP is lowered prior to their exposure to EDKC and radiation, cells become more vulnerable to the dye mediated photosensitization.

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