TARGETABLE PHOTOACTIVATABLE DRUGS .3. INVITRO EFFICACY OF POLYMER-BOUND CHLORIN-E6 TOWARD HUMAN HEPATOCARCINOMA CELL-LINE (PLC PRF/5) TARGETED WITH GALACTOSAMINE AND TO MOUSE SPLENOCYTES TARGETED WITH ANTI-THY1.2 ANTIBODIES/

Chlorin e6 and HPMA copolymer-bound chlorin e6 were compared with chlo rin e6 polymer conjugates containing galactosamine or anti-Thy 1.2 ant ibody as targeting moieties. Galactosamine recognizes asialoglycoprote in receptors on the human hepatocarcinoma cell line PLC/PRF/5 and the anti-Thy 1.2 antibody interacts with Thy 1.2 alloantigens on mouse spl enic T cells. The efficiency of photodynamic injury as a function of i ncubation time and temperature, and irradiation time was studied. Two- day-old cultures of PLC/PRF/5 cell line were most sensitive to HPMA co polymer bound chlorin e6 (targeted or nontargeted), whereas no differe nces were observed when free drug was tested on 1-, 2-or 3-day-old cul tures. Dark toxicity of the free drug was observed at concentrations a s low as 2 X 10(-6) M. Dark toxicity decreased when chlorin e6 was bou nd to HPMA copolymers, especially to conjugates containing targeting m oieties. The effect of incubation time was seen only in the hepatocarc inoma cell culture. For galactosamine-targeted HPMA copolymer bound ch lorin e6, 2-3 h were necessary to induce a pronounced killing effect. For anti-Thy 1.2 targeted polymeric drug and for free chlorin e6, 1 h of incubation was sufficient to load the cells with a photolytic dose of chlorin e6. Dependence on the time of irradiation was observed in b oth targeted conjugates. One hour of irradiation induced only limited photolysis, whereas 7.5 h of irradiation was necessary for substantial photodynamic injury. Photodynamic destruction of cells exposed to fre e drug was similar for irradiation periods of 1-7.5 h. In accordance w ith the mechanism of cellular uptake of polymeric conjugates by recept or-mediated endocytosis, the conjugates were less photodynamically act ive when incubated with cell cultures at a lower (4-degrees-C) tempera ture. Nontargeted polymeric chlorin e6 was always considerably less ph ototoxic when compared to targeted HPMA copolymer conjugates. Antibody response to thymus-dependent antigen (SRBC) induced in vitro is more sensitive to the targeted photosensitizer, if compared with the estima tion of cell viability. It suggests that lower concentrations of the p hotosensitizer do not destroy (desintegrate) the target cells, but the ir function and/or proliferation may be impaired. Binding of antibodie s via carbohydrate moieties in the Fc portion of the anti-Thy 1.2 mole cule increases the photodestructive capacity of the antibody targeted photosensitizer, when compared to conjugates where the antibody was bo und via N(epsilon)-amino groups of lysine residues. A concentration of 1 X 10(-7) M of chlorin e6 in the former conjugate kills 40%, and a c oncentration of 1 X 10(-8) M 30% of target T cells while the latter co njugate and free drug are ineffective at the above mentioned concentra tions. The results obtained from these two in vitro models allowed us to compare the photodynamic effect of targeted HPMA copolymer bound ch lorin e6 on a hepatocarcinoma cell line (model of anticancer treatment ) and on normal lymphocytes (model of immunosuppression).