Nobuo Matsumoto

Spectroscopic study of haematoporphyrin oligomers in tumour tissue

Fluorescence spectra of modified haematoporphyrin (Hp) oligomers (of mean molecular weights 3000, 5000 and 12000) and Photofrin II in tumour tissue were semi-quantitatively measured by a reflection method. Characteristic peaks of the emission spectra, obtained by excitation at 370 nm, appeared at 677 nm and also at 634 and 695 nm. The intensity ratio of the emission band (677 nm) to the other emission bands (633 nm) increased in accordance with increases in the oligomer molecular weight. The integral intensity of the fluorescence emission spectra of oligomer 3000 (i.e., the pentamer of Hp in tumour tissue) was about two times greater than that of Photofrin II.The solutions extracted from the tumour tissues following injection of these porphyrins were analysed by fluorescence spectrophotometry. The integral fluorescence intensity of the pentamer solution was about three times larger than that of the Photofrin II solution. The extracted solution was fractionated by high-pressure liquid chromatography (HPLC), and the fluorescence of each fraction was measured by fluorescence spectrophotometry. The chromatography fraction of each oligomer had the same long elution time and a single sharply peaking fraction. The fluorescence spectrum pattern of the eluted oligomer fraction showed a shoulder at 677 nm and the same spectrum as that in the extraction solvent before injection. The fraction corresponding to dihaematoporphyrin ether (DHE) or its dissociated molecules showed a new emission peak at 647 nm in addition to the other monomer emission bands.From these spectroscopic analyses it was considered that Photofrin II and Hp oligomers had a tendency to aggregate in tumour tissue and that the pentamer was more readily incorporated in tumour tissue than was Photofrin II. Accordingly, it was concluded that the modified oligomers were stable compounds and that these oligomers—especially the one consisting of 5±2 molecules of Hp—were effectively incorporated into, and accumulated within, tumour tissue. It is suggested that the oligomer may be useful as an indicator of tumour localization.

Temperature-dependence of haematoporphyrin derivative uptake in vitro

The uptake of haematoporphyrin derivative (HPD) by Ehrlich ascites tumour cells was investigated by measuring the fluorescence intensity of HPD bound to the cells at various temperatures. Analysis of the HPD fluorescence emission patterns suggests that there are two steps in the HPD binding process. First, a weak binding mode: the fluorescence peak appeared at 617 nm in the early incubation period below 25°C. Secondly, a stable binding mode: the fluorescence peak appeared at 630 nm in the late incubation period above 30°C, and it was considered that the HPD uptake was due mainly to facilitated diffusion. The Arrhenius plots of logarithmic values of the initial velocity and the reciprocal of the absolute temperatures in the HPD-uptake reaction showed a biphasic linear relationship having an inflexion point at 29±5°C. From the plots, the lower activation energy corresponded to the former mode, and the higher one to the latter mode. It was considered that the critical temperature might correlate with the phase change of the membrane lipid to which HPD molecules bound and by which they are transported. Furthermore, from the results of double vital staining with Hoechst 33258 and HPD it was shown that the uptake by late G1-, S- and early G2(+M)-phase cells increased somewhat in this order.