Photosensitizer targeting in photodynamic therapy. I. Conjugates of haematoporphyrin with albumin and transferrin.
Conjugates of haematoporphyrin (HP) with serum albumin and transferrin were prepared, purified by gel filtration and characterized by high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE) and spectroscopy. Although the fluorescence was somewhat quenched, the conjugates had similar singlet oxygen quantum yields to free porphyrin. The albumin conjugate (HP-BSA) could be divided into monomeric and cross-linked fractions. In NIH 3T3 and HT29 cells, native albumin could not compete with the uptake of HP-BSA and the uptake was greatly enhanced in the absence of serum and in the presence of poly-L-lysine. We infer that the conjugate was mostly associated with the plasma membrane in these cells. The uptake of HP-transferrin showed evidence of a receptor-mediated component in that it was partially inhibited by native protein and increased when transferrin receptors were upregulated by an iron chelator. J774 macrophage-like cells accumulated fluorescence from HP-BSA to a much higher degree than HT29 cells, even though the protein was extensively degraded (HT29 cells did not appear to degrade the protein). The time course of the photocytotoxicity of HP-BSA was prolonged in J774 cells, although their response to free porphyrins was similar to that seen in HT29 cells. Chloroquine inhibited protein degradation without having an effect on the fluorescence uptake. J774 cells acquired more fluorescence and degraded more protein when supplied with cross-linked HP-BSA compared with monomeric fraction. For a given fluorescence uptake, the cross-linked fraction was also more photocytotoxic. We conclude that macrophages can acquire photosensitizer-protein conjugates avidly and that these are delivered to the lysosomes. These types of conjugate may have applications in targeting fluorescent molecules for diagnostic imaging and for the photodynamic treatment of macrophage malignancies.
Hamblin
M R
MR
Department of Surgery, University of Dundee, Ninewells Hospital and Medical School, UK.
Newman
E L
EL
D016475:3T3 Cells; D000230:Adenocarcinoma; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms; D004591:Electrophoresis, Polyacrylamide Gel; D006415:Hematoporphyrins / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010778:Photochemotherapy / Q000379:methods*; D017319:Photosensitizing Agents / Q000138:chemical synthesis*; D012709:Serum Albumin; D012710:Serum Albumin, Bovine / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014168:Transferrin / Q000138:chemical synthesis* / Q000493:pharmacokinetics / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D006415:Hematoporphyrins; D017319:Photosensitizing Agents; D012709:Serum Albumin; D014168:Transferrin; C091193:haematoporphyrin-bovine serum albumin conjugate; C091194:haematoporphyrin-transferrin conjugate; D012710:Serum Albumin, Bovine