Morgan, Janet; (1990) Antibody directed photosensitive liposomes: Their potential for cytotoxic action. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The purpose of this study was to design a tumour specific cytotoxic system which will act rapidly and with few side effects. The water soluble sulphonated derivative of the photosensitive dye aluminium phthalocyanine (AlSPc), which is known to be activated by light to produce singlet oxygen, was tested as a cytotoxic agent in antibody targeted liposomes. The methods used to purify the AlSPc, to encapsulate it in liposomes and to attach targeting ligands, antibody or Protein A, covalently to the liposomes were described. Preliminary experiments optimised conditions for the greatest differential of cytophototoxicity between targeted and untargeted liposomes. Further series' of experiments using a variety of cells showed that the phototoxic effect was dependent on the concentration of liposomes, antibody and AlSPc as well as the light exposure at specific wavelengths. Results were similar for both directly and indirectly targeted liposomes. The former had specific antibody attached to the liposomes, and the latter had anti-muse immunoglobulin or Protein A attached to the liposomes which then bound to cell specific antibody in a 'sandwich' technique. Target cells included human T lymphocytes, B cell lines, and human osteosarcoma and colorectal carcinoma cell lines. Specific killing was demonstrated in all cases when relevant antibodies were used. When cells did not express antigen for the antibody or when irrelevant antibody was used there was no phototoxicity. Free AlSPc incubated under the same conditions as liposomes also had no toxic effects. Diffarent concentrations of AlSPc were encapsulated in liposomes to deteonine the degree of amplification required for maximum photocytotoxicity. Similarly, purified fractions of di-, tri-, and tetra- sulphonated phthalocyanine encapsulated in liposomes were compared for their phototoxic effects, and the differences observed were explained. Using a cooled charge coupled device (CCD) camera, a sensitive method for the detection of AlSPc fluorescence, it was demonstrated that the phototoxic effect of AlSPc liposomes was effective without internalisation of the liposomal contents, and also that cell binding in cell mixtures was antibody specific. Some non specific binding of lioosomes was also shown but it was insufficient to cause toxicity. Fluorescent analysis of bone marrow samples incubated with CDS and anti-B cell antibodies and AlSPc liposomes showed specific targeting of subpopulations of cells and a small population of cells also took up liposomes nonspecifically. When subpopulations of bone marrow were targeted by AlSPc liposomes and also treated with red light, growth of CFU-GM colonies from untargeted progenitors was the same as untreated controls. The mechanism by which cell death was photodynamically induced by targeted AlSPc liposomes is discussed. The potential of the techniques used here as a treatment for specific clinical situations such as bone marrow purging and photodynamic therapy of bladder and ovarian carcinoma are discussed.

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