THE DESIGN OF A NOVEL ANTI-TUMOUR DRUG DELIVERY SYSTEM USING IMMUNE-DERIVED CELLS.

Exploitation of the natural ability of some immune-derived cells to target and kill tumour cells has been approached by use of the cells in conjunction with liposomes which have been used to carry a wide variety of therapeutic agents. The proposed delivery device combines the two approaches of targeting liposomes specifically to tumours and metastases and the adoptive immunotherapy of monocytes and/or macrophages. It involves encapsulation of chemotherapeutic drug within a liposomal matrix which affords protection from the host-immune response by uptake of the drug-liposome, combination into macrophages. Liposomes were prepared containing either the fluorescent probe carboxyfluorescein located in the liposomal aqueous phase, or the anti­cancer drugs doxorubicin or mitoxantrone which located in the lipid bilayer. Unelicited murine peritoneal macrophages were chosen as the m􀀂crophage model for the present study and attempts were made to isolate them from murine peritoneal exudate. The isolation of monocytes from human whole blood was also conducted since it would be necessary to carry out further studies in a human model. The number of unelicited murine peritoneal macrophages available for injection was maximised by culture of the macrophages in polyallomer centrifuge tubes- a substratum to which minimum adherence and a similar level of phagocytosis occurred compared to plastic or Teflon film. The effect of lipid type, charge, pre-incubation of liposomes in serum and liposomal concentration upon uptake of carboxyfluorescein liposomes by murine peritoneal macrophages in vitro was found to be negligible with respect to the concentration of carboxyfluorescein ingested. However, uptake in vivo of i.p. injected mitoxantrone and doxorubicin liposomes was considerably higher than that observed in vitro and the many liposomes ingested occupied a large volume of intracellular space within the macrophages. Furthermore, the macrophages were observed to retain their viability after liposome internalisation. Observation of cells using a laser light confocal microscope revealed that mitoxantrone was retained in the internalised liposomes compared to the doxorubicin of which much was released and collected at the nuclear membrane as was also observed with the free doxorubicin. The mitoxantrone liposomes were considerably less toxic to a human breast cancer cell line (MCF-7) in vitro than free mitoxantrone but more toxic than the drug­free liposomes which had a negligible cytotoxic effect. The murine peritoneal macrophages (MPM) containing mitoxantrone liposomes were more toxic in a 10:1 than a 1:1 MPM:MCF-7 ratio. This suggested that the toxicity was dependent upon the concentration of available free mitoxantrone which is consistent with observations by others for the intact release of doxorubicin from macrophages. Liposomes were labelled with [3H]-mitoxantrone and their distribution was observed in mice alone and within murine peritoneal macrophages loaded in vivo. The label was located predominantly in the liver, spleen, gut and carcass one hour after i.v. injection into a litter mate which revealed that the macrophages containing the liposomes were not trapped in the lungs and were therefore capable of reaching a tumour target.