Rat RBCs loaded with 125I-CA by hypotonic dialysis and isotonic resealing were evaluated as a carrier system. Loaded RBCs stored at 4 degrees C remained unlysed (90% survival) allowing release of encapsulated 125I-CA for up to 4 days. Thereafter, cellular lysis increased significantly. IP-injected loaded RBCs reached the maximum level (50%) in circulation at 24 h post-injection. Circulating loaded RBCs showed a half-life of 8-10 days, which was advantageous for carrier function. In contrast to IP-injected free CA, which remained in circulation for only a short time, encapsulated CA showed significant levels in circulation up to 10 days post-injection. The profile of organ uptake with time is essentially not altered for loaded with respect to native cells, being higher the removal of loaded cells and mainly localized in spleen. Nevertheless, liver is the organ with highest elimination capacity for both native and loaded cells, showing its maximum at 24 h post-injection. Concomitantly, the concentration of 125I-CA in all organs studied was highest at this time. These data demonstrate that rat loaded RBCs can potentially be used as a carrier system for long-term dissemination of drug into the organism, with specially increased delivery to the spleen. They also support the use of the rat as an experimental model for biochemical and pharmacological studies in these therapeutic systems.

Summary Levels of glutathione (GSH) in tumour tissue may be important in determining the clinical response to certain anticancer agents. Recent reports have suggested that D,L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of GSH synthesis, may be used to deplete tumour cell GSH and thus increase the therapeutic ratio of these agents. We have previously shown that 1-naphthol is a potential antitumour agent, and that its possible metabolite 1,4-naphthoquinone is thiol reactive and capable of redox cycling. It was therefore of interest to investigate the effect of pretreatment with BSO, on the toxicity of these agents, to tumour cells. For comparison we included three other cytotoxic agents, melphalan, helenalin and menadione, the toxicities of which are reported to be modulated by intracellular GSH. Depletion of GSH using BSO did not effect the toxicity of 1-naphthol, or 1,4-NQ but did produce slight potentiation of the cytotoxicities of menadione, helanalin and melphalan. The lack of effect of BSO on 1-naphthol and 1,4-NQ is not easily explained but if one also considers the modest potentiation of cytotoxicity achieved with the other agents studied, the potential use of BSO in combined chemotherapy is at best rather modest.Based on our findings that l-naphthol is selectively toxic to short term organ cultures of human colonic tumour tissue compared to normal colonic tissue from the same patients, we suggested the potential use of l-naphthol or related compounds in cancer chemotherapy (Cohen et al., 1983;Wilson et al., 1985). Recently we have also shown an antitumour activity of l-naphthol against Ehrlich ascites tumour cells (Jones et al., 1987) and it therefore is of interest to elucidate its mechanism of toxicity and formation of possible reactive metabolites. l-Naphthol may be metabolised by a microsomal mixed function oxidase to cytotoxic naphthoquinones, primarily 1,4-naphthoquinone (d'Arcy Doherty et al., 1984a, b, 1985). The toxicity of both 1-naphthol and its possible metabolites 1,2-naphthoquinone and 1,4-naphthoquinone, to isolated hepatocytes, is prececed by a rapid depletion of intracellular glutathione (GSH) (d 'Arcy Doherty et al., 1984b).GSH is the major nonprotein thiol in the cell and plays a critical role in cellular defences against oxidative stress, free radicals and alkylating agents (Meister & Anderson, 1979). One of the problems associated with chemotherapy is the wide range of sensitivities to treatment with any or one agent, which is thought to be, in part, due to the differences in sulphydryl levels in tumours. Several recent reports, have therefore considered the potential of modulating intracellular GSH levels in order to increase the chemotherapeutic efficacy of certain antitumour agents, whose toxicity is modulated by GSH (Akman et al., 1985;Arrick et al., 1983;Capranico et al., 1986;Hamilton et al., 1985;Russo et al., 1986;Suzukake et al., 1982Suzukake et al., , 1983.The cytotoxic and antitumour effects of certain quinones e.g. adriamycin and menadione, are thought to be r...

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Chemical alteration of red blood cells (RBCs) can induce increased phagocytosis of modified cells by macrophages. In this study we have used different chemical treatments for the modification of the mouse red-blood-cell membrane surface, namely oxidant compounds, such as ascorbate/Fe(+2) and diamide [azodicarboxylic acid bis(dimethylamide)], or Band 3-cross-linking reagents. We monitored the phagocytosis of oxidized or Band 3-cross-linked mouse red blood cells by peritoneal macrophages. The extent of phagocytosis of RBCs is not affected by oxidation with ascorbate/Fe(3+), but it is increased (up to 10%) by oxidation with 2 mM diamide. Furthermore, phagocytosis is greatly increased (up to 40%) as a result of cross-linking with either of two Band 3 bifunctional reagents [bis(sulphosuccinimidyl) suberate (BS(3)) and 3,3'-dithiobis(sulphosuccinimidyl propionate) (DTSSP)]. To evaluate targeting towards macrophages of such modified RBCs for therapeutical purposes, we have determined the phagocytosis of Band 3 carrier RBCs loaded with carbonic anhydrase. In this case phagocytosis is high enough (25%) to deliver the enzyme into macrophages. We have also assayed the influence of serum components and IgG on the efficiency of phagocytosis and discuss the possible phagocytosis mechanisms. In the case of BS(3)-cross-linked carrier RBCs, phagocytosis is markedly enhanced (from 12% up to 25%) by serum components. This opens a way for therapeutic application of these carrier RBCs, with special relevance in short-term delivery to cells of the mononuclear phagocytic system.

Previous work has shown increased uptake of hypotonically loaded rat RBCs by the spleen and liver "in vivo," suggesting that the cells of MPS are involved in their elimination from the circulation. In order to elucidate the mechanism of such elimination, we have undertaken studies on the interaction of such loaded RBCs, in comparison with native RBCs, with peritoneal macrophages. Erythrophagocytosis assays were performed in well plates to which thioglycollate-induced peritoneal macrophages had adhered. Native or loaded 51Cr-RBCs were added under different opsonization conditions to monolayer adherent macrophages, and then the amount of RBCs that were recognized was determined, with separation into adhesion and phagocytosis fractions. Native RBCs are slightly recognized by peritoneal macrophages, about one RBC per macrophage (Mphi). Osmotic treatment of rat RBCs used for encapsulation (independently of the encapsulated substance, 125I-CA or FITC-dextran) produces some modification in the erythrocyte membrane that induces higher recognition of these cells, about three loaded RBCs per macrophage. Consequently, both fluorescent (FITC-Dx) and radioactive (125I-CA) substances previously encapsulated in RBCs were transferred to M(phi)s. The fluorescence microscopic observations confirmed these results. Moreover, in the case of carrier 51Cr-cells loaded with 125I-CA, the amount of 125I-radioactivity delivered into M(phi)s was relatively higher than that of 51Cr. The highest ratio, 125I-CA (encapsulated substance)/51Cr-RBCs (carrier cells), present in M(phi)s means there was a stronger interaction with macrophages of RBCs that carry a higher amount of encapsulated CA, as a function of the heterogeneity of the loaded rat RBCs population previously reported. Finally, the adhesion and phagocytosis of loaded RBCs seem not to involve complement receptors or Fc receptors on the macrophages.