Roy Harris

Activity of Doxorubicin Covalently Bound to a Novel Human Serum Albumin Microcapsule

Doxorubicin is widely used in the treatment of human malignancies, however is associated with significant cardiac, bone marrow and gastro-intestinal toxicity. Delivery systems may ameliorate this toxicity and increase treatment specificity by increasing the proportion of drug delivered to sites of disease. We have developed a novel preparation of doxorubicin (Dox) covalently linked to a heat stabilised human serum albumin microparticle (HSAM) carrier (median particle diameter of 4 μm) and assessed its activity in 4 malignant cell lines.Materials and methods. Doxorubicin microcapsules were compared with free doxorubicin in the rat carcinoma cell line, WRC256, and the human lines, OVCAR3, MCF7 and the Dox resistant MCF7/Dox, using a cell counting technique. IC50 were calculated from regression analysis of the resulting survival curves. Endocytosis of the microcapsules by cells in culture was observed. The rate of microcapsule uptake was assessed using dual wavelength filtered fluorescence microscopy and flow cytometry.Results. The mean IC50 following incubation with the Dox microcapsules was around 5 times greater than Dox for WRC256 (p < 0.001), MCF7 (p < 0.01) and for OVCAR3 (p < 0.01). MCF7/Dox was significantly more sensitive to Dox microcapsules than free Dox (p = 0.034). A negative correlation between the rate of microcapsule uptake and the IC50 values for each cell line in culture exists (r = −0.96, p = 0.04).Conclusions. We conclude that: 1) Doxorubicin microcapsules retain activity in vitro and appear to overcome p-glycoprotein mediated Dox resistance. 2) The observed activity of Dox microcapsules correlates with the rate of particle uptake. Further studies in animal tumour models are in progress.

Production of recombinant human hemoglobin A in Saccharomyces cerevisiae

Publisher Summary The ability to produce human hemoglobin (Hb) in a genetically engineered host microorganism offers a number of valuable opportunities. First, a recombinant microorganism provides an attractive alternative to outdated stocks of red blood cells as a source of Hb for formulation into an Hb-based red cell substitute. Second, specific mutant Hbs can be synthesized and produced in the host for subsequent detailed structural and functional studies. Both Saccharomyces cerevisiae and Escherichia coli have been employed for the production of recombinant human Hb. This chapter presents a straightforward method for producing recombinant Hb (rHb) in S. cerevisiae and E. coli that avoids the refolding and reconstitution steps. It also describes methods for expression of recombinant human Hb A (rHb A) in S. cerevisiae, the purification of the protein, and techniques used for subsequent structural and functional characterization. Coexpression of α- and β-globins to produce rHb A can be achieved either by using a single plasmid carrying both α- and β-globin expression cassettes, or by cotransformation with two plasmids, each carrying α- and β-globin expression cassettes and complementary auxotrophic markers. Examples of both of these approaches are also discussed in the chapter.