Abstract

Macromolecular therapy such as treatment with enzymes is often limited by the fact that not much is absorbed into cells. Even more difficult is the task to reach occluded target destinations such as the brain. Chitosans are a range of different polysaccharides that are derived from the shell-protein chitin which is processed by varying degrees of deacetylation to yield chitosans. It has previously been described to self-assemble into micelles with a net positively charged outer surface, possibly acting as a carrier, which could explain the apparent increase in uptake of different compounds into cells when incubated together with chitosans. A 20% deacetylated chitosan is known to loosen the tight junctions of epithelial cells, decreasing occludin and ZO-1 (a tight junction-associated protein) at the cell periphery. It is a promising compound because of its modifiability and low toxicity. Here, we show a number of different chitosan types that increase the cellular uptake of a labeled Glutathione S-Transferase (GSTA4-4) in varying degrees in the U2OS cell-line. The uptake of GSTA4-4 and a suitable substrate could lead to depletion of intracellular Glutathione (GSH) which can cause irreversible damage to cells, especially if colocalized with mitochondria, which contain vital GSH-pools. Oregon Green-labeled GSTA4-4 alone did however not colocalize with mitochondria but with increased uptake through addition of chitosan we hope it might colocalize spontaneously to a higher degree. If successful, this treatment, in combination with antibodies against cancer epitopes such as CD19, could serve as an alternative to other cancer therapies that cause systemic damage. Two potential routes are to either use a fusion protein composed of GST and a suitable antibody’s variable fragment, or to produce chitosan vehicles with integrated antibodies (that have hydrophobic Fc-regions, e.g. BCRs) if these self-assemble, if the antibodies do not interfere with the membrane permeabilization of chitosan and if the integration of the hydrophobic region does not affect the binding properties of the antibody. Otherwise it is possible to link antibodies to the surface of the micelles through chemical procedures, although this option is most likely more time and resource consuming. Intracellular excess of GST can still cause reversible damage and it is worth investigating if free distribution in the cytosol is hindered by encapsulation of GST inside endosomes and/or chitosan-micelles by measuring GST-specific markers. It is possible that there is adsorption of GST onto the micelles as well and this could be measured by monitoring GST-specific-antibody colocalization with the micelles after encapsulation.