Red Blood Cell as an Elastic Probe: Interaction with Drugs and Toxins
Thesis or dissertation
University of Exeter
In this thesis we investigate the interaction between drugs and toxins with membranes using red blood cells (RBCs) as morphoelastic probes. Using fluctuation spectroscopy, we were able to probe the RBC mechanical response to a simulated diabetic environment and to investigate the effect of metformin, one of the most widely used medicines to treat diabetes. Healthy RBCs were incubated in high levels of glucose or glucose and metformin and their mechanical properties were tested upon the exposure to oxidation with hydrogen peroxide (H2O2). My results show that the response to oxidation and glycation is different for different donors, with some donors more susceptible to oxidation than others. I have also found that glycated cells are more susceptible to oxidation with H2O2 than control and metformin treated RBCs. Metformin treated RBCs show a response to oxidation similar to control cells which suggests that metformin may have some antihyperglycaemic and antioxidant effects which could preserve the RBCs membrane elasticity within the normal limits, counteracting the adverse effects of oxidative stress. The interaction between the RBC membrane and two of the Clostridium perfingens toxins, $alpha$ -toxin and NetB, is next studied in this thesis. Using fluctuation and absorbance spectroscopy, changes in the RBCs morphology caused by the toxins can be monitored allowing us to describe the course of the toxin membrane interaction. I conclude that the two toxins studied in this thesis have two different mechanisms of action. Both toxins produce a decrease in the cell radius but through two different mechanisms. NetB causes a decrease in the cell radius by forming large pores in the red cell membrane allowing for quick lysis and the exchange of material across the membrane. Whereas $alpha$-toxin causes a decrease in the cell radius by hydrolysing specific lipids in the cell membrane without necessarily causing the formation of membrane pores. These differences in the interactions between the two toxins and the red cell membrane have distinct fingerprints in the evolution of the cell shape and membrane thermal fluctuation dynamics. Fluctuation and absorbance spectroscopy were also used to investigate the effect of nitroglycerine (GTN) on the RBC morphology and mechanical properties. This study was prompted by a recent report in the literature that related decreases in the viscosity of whole blood to changes in the membrane surface charge. My results show that changes in the electrophoretic mobility of GTN-treated RBCs strongly depend on the incubation time. Cells incubated in GTN for 5 minutes decreased their mobility by about 20% whereas cells incubated for 20 minutes increased their mobility by about the same amount. Further investigations on the RBC morphology showed that GTN causes changes in the RBC shape. The matching times scales between those experiments and the electrophoretic experiments made me conclude that RBCs shape may play a role in the electrophoretic mobility of the RBCs treated with GTN. The main results obtained in this thesis demonstrate the viability of the idea of using RBCs as morphoelastic probes, which can provide detailed information about the interaction of solutes of interest and the plasma membrane. At the end of this thesis I propose use of RBCs in such a capacity to monitor the progression of disease by comparing the cell elastic state to clinical markers of disease.
PhD in Physics