Research The Biomechanical Properties Of Disease-causing Red Blood Cells Based On Atomic Force Microscopy Technology

Erythrocyte is unique among human cell type in that the plasma membrane,its only structural component,accounts for all of its diverse antigenic,transport,and mechanical characteristics.Erythrocyte membrane is remarkably soft,elastic and deformability.Dynamic membranes of the erythrocytes are strongly related to the membrane structure and mechanical properties.The properties can be changed by human disease states,in turn,influence the ability of erythrocyte to transport oxygen in circulation.Quantifying the biomechanics of erythrocytes promises more sensitive probes of their structure at the nanoscale and suggests new insights into the etiology of a number of human diseases.In the healthy individual,the cells withstand repeated,large-amplitude mechanical deformations as they circulate through the microvasculature.Certain pathological conditions cause changes in both the equilibrium shape and mechanics of erythrocytes,which can result in breakdown of their physiological functions at both the tissue and organ levels.These modifications provide information on critical interactions among membrane structure,mechanical stress,and biochemical links between the cell interior and the external pathological environment.The changes of biomechanical properties of erythrocytes can potentially be used to quantitatively reflect the state of their health and pathological.It is possible to gain useful applications in clinical diagnostics and even provide suitable strategies towards effective therapeutic treatments of human diseases.We seeked to investigate the structure-property-function relationship of healthy erythrocytes so as to comprehensive understand their important physiological functions,and establish possible connections to certain human diseases.In the thesis,we used Atomic force microscopy(AFM)experimental and force-distance curve theoretical techniques to investigate a variety of problems relating to biomechanics of pathological erythrocytes.There are four aspects about this research thesis:(1)Influence of Lead Poisoning to the Mechanical Properties of Erythrocytes by Means of AFM.AFM was used to probe the deformability of healthy erythrocytes and the abnormal erythrocytes from the different concentration of lead poisoning solutions,and employed to measure the stiffness of abnormal Pb-RBCs by force-indentation-curves.The determined Young's modulus was compared with that obtained from measurements of RBCs from healthy subjects.The results showed that lead poisoning caused loss of RBC deformability and increased osmotic fragility.The Young's modulus of pathological Pb-RBCs was approximately four times higher than in normal cells.These changes could provide insights into possible the dynamics of RBCs membrane and cytoskeleton network,because the mechanical properties of RBCs can be influenced by the poisoning degrees of Pb exposure.(2)Membrane Biomechanics of Erythrocytes Parasitized by Salmonella Paratyphi B Studied Using AFM.Parasitization by Salmonella paratyphi B leads to morphology structural,biochemical component,and mechanical deformation alters to the host erythrocytes.To study these modifications,we applyed AFM to measure the mechanical properties of the membrane of RBCs-infected by Salmonella paratyphi B,including stiffness,shear modulus and bending modulus.Combining the technique with a new mathematical model describing RBC membrane was very sensitive to the invader of parasitic Salmonella paratyphi B.The measurements indicated that the growing parasite in their host RBCs caused loss of cell volume and deformation decreased.Coincident with the morphological transition,it was significantly increased in the membrane's shear and bending module.The mechanical transition could alter cell circulation and impede oxygen delivery.Proteins transported from invading organisms,such as the virulent parasitic Salmonella paratyphi B,to specific binding sites in the spectrin network were considered to introduce significant alterations to RBC membrane dynamics and mechanical response.The changes could provide insights into possible mechanistic pathways in the pathogenesis of parasitic Salmonella paratyphi B,because the parasite altered biophysical and mechanical properties of RBCs during the intraerythrocyte stage.(3)Mechanical Properties of Membrane Tethers Pulling from Sickle Red CellsAnalyzed by Force Spectroscopy.AFM probed the pull-out mechanics of lipid tethers from the sickle cell disease(SCD)of erythrocyte.GLA and Band 3 on the cell membrane,the pull-out force curves determined by AFM could distinguish the presence or absence of their cytoskeletal associations.The measurements indicated that interaction of GLA with two lectins,wheat germ agglutinin(WGA)and concanavalin A(ConA),was strong enough to pull out lipid tethers with different stationary plateaus of the tensile force.After removal of glycocalyx layer from the pathological SCD erythrocytes by deglycosylation,the probability of bond formation between covalent crosslinkers and membrane proteins was decreased.Tether breakdown and unbinding of cytoskeletal components took place simultaneously.Based on the observation results,the specific protein of SCD cell in pathological state was identified by the difference distribution of the tethers of sugar-membrane protein.(4)Force Spectroscopy Detected Ramos Cells and Interacted with Erythrocyte-Membrane-Coated Drugs Nanoparticle.We used the trace detection method of single molecule and single cell probe,to observe the membrane microstructure of Burkitt lymphoma Ramos cells.AFM force curve measured the viscoelasticity of Ramos membrane.Adopting extrusion RBC-membrane-derived vesicles,and to parcel anti-cancer drugs of Rul-NPs polymer nanoparticles,which were co-incubated with Ramos cells.We analyzed the physical characteristics of RBC-vesicles encapsulated Rul-NPs drug,the phy-chemical properties of NPs particles and pharmacokinetics.The results showed the invading Ramos cells were viscosity plasma cells.RBC-vesicle-coated Rul-NPs drug capsules can combine with Ramos cell membranes.RBC-membrane-coated nanoparticles simulated clinical drug delivery system.The translocation of natural cellular membranes,the associated proteins,and the corresponding functionalities to the surface of synthetic particles represents a unique approach in nanoparticle functionalization.