Interactions Between Chitosan And Model Biomembranes

Chitosan is an important natural polysaccharide,which has unique physiological functions such as excellent biodegradability,biocompatibility,bioadhesion,permeability and antibacterial property.It remains the subject of intensive research as an ideal material for biomedical applications.The interaction between chitosan based biomedical materials and biological cells is the key factor determining its performance.It is not only of great scientific significance to study the interaction between chitosan or its derivatives and biomembranes,but also lays the theoretical foundation for the design and application of chitosan based biomedical materials with excellent properties.In this thesis,the chain structure and conformation of chitosan were studied,then quaternized chitosan with different molecular weights were synthesized and characterized,and the interaction between quaternized chitosan and various model biomembranes and its effect on their conformation and morphology were studied.The main results are the following:1.The molecular weight distribution and chain conformation of a series of chitosan samples with different molecular weights were characterized by size exclusion chromatography or asymmetrical flow field-flow fractionation coupled with multi angle laser light scattering(SEC-MALLS,AF4-MALLS).The effects of various experimental conditions,such as the eluent composition,the polymer concentration,the flow rate of eluent and the characteristics of SEC columns,on the separation efficiency were studied in detail.It was found that SEC experiment of chitosan must be carried out under suitable conditions:a sufficient amount of salt(cs>200 mm)must be added to the eluent to avoid chitosan adsorption on the SEC columns,the concentration of chitosan must be in the range of dilute solution(0.125?0.25 mg/mL)to avoid overloading the SEC columns,the flow rate of eluent must be low enough to avoid the chromatographic mode transition.The delayed elution of high molecular weight chitosan at high flow rate is explained by the chromatographic mode transition from SEC to slalom chromatography,which originates from the coil-stretch transition of chitosan chains in elongational flow through the columns.The persistence length Lp of chitosan chain in 200 mM acetate buffer(pH=4.5)was 10 nm with semi-flexible chain structure.AF4 is a complementary technique for the separation of chitosan in aqueous solution down to millimolar ionic strength,thereby overcoming the problem of polymer adsorption in SEC.The persistence length of chitosan in acetate buffers,obtained by fitting the conformation plot by the wormlike chain model,is linearly proportional to the Debye length:Lp(nm)=4.1 k-1+7.7.With increasing salt concentration from 1.25 mM to 800 mM,Lp of chitosan decreased from 45 nm to 9 nm,and its intrinsic persistence length Lp,0=7.7 nm.2.N,N,N-trimethyl chitosan(TMC)samples with different molecular weights were prepared by chemical modification of chitosan.The degree of quaternization is between 70%and 82%,and the molecular weight ranges from 29 to 136 kg/mol.The TMC chain took a random coil conformation in 200 mM acetate buffer(pH=4.5),and the persistent length Lp was about 3.2 nm,which is smaller than that of chitosan.It is due to the introduction of methyl groups on the amino group of chitosan,which increases the volume of side group and destroys the intramolecular hydrogen bonds between chitosan repeating units,resulting in the increase of the chain flexibility.TMC showed excellent antibacterial activity against Escherichia coli and Staphylococcus aureus with resistence close to 100%.3.Dynamic laser light scattering(DLS)and Zeta potentiometry showed that DOPG/DOPC liposomes with different negative charge contents were successfully prepared by the extrusion method.The optimal conditions for the separation and characterization of liposomes by AF4-MALLS were the following:an injection concentration of 0.2 mg/mL and an injection volume of 50 ?L.The detector flow rate was kept at 0.5 mL/min,while the cross flow rate decreased exponentially from 1.0 mL/min to 0.1 mL/min in 20 minutes and kept at 0.1 mL/min for another 10 minutes.It was found that the results obtained by AF4-MALLS agreed well with those measured via DLS,indicating it is an effective method for the separation and characterization of liposomes.4.The interactions between quaternized chitosan and liposomes were studied by DLS and Zeta potentiometry.The size of low charged(5%)DOPG/DOPC liposomes did not change upon modification with TMC,indicating a weak interaction between TMC and low charged liposomes.The sizes of 20?40%charged DOPG/DOPC liposomes increased drastically after decoration by TMC with increasing the TMC/Lipid mole ratio until large aggregates appeared,indicating a strong interaction between TMC and high charged liposomes.Interestingly,the size of 10%DOPG/DOPC liposomes did not change upon decoration with TMC 95-5,but increased upon decoration with TMC 95-50 and 95-200.Therefore,the interaction between TMC and liposomes increase with the increase of both liposome charge and TMC molecular weight.AF4-MALLS was successfully used to separate and characterize the size distribution of TMC-liposome complex,and the results were consistent with those obtained by DLS.5.The adsorption of TMC with different molecular weight on supported lipid bilayer(SLB)was studied via quartz crystal microbalance with dissipation(QCM-D).No adsorption of TMC on DOPC SLB was found,indicating no interaction between TMC and SLB of 100%DOPC.In contrast,strong adsorption of TMC on 5?20%charged DOPG/DOPC SLB via electrostatic interaction was observed.It was found that the interactions between TMC and SLB increase with the increase of both SLB charge density and TMC molecular weight.While the adsorption of TMC on SLB gradually saturated at biomembrane charge density larger than 5?10%,depending on the molecular weight of TMC.