| Studying the dynamic behavior of molecules on lipid membranes is of great significance for understanding the mechanism of drug molecules interacting with real cell membrane and advancing rational design of drug molecules.Accurate detection of the molecule-membrane interaction requires a detection technique that can observe the membrane surface in situ,in real time,and specifically.Second harmonic generation(SHG)has been widely used to study various biological surface interfaces,such as vesicles,Escherichia coli membrane and erythroleukemia cell membrane,due to its advantages of surface selectivity and sensitivity.Hemicyanine,malachite green dyes and anthraquinone anticancer drugs are non-centrosymmetric molecules with large π conjugate structures,so SHG method can be used as a detection technology to study the interaction between such molecules and lipid membrane.Among them,hemicyanine and malachite green dye molecules are commonly used as model dyes in the field of SHG.However,the details of dynamic behavior and the quantitative information on the transportation kinetics need to be further explored.In recent years,in order to extend the application of SHG method and show the advantages of this method,researchers have started to extend the molecular systems studied from hemicyanine and malachite green dyes to real biological or drug molecular systems,however,the direct study of the interaction between these molecules and lipid membranes is still lacking.Therefore,in order to enrich the application fields of SHG method,anthraquinones with SHG response are chosen as another molecular system to explore in this thesis.Based on the above research background,this thesis mainly used SHG spectroscopy,combined with two-photon fluorescence(TPF)spectroscopy and molecular dynamic(MD)simulation methods,starting from the model dye system,then extending to anthraquinones,to study the kinetic process of the interaction between molecules and vesicle membrane.Firstly,the interaction bwteen two model dye molecules,4-(4-diethylaminostyry)-1-methyl-pyridinium iodide(D289)and malachite green(MG)with 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)(DOPG)vesicles were investigated using SHG and MD simulation.The MD simulation results revealed the orientational flipping behavior of D289 and MG molecules on the DOPG membrane surface,that is,the orientation angle ＜ 90° during adsorption and the orientation angle > 90° when they embedded in the outer membrane.This work revealed that the decrease of SHG signal can be attributed not only to the commonly recognized transmembrane transport process,but also to the orientational flipping process of molecules on the membrane surface,thereby enhancing researchers’ understanding of the dynamic behavior of model dye molecules on the membrane surface.In addition,a cross-membrane transport model was applied,which demonstrates that the difference in concentration-dependent regular during the transportation of D289 and MG is related to the magnitude of the inside and outside transport rate constants,thus further enriching the quantitative information of the molecular transportation and providing a simplified theoretical model.Then,the interaction between anthraquinones with different side groups and DOPG vesicles were investigated by the combined SHG and TPF spectroscopies and MD simulation.Experimental and MD simulation results revealed that adriamycin/doxorubicin(DOX),daunorubicin(DNR)and demethoxydaunomycin/idarubicin(IDA)drug molecules with a charged side chain have the same adsorption and embedding kinetic behavior on the surface of DOPG membranes,that is,the molecular orientation angle is always > 90°.On the other hand,mitoxantrone(MIT)molecules with two charged side chains have a different kinetic behavior including the orientation flipping and embedding on the membrane surface,that is,the molecular orientation angle changes from ＜ 90° to > 90°.In addition,by quantitatively analyzing the time constant and the electrostatic interaction energy between the drug molecules and the lipid molecules,we revealed that the speed of the embedding and transporting process of anthraquinone molecules was not only related to the hydrophilicity of the molecules,but also related to their charge and embedding mode.This study revealed the orientation changes of drug molecules with different structures during their adsorption and embedding on lipid membranes,thus obtained a clearer physical picture of their behavior on the membrane surface,which is expected to provide theoretical guidance for exploring the mechanism of interaction between real cells and anthraquinones,and to promote rational design of drug molecular structures.Finally,the effects of lipid membrane composition on the dynamic behavior of anthraquinone molecules on the membrane of vesicles were studied using SHG and TPF spectroscopy and MD simulation.The selected lipid molecules other than DOPGare 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-pho-(1’-rac-glycerol)(POPG)and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine(DOPS).The results revealed that compared with PG lipids,DOPS lipids significantly reduced the surface area due to the formation of inter-lipid hydrogen bonds in the head group region,resulting in a shallower embedding depth of DOX,DNR and IDA on its surface and prevented the flipping embedding of MIT.In addition,the formation of complex hydrogen bond networks in the head group region of DOPS phospholipid and the decreased unsaturation of the tail chain in POPG phospholipid make the order of the lipid tails higher than that of DOPG lipids,thereby hindering the transmembrane transport of IDA molecules.This study elucidates the effects of phospholipid head groups and tail chains on the embeddingand cross-membrane transporting behavior of drug molecules,which is expected to provide clues for controlling molecular delivery efficiency by manipulating lipid film components.In addotion,it further demonstrates the effectiveness and universality of combining SHG and TPF spectroscopy in studying the molecular behavior of membrane surfaces. |
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