Studies On The Interaction Of Poly-D-Lysine And Cell Plasma Membrane

Chirality is a ubiquitous phenomenon in natural life.Except for glycine,amino acids have two chiral forms,the L and D enantiomers.Protein and peptides in nature are essentially composed of the L-type amino acids.The side-chain direction of D-amino acids is different from L-amino acids,so the replacement of L-amino acids with D-amino acids would cause changes in the structure,hydrophobicity,aggregation and other physical and chemical properties for amino acid-based biomolecules,which would lead to changes in the behavior of interactions with cells.As a polycation polymer,poly-lysine has attracted wide attention for its high hydrophilic nature,large positive charge and flexible molecular structure,and is often used in drug delivery,antibacterial and cell adhesion studies.In this thesis,we explored the difference between poly-L-lysine（PLL）and poly-D-lysine（PDL）effect on cells at the molecular and cellular levels.The main study results are as follows:（1）The interaction between poly-lysine（PLL/PDL）and model cell membranes was examined using sum frequency spectrum（SFG）.The molecular and interfacial hydration structures were studied when PLL/PDL adsorbed on the surfaces of the electroneutral1,2-distearoyl-sn-glycero-3-phosphocholine（DSPC）and electronegative 1,2-dipalmitoyl-sn-glycero-3-phospho-（1’-rac-glycerol）（DPPG）lipid bilayers,respectively.It was found that both PLL and PDL hydration had significantly different structures on the phospholipid surface,even the global orientation of water molecules on the PC surface was opposite,and secondary structures of PLL and PDL on the PG surface were also obviously different.（2）We used cell imaging,MTT,and flow cytometry to observe the cell apparent differences caused by the interaction between PLL/PDL and cells including A549（human lung cancer cell line）cells and HPAEpi Cs（human alveolar epithelial cell line）.The site of action on the cell membrane was also explored.Meanwhile,apoptosis,autophagy and necrosis were further investigated when PLL/PDL interacted with the cell plasma membrane.We found that PDL could be anchored onto the plasma membrane and interact with the lipids,leading to the rapid morphological change and death of A549 cells and HPAEpi Cs.In contrast,PLL exhibited good cytocompatibility and was not anchored onto the plasma membranes of these cells.Unlike PLL,PDL could trigger protective autophagy to prevent cells from necrosis in a certain degree,and the PDL-caused cell death occurred via intense necrosis(featured by increased intracellular Ca²⁺content and plasma membrane disruption),indicating that chirality can affect the interaction between poly-lysine and the plasma membrane.（3）We also studied the interaction behaviors of short chain poly-lysines with cells.The localization of short-chain PLL with a repeat unit number of 9（termed LL9）and DL9 in cells and the occurrence of autophagy and necrosis were investigated.It was found that DL9 could locate in lysosome and induce autophagy at high concentrations,but it could not elicit drastic cell death.LL9 only contains lysosomes that can enter cells but does not start from cellular autophagy,which proved that the repeat unit number of poly-lysine can affect its cellular action with chiral differences.This study confirmed that chiral poly-lysine interacted with the plasma membrane,which can induce protective autophagy and strong necrosis.In addition,this effect was correlated with the chirality and chain length of lysine,which has guiding significance for future understanding of chiral molecules interacting with plasma membrane and designing chiral drugs.