Studying The Interaction Between Surfactants And Cells Via Fluorescence Tracing Method

Surfactants are extensively applied in various industries.Due to their frequent and close contact with human,the safety of surfactants has aroused widely public attention.Various methods have been applied to study the interaction between surfactants and cells.However,it has never been visually observed.Hence,we synthesized a variety of fluorescent surfactants,which are visible under fluorescent microscope,to study their interaction mechanism with cells.The involved surface activity into cytotoxicity would also be clarified.Firstly,we designed and synthesized several anionic and non-ionic fluorescent surfactants based on different fluorescent groups including fluorescein,8Hydroxypyrene-1,3,6-trisulfonic acid,trisodium salt(HPTS),coumarin and fluorescein isothiocyanate(FITC).By comparing the fluorescence properties as well as the surface activity,anionic fluorescent surfactants HPTS-Cn with HPTS as the fluorescent hydrophilic group and varying alkyl chain as the hydrophobic group were selected to represent the anionic surfactants.The synthesized HPTS-Cn showed the maximum emission wavelength at 440 nm as well as the stable fluorescence intensity at pH 4.58.Within the range of 0.5-60 μM,HPTS-Cn showed a good linear relationship between the fluorescence intensity and the concentration.Moreover,HPTS-Cn in the buffer exhibited CMC as low as 0.05 mM and their surface activities were closely related to the hydrophobic chain length at a sequence of HPTS-C16>HPTS-C18>HPTS-C12>HPTS-C8.Similarly,non-ionic fluorescent surfactant FITC-Pluronics with FITC as fluorescent group and polyethylene glycol-polypropylene glycol block copolymers as surface active groups were selected to represent the non-ionic surfactants.The synthesized FITC-Pluronics showed fluorescence with maximum emission wavelength of 525 nm and good surface activity,among which FITC-P123 has better surface activity than FITC-F127.Secondly,the fluorescent surfactant above was applied to study their interaction with human cells.Human skin fibroblasts(HF),human hepatoblastoma HepG2 cells,and human hepatic stellate cell line LX-2 were respectively used to evaluate the cytotoxicity of fluorescent surfactants.It was found that their cytotoxicity were positively correlated with their surface activities as well as their binding on cells,whereas HPTS-C16 exhibited the strongest functions.Through fluorescence microscopy,it was dynamically observed that HPTS-C16 initially inserted into the cell membrane,then entered the cell and gathered in the endoplasmic reticulum which turned into swollen.At the same time,the destruction of the cytoskeleton,the rupture of tunnel nanotubes(TNTs)and the generation of vesicles were also observed.Overall,this thesis,for the first time,intuitively and dynamically demonstrated the complete process of surfactant-induced cytotoxicity.The synthesized non-ionic fluorescent surfactant FITC-Pluronics were non-cytotoxic and could cross cell membrane by endocytosis before finally located on the lysosome.Similarly,the interaction between fluorescent surfactants and bacteria/fungi were also studied.It was found that the non-ionic surfactant FITC-P123 had no activities against bacteria/fungi along with no attachment on cytoplasm membranes.In oppsite,the anionic surfactant HPTS-Cn had obvious antibacterial and antifungal activities.Similar to their performances on human cells,HPTS-Cn showed the antimicrobial activities which were closely correlated with its surface activity,whereas HPTS-C16 exhibited the most distinguished functions.Taking Alternaria alternata as an example,HPTS-C16 presented both inhibitory effect on fungal spores and hyphae.It was found that HPTS-C16 could interact with the plasma membrane of fungi before causing the leakage of intracellular substance and the eventual death.Relatively,HPTS-C16 exerted a stronger inhibitory effect on the spores than on hyphae.Finally,considering that the fluorescent surfactants demonstrated the toxicity on human cells and microorganisms via interaction with their membranes,DPPC-GUV were used as a simplified model of membrane to illustrate the interaction of surfactants with cells,with the aid of the molecular dynamic simulations.It was found that the anionic surfactant HPTS-C16 could insert the phospholipid bilayer with a hydrophobic tail chain,destructing the phospholipid bilayer and enhancing membrane permeability.Nevertheless,the non-ionic surfactant FITC-P123 could only approach to the surface of membrane,and then bind with phospholipid bilayer through hydrophobic interaction,causing a slight increase of membrane permeability and a weak membrane damage.In conclusion,this thesis investigated the interaction between surfactants and cells/microorganisms/membranes via a visual observation,providing new aspect for studying the toxicology or functions of surfactants.