Analysis Of Natural Clam Soup Nanoparticles And Macrophages By Capillary Electrophoresis

In recent years,the natural nanoparticles have increasingly drawn attention of researchers.Compared with chemically synthesized nanoparticles,natural nanoparticles have better biocompatibility,less environmental toxicity,greener manufacture methods,and broader pharmaceutical applications.Although the nanoparticles can be separated with the commonly used methods like ultrafiltration and size exclusion chromatography,the defects include the separation was time-consuming,the granular characteristics are easily altered by the filtration membrane or separation resin.Therefore,the capillary zone electrophoresis method is used for the fast and non-invasive separation and analysis of the nanoparticles.Meanwhile,cells can also be viewed as a natural microparticle to be separated with the capillary electrophoresis.In this study,the capillary electrophoresis separation of murine mucosal macrophages was conducted and optimized,which was then taken to study the interactin between the clam soup nanoparticles and mucosal macrophages.The main findings were summarized as below:1.A capillary zone electrophoresis method for the direct and rapid separation and quantification of nanoparticles derived from the freshwater clam soup was established.The method was verified to be effective and non-invasive by comparing with nanoparticles separated with ultrafiltration,dialysis and size-exclusive chromatography.2.The CZE conditions of nanoparticles separation,such as types of buffer,pH,applying voltage,were optimized.The optimal conditions are:detection wavelength 214 nm,the separation voltage 20 kV and 50 mmol/L boric acid-borax buffer(pH=8.7).The concentration of clam soup nanoparticles was linear with the peak area of the electropherogram(R~2=0.998)in the range of 2.6×10~9 to54.9×10~9 particles/mL.The relative standard deviation of the instrument precision test and method precision test is less than 5%(n=6).As a method for quantitative analysis of the colloidal nanoparticles,it is simple and rapid,highly sensitivity with good reproducibility.3.Established a capillary zone electrophoresis analysis method for murine peritoneal macrophages.The optimal conditions were:detection wavelength 280nm,separation voltage 15 kV,150μm capillary(effective length 40 cm),20mmol/L pH=7.4 phosphate buffer added with 4.6%glucose.The macrophages exhibited several different electrophoretic migration times(multiple peaks)possibly due to different cell size,shape and surface charge.The concentrations of cells affected the migration time,the higher cell numbers elongated the migration time.Ultrasound pretreatment could weaken the influence of cell number on the migration time.4.Capillary zone electrophoresis analysis of interaction between the macrophages and clam soup nanoparticles.The clam soup NPs were introduced to macrophage at 1 mg/mL.Cellular differentiation was observed after 2 h.The engulfment amount of nanoparticle was 8.7×10~2 particles/cell at 24 h,and the number of phagocytic nanometers at 48 h was 2.13×10~3 particles/cell.Engulfment of the NPs shorten the migration time of cells,indicating the NPs have affected the cellular membrane potential,increased the surface charge of cells and therefore altered the migration rates.In addition,the CZE can identify both the clam soup compositions absorbed by the cells and the new compositions presented in the soup dispersion,most possibly secreted by the cells.To sum up,capillary zone electrophoresis is an effective method for rapid separation and quantification of natural nanoparticles,and for separation of macrophages,as well as monitoring the interaction between the NPs and the cells,revealing the subsequent changes in the soup compositions,particles number and differentiation of macrophages.It provides a novel,easy and effective approach for monitoring the physiological response and function alteration of macrophage triggered by food compositions.