| As a natural polysaccharide polymer,starch is not only biodegradable,but also has a wide range of sources,low price,no biotoxicity and immunogenicity,and has become a key research project in the field of nanotechnology.Throughout the research history of nano-starch particles,most of the literature mainly focuses on the optimization of preparation methods and the application of drug-targeted therapy,while the research on the amount of nano-starch particles phagocytized by cells is rarely reported in the literature.In order to make up for this gap,the laboratory has prepared different levels of nano-starch particles by different methods based on the relevant research background,and further expanded the application of nano-starch particles in the cell field by fluorescent labeling technology.The research work mainly includes three aspects:First,the preparation of nano starch granules.In this chapter,rice is used as raw material,and the rice starch is extracted by alkali method,the purity is 89.17%,and the protein content is only 0.23%,which is negligible,and has no effect on the subsequent fluorescent labeling experiment.The above rice starch is subjected to miniaturization treatment by a precipitation method,an alkali freezing method,a crosslinking method,and an acid hydrolysis method to obtain starch granules of 100,200,400,and 800 nm,respectively.The water absorption,solution stability,gelatinization properties,morphology,infrared detection,X-ray diffraction and other properties of nanostarch were compared with the original rice starch.Second,the preparation of fluorescently labeled starch granules.Using amino acid as a raw material of different grades,the amino-rich polylysine molecule was modified on the nano-starch particles by electrostatic bonding,and some amino groups on the poly-lysine and the fluorescent molecule isothiocyanate were fluorescent.A fluorescent probe with a FITC fluorescent label was prepared,and the probe was subjected to fluorescence spectrum scanning,fluorescence substitution,fluorescence stability and cytotoxicity analysis.The results showed that nano-starch could be combined with FITC after amination to achieve the purpose of fluorescent labeling.The degree of substitution satisfied the test conditions and did not affect the overall structure of nano-starch,and the biological activity was high.When the concentration of fluorescent nano-starch is less than 100 ?g/mL,HeLa cells have low lethality and are a low-toxic carrier material,which provides a certain safety guarantee for its application in cell biology.Third,HeLa cells phagocytized the amount of fluorescent nano-starch particles and lysosomal labeling.Taking the 200 nm grade starch granules as the representative,the initial concentration of nano-starch,incubation time,incubation temperature,and the presence or absence of serum in the medium on the amount of HeLa cells phagocytizing nano-starch particles were discussed.The optimal conditions for phagocytosis of nano-starch particles:in the absence of serum,the initial concentration of nano-starch was 200?g/mL,the incubation time was 6 h,and the incubation temperature was 37?,the amount of phagocytic fluorescent nano-starch particles was the largest.It is also a process that consumes energy and gradually reaches saturation over time.In addition,based on the optimized conditions,the differences in the phagocytosis of different levels of fluorescent nano-starch were compared between HeLa cells,and the transport and localization of 200 nm starch granules in cells were investigated.The results showed that the phagocytosis of HeLa cells to 100,200,400,800 nm fluorescent starch and fluorescent raw rice starch granules were 296.37,322.14,125.52,47.66,3.42 ?g/mg,respectively,indicating that the nano-starch with smaller particle size is easier.After entering the cell,and the nanostarch particles enter the cell through the surface of the cell membrane,they can be well located in the lysosome and can be used as a novel lysosomal marker material. |
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