Tea Polyphenols Nanoparticles For Nucleic Acids Delivery:A Preliminary Study

Objective:This study is the first to explore the use of Mn²⁺-catalyzed functional tea polyphenols nanoparticles（TPNs）as nucleic acids delivery vehicles,furthermore,by investigating their effectiveness in the delivery of nucleic acids in vitro and in vivo,it provides new ideas and directions for the study of gene carriers.Methods:（1）NA@TPNs were prepared;the nucleic acid loading capacity,protection capacity,mounting mode and particle size,ζ-potential,PDI,stability,particle morphology and elemental composition of NA@TPNs were examined by Malvern particle size meter,fluorescence spectrometer,transmission electron microscopy and agarose gel electrophoresis.（2）Fluorescence microscopy,flow cytometry,q RT-PCR and Western-blot to detect nucleic acid delivery,entry pathways and lysosomal escape of NA@TPNs in vitro.（3）Biosafety of NA@TPNs tested by MTT assay and live-dead cell staining.（4）Various intrinsic biological activities of NA@TPNs such as anti-inflammatory,antioxidant and macrophage repolarization were analyzed by fluorescence microscopy,flow cytometry,q RT-PCR and Western-blot.（5）Animal live imaging technique to observe the nucleic acid delivery and organ targeting of NA@TPNs in vivo;hemolysis assay to detect the biocompatibility of NA@TPNs;q RT-PCR assay to screen for the best sicaspase-3 gene fragment.（6）8-week-old BALB/c mice were injected with Concanavalin A to induce an acute hepatitis model.Survival rates,liver morphology and weight were recorded,and blood biochemical parameters and oxidation levels in serum and liver were measured;The levels of inflammatory factors（TNF-α,IL-1βand IL-6）in mouse serum and liver were measured by ELISA;the apoptosis of mouse hepatocytes in different treatment groups and the expression of apoptosis-related factor caspase-3 were detected by TUNEL and immunofluorescence staining;the pathological changes of important organs（heart,liver,spleen,lung and kidney）in mice were observed by H&E staining.（7）Statistical analysis and graphing were performed using Graph Pad Prism 8.0 software,and one-way ANOVA was used to compare multiple samples.P<0.05 indicates a statistically significant difference.Results:（1）NA@TPNs can efficiently encapsulate a wide range of nucleic acids,with a high encapsulation rate（>80%）and good nucleic acid protection at a mass ratio（EGCG:NA）of<10:1.They are irregularly spherical in appearance,with a particle size of about 220 nm and good stability.（2）NA@TPNs efficiently deliver nucleic acids into cells and perform their corresponding biological functions by means of giant cellular drinking.（3）NA@TPNs have high biosafety,good anti-inflammatory,antioxidant and macrophage repolarization intrinsic biological activities.（4）NA@TPNs successfully delivered sicaspase-3 to mouse liver,combined with the intrinsic biological activity of the vector,exerted anti-inflammatory and antioxidant effects,reduced hepatocyte apoptosis,and efficiently alleviated liver injury in acute hepatitis,significantly increasing the survival rate of mice（80%）.Conclusion:TPNs are nucleic acid vectors with high gene delivery efficiency and biosafety,suitable for the in vivo/ex vivo delivery of a wide range of nucleic acids.With its intrinsic biological activities such as anti-inflammation and anti-oxidation,NA@TPNs can be more effective in disease treatment when equipped with nucleic acids.