Effective Delivery Of FGF21 And Liraglutide In HEPA1-6 Cells And Insulin Resistance Animal Model Via Multi-Stage Porous Silica Nanomaterials

Part 1 The cytotoxicity of NH₂-HPSNs and liraglutide in Hepa1-6 cells and themaximum loading capacity of NH₂-HPSNs for pFGF21 and liraglutide.Objective:To verify the construction of FGF21 plasmid.To explore the toxic effects and maximum carrying capacity of NH₂-HPSNs.Methods:FGF21 plasmid was constructed and verified by sequencing.CCK8 was used to detect the toxic effects of NH₂-HPSNs and liraglutide on model cells.UV spectrophotometry was used to detect the maximum drug loading of NH₂-HPSNs on Liraglutide.Agarose gel electrophoresis was used to detect the maximum loading of NH₂-HPSNs on FGF21 Mass ratio.Fluorescence expression was observed under the fluorescence microscope.Results:The sequencing results showed that the FGF21 plasmid was successfully constructed.The CCK8 test found that with the increase of the concentration of NH₂-HPSNs,the survival rate of the cells decreased.After two days of treatment,the survival rate of the cells reached a peak at low concentrations of NH₂-HPSNs（≤300μg/ml）,and when the concentration reached 600μg/ml,The survival rate is significantly reduced（survival rate dropped to 70.47%）.The concentration of liraglutide did not have a significant toxic effect on the cells in the range of 1-100 nM/L.The maximum drug loading of NH₂-HPSNs carrying liraglutide was up to 13.8%by UV spectrophotometry.Agarose gel electrophoresis showed that the loading of plasmid increased with the increase of NH₂-HPSNs.When the ratio of NH₂-HPSNs/pFGF21reached 60:1,pFGF21 was completely encapsulated and the absorption rate reached100%.The ratio does not affected by the concentration of liraglutide（0%,4%,13.8%）already carried in the solution.Conclusions:The FGF21 plasmid was successfully constructed.NH₂-HPSNs and liraglutide had low cytotoxicity to Hepa1-6 model cells.The maximum drug loading of NH₂-HPSNs carrying liraglutide is 13.8%（32nmol/mg）.The maximum binding mass ratio of pFGF21 is 60:1.PART 2 BIOLOGICAL EFFICIENCY OF NH₂-HPSNs/LIRAGLUTIDE/p FGF21 TRANSFECTION IN VITROObjective: To explore the optimal mass ratio and time of NH₂-HPSNs/p FGF21 transfected in vitro.To verify the transfection efficiency of NH₂-HPSNs.To detect whether NH₂-HPSNs carrying both p FGF21 and liraglutide can fully exert biological effects in vivtro and whether the combination of FGF21 and liraglutide can improve insulin resistance more effectively.Methods: The NH₂-HPSNs/p FGF21 were expanded in equal proportions（150/2.5,300/5,600/10）added to Hepa1-6 cells.The cells were collected after about 60 hours.Then used Q-PCR and western blot to detect the m RNA and protein levels of FGF21.The cells were transfected with the optimal mass ratio,and the cells were collected for different times（0h,24 h,48h,and 72h）,then compared the expression of FGF21.Set four control groups(blank group,p FGF21 group,lipo2000 group,NH₂-HPSNs group and using lipo2000 and NH₂-HPSNs to transfect p FGF21 into Hepa1-6 cells,after 48 h,cells were collected to detect the m RNA and protein levels of FGF21.Four groups of cells were set up as control group,liraglutide group,p FGF21 group and liraglutide+p FGF21 group.Each group was divided into with/without transfection by NH₂-HPSNs.Western blot was used to detect PEPCK,p-AKT/AKT and changes in the insulin signaling pathway p-IR/IR.Results: The mass expression ratio was 300 μg/5 μg,and FGF21 was optimally expressed at 48 h.The performance comparison experiments showed that the control group with almost none expression,and the expression of FGF21 after transfection with NH₂-HPSNs was higher than that with lipo2000.Functional verification tests suggest that the expression of biological indicators is more significant when NH₂-HPSNs are added,whether it is the liraglutide group,the FGF21 group or both.At the same time,the efficiency of the two interactions is far greater than their individual effects.Conclusion: The optimal conditions for carrying p FGF21 through NH₂-HPSNs are 300μg: 5μg,48 h.The transfection efficiency of carrying p FGF21 with NH₂-HPSNs was higher than that of the conventional transfection reagent lipo2000.Whether it is p FGF21 or liraglutide or both,the efficiency of loading through NH₂-HPSNs is higher than when used alone,and their combined effect on improving insulin resistance is superior to their individual effects.PART 3 BIOLOGICAL EFFICIENCY OF NH₂-HPSNs/LIRAGLUTIDE/p FGF21 TRANSFECTION IN VIVOObjective: To explore the optimal time for the NH₂-HPSNs carrying p FGF21 transfected into mice.To confirme whether NH₂-HPSNs/p FGF21/liraglutide can fully exert biological effects in vivo.Methods: In this part of the experiment,8 weeks C57BL/6J mice were randomly divided into 6 groups,5 in each group,and fed with high fat diet for 12 weeks to construct insulin resistant obese mice.After fasting for 10-12 hours,NH₂-HPSNs/p FGF21 were continuously injected into the tail vein,and the liver of the mice was taken at 0,1,3,5,7,and 9 days respectively.The FGF21 m RNA level was detected by real-time fluorescence quantitative analysis,and the FGF21 protein was detected by western blot.Then fed eight groups of mice: blank control group,material control group,liraglutide group,NH₂-HPSNs/liraglutide group,p FGF21 group,NH₂-HPSNs/p FGF21 group,p FGF21+liraglutide group,H2-HPSNs/p FGF21/liraglutide group,the liver of the mice was taken 7 days after continuous injection,and the changes of liver glucose metabolism and insulin signaling pathway were detected by Western blot.Results: The m RNA of FGF21 peaked on day 5 and the protein peaked on the seventh day.Compared the groups without NH₂-HPSNs,those with NH₂-HPSNs are more efficiency even when they carried liraglutide,p FGF21 or both.At the same time,the PEPCK was reduced by 40.54%,and the AKT phosphorylation level was increased by 95.41%.The phosphorylation level of InsR is also similar to the change in AKT phosphorylation levels.Conclusion: NH₂-HPSNs can carry liraglutide and p FGF21 in vivo as a vector effectively.The improvement of insulin resistance was better than that those without NH₂-HPSNs.