Preparation And Characterization Of Scutellarin Loaded On Flexible Nano-liposomes And In Vitro Study Of Its Osteogenesis

Objectives:To prepare scutellarin loaded on flexible nano-liposomes(S-FNL)and compare the osteogenic effect in vitro of S-FNL and scutellarin(S).Methods:1.Preparation and scheme optimization of S-FNL: using membrane hydration method,setting different temperature and rotation speed with rotary evaporation instrument,and taking soybean lecithin and cholesterol as raw materials,the blank nano-liposomes(NL)was prepared.The particle size and PDI of liposomes were measured by Malvern laser particle size analyzer,and the morphology of liposomes was observed by TEM in order to optimize the preparation temperature and speed of liposomes.S-FNL was prepared by membrane hydration method with soybean lecithin,cholesterol,stearamide,sodium cholate and scutellarin as raw materials according to the optimized preparation temperature and rotation speed.Through single factor experiment and orthogonal experiment analysis,the entrapment efficiency of scutellarin was compared and the preparation scheme of S-FNL was optimized.The particle size,PDI and zeta potential of liposome were measured by particle size analyzer,and the morphology of liposome was observed by TEM.2.Culture and identification of primary osteoblasts: the osteoblasts of SD rats' skulls were extracted by enzyme digestion combined with tissue block method,cultured and transferred to the third generation,and identified according to morphological observation,growth curve drawing,alkaline phosphatase(ALP)staining and calcium nodule staining.3.Study on osteogenesis in vitro: cell proliferation experiment(CCK-8 experiment)measured and compared the relative OD value of cells under different concentrations,and screened the best drug concentration.According to the different administration,the experiment was divided into three groups: S-FNL group(scutellarin loaded on flexible nano-liposomes group),S group(scutellarin group)and BC group(blank control group).According to the groups,CCK-8 experiment and ALP activity test were carried out with the best drug concentration,OD value was measured,cell survival rate and ALP activity were calculated.Using GraphPad prism 8.0.2 software for statistical analysis,P < 0.05 was statistically significant.Alizarin red staining was used to detect calcium nodules in each group,and the formation of calcium nodules in the three groups was observed and compared under microscope.Result:1.NL was successfully prepared by thin film hydration method.The optimal preparation scheme was: spinning temperature 40?,rotational speed 66 r/min;hydration temperature 48?,rotational speed 88 r/min.The average particle size of NL was(70.5 ± 5.39)nm and PDI was(0.191 ± 0.01).It can be seen that NL is spherical like under TEM.The best ratio of S-FNL is 2:1 of phospholipid and cholesterol,20:1 of phospholipid and scutellarin,8:1 of phospholipid and rigid amide,8:1 of phospholipid and sodium cholate,and 1 mL of hydrated volume(Phosphate-Buffered Saline).The average particle size of S-FNL was(153.2 ± 3.35)nm,PDI was(0.188 ± 0.02),Zeta potential was(-32.1 ± 1.57)mV,and encapsulation efficiency was(47.2 ± 0.30)%.The morphology of S-FNL was spherical like under TEM.2.The primary osteoblasts showed different shapes,such as triangle,fusiform and polygon.Osteoblasts are connected by protuberances,and grow in contact and whirlpool.Osteoblasts grew rapidly within 3-5 days after inoculation,and were in the logarithmic growth stage;the growth was the highest on the 5th day,and then slowly entered the platform stage.After ALP staining,the active site of ALP was dark blue or blue purple.After alizarin red staining,the calcium nodules stained orange red could be seen clearly under the microscope.3.The best concentration of CCK-8 was 15 ?g/mL.Compared with BC group,S-FNL group and S group were better in promoting cell proliferation and ALP expression(P < 0.05),and S-FNL group was better than S group(P < 0.05).The cells of the three groups were stained with alizarin red,and the calcium nodules stained with orange red could be seen under the microscope.Conclusion:In this experiment,S-FNL was successfully prepared by film hydration method,and the preparation scheme was optimized.S-FNL has better osteogenic effect on osteoblasts than S alone.However,the specific osteogenic mechanism of S-FNL and its osteogenic effect in vivo need to be further explored and studied.