Preparation Of Quercetin Solid Lipid Nanoparticles And Study Of Estrogen Like Neuroprotection

Solid lipid nanoparticles is a solid colloidal drug delivery system,which is made of natural or synthetic solid lipids as carriers,inlaid drugs in the lipid core or adhered to the lipid surface.The particle size is between10-1000nm.The carriers used in SLN are non-toxic or low toxic,biocompatible and biodegradable lipids,which is a new generation of submicron drug delivery system developed in the early 1990s.Solid lipid nanoparticles have the advantages of controlling drug release,avoiding drug degradation or leakage,good cell affinity and histocompatibility,and SLN is considered to be the most promising formulation through the blood brain barrier.In this paper,quercetin standard was used as the drug substance,glycerin monostearate as the carrier,lecithin and poloxamer 188 as the emulsifier.Quercetin solid lipid nanoparticles were prepared by melt ultrasonic method on the basis of single factor and orthogonal test to improve the poor water solubility of Quercetin.The physical and chemical properties,in vitro release characteristics,estrogen like neuroprotection of que-sln were investigated Cell uptake rate and blood-brain barrier permeability were used to evaluate Que-SLN.The research methods and results are as follows:1.Preparation of quercetin solid lipid nanoparticlesThe drug content was determined by UV spectrophotometry,and solid lipid nanoparticles were prepared by melt ultrasound.The effects of dosage,the ratio of glycerin monostearate to egg yolk lecithin,the content of poloxamer 188 and ultrasound time on the formulation were investigated.The optimized formulation was obtained by（L9（3⁴））orthogonal experiment.The dosage was 5mg,the ratio of glycerin monostearate to yolk lecithin was 100:100,the content of P188 was 1%and the ultrasonic time was 5 minutes.The encapsulation efficiency of Que-SLN was（90.54±1.36）%,and the drug loading was（4.33±0.06）%.2.Physical and chemical properties and in vitro evaluation of quercetin solid lipid nanoparticlesTransmission electron microscopy was used to observe the appearance of quercetin solid lipid nanoparticles;Malvern particle size analyzer was used to measure the particle size and potential of the nanoparticles;dialysis method was used to investigate the in vitro release characteristics;MTT method was used to detect the size of cell viability in different treatment groups;Transwell chamber was used to investigate the permeability of Que-SLN blood-brain barrier in vitro,and fluorescence probe labeling was used to investigate the difference of cell uptake rate.The prepared Que-SLN was spherical in appearance,round in shape,uniform in size,with particle size of（191.23±3.25）nm,PDI of（0.19±0.04）,potential of（-24.40±1.24）m V and quercetin concentration of 452.70 mg·L^-1.The release rate was（87.60±0.66）%in 72 hours.MTT results showed that Que and Que-SLN can promote the proliferation of PC12,Que-SLN has a pro-proliferative effect on PC12 cells at a concentration below 200μmol·L^-1（P<0.05）,and Que has a pro-proliferative effect on PC12 cells at a concentration below 50μmol·L^-1（P<0.01）;The survival rate of cells in Que-SLN(50,100,200μmol·L^-1)concentration group was（116.79±2.75）%（P<0.001）,（119.46±1.89）%（P<0.001）,（107.55±3.17）%（P<0.05）,compared with Aβ_25-35 group,the survival rate of cells in Que-SLN(50,100,200μmol·L^-1)concentration group was significantly increased（P<0.05）;at 50μmol·L^-1,at the concentration of 50μmol·L^-1,the protective effect of Que on PC12 cells injured by Aβ_25-35 was basically the same at 24 hours and 48 hours（P>0.05）,while the protective effect of Que-SLN at 48 hours（80.14±3.96）%was significantly better than that at24 hours（67.28±3.10）%（P<0.01）,the difference was statistically significant;In the simulated blood-brain barrier experiment in vitro,Que and Que-SLN can both pass through the simulated blood-brain barrier.At the same concentration,Que-SLN has stronger permeability of blood-brain barrier than Que（P<0.01）.The cell uptake test shows that in the qualitative analysis,the cell uptake increases with the increase of concentration,at the same concentration,the cell uptake of coumarin-6-SLN was significantly higher than that of coumarin-6,and the difference was significant（P<0.05）.In the quantitative analysis,when the concentration was 20,40,60,80μmol·L^-1,the cell uptake of coumarin-6and coumarin-6-SLN were（16.55±1.26）%,（35.22±2.34）%（P<0.01）;（25.65±4.12）%,（40.15±2.45）%（P<0.05）;（30.25±5.11）%,（57.27±1.69）%（P<0.05）;（22.35±1.25）%,（34.75±2.47）%（P<0.05）.At the same concentration,compared with coumarin-6,the cell uptake rate of coumarin-6-SLN group was significantly higher,the difference was statistically significant（P<0.05）.Combined with the results of qualitative and quantitative analysis,with the increase of coumarin-6 concentration,the cell uptake increased,but the cell uptake rate increased first and then decreased,indicating that PC12 cells have saturated with coumarin-6.3.Study on the estrogen like neuroprotection of quercetin solid lipid nanoparticlesIn this study,PC12 cells were injured by Aβ_25-35 to establish Alzheimer’s disease model in vitro,and explore the mechanism of Que-SLN’s neuroprotective effect.PC12 cells were treated with Aβ_25-35 for24 hours,and then added with 17β-E2(0.1μmol·L^-1),Gen(50μmol·L^-1)and Que-SLN(50,100,200μmol·L^-1)for 24 hours.MTT method was used to detect the effect of each group on the activity of PC12cells injured by Aβ_25-35.Immunofluorescence staining and Western blot was used to detect the expression of ERαand ERβprotein of two subtypes of estrogen receptor in PC12 cells damaged by Aβ_25-35.The results showed that 20μmol·L^-1 concentration of Aβ_25-35 could significantly reduce the survival rate of PC12 cells after 24 hours（P<0.01）,and 17β-E2(0.1μmol·L^-1),Gen(50μmol·L^-1)and Que-SLN(50,100,200μmol·L^-1),could significantly increase the survival rate of PC12 cells compared with the model group（P<0.05）.The results of immunofluorescence staining and Western blot showed that compared with the model group,quercetin solid lipid nanoparticles significantly increased the expression of ERαprotein（P<0.01）,but there was no significant difference in the expression of ERβprotein（P>0.05）.