| Objective: This work focuses on preparation the resveratrol nanoparticles modified lactoferrin(Lf-Res-NPs),and investigating the pharmacokinetics in vivo of mice.We aim to observe the therapeutic effect on Alzheimer's disease(AD)model rats,and to evaluate the Brain-Targeted effect.Methods: Resveratrol nanoparticles(Res-NPs)were prepared by interfacial polymerization and designed the optimum formulation and process by central composite design method.Lf was thiolated and conjugated to the distal maleimide functions of the Hydroxyl-PEG-Maleimide,which the side of-OH functions linked the Res-NPs to form the Lf-Res-NPs.Laser particle size analyzer detected nanoparticles size and the morphology of nanoparticles was observed by transmission electron microscopy.(2)270 SPF Kunming male mice were randomly divided into three groups as Res Solution group,Res-NPs group and Lf-Res-NPs group.Routine feeding one week,blood samples were collected after 0.17,0.5,0.75,1,2,6,10,12 and 24 hours after intraperitoneal injection with ten mice at each time point.The concentrations of Res in the heart,liver,spleen,lung,kidney and blood were determined by HPLC.Calculated the pharmacokinetic parameters and the target indexes in each tissue.(3)50 SPF SD rats,half male and half female,were randomly divided into five groups as normal group,AD model group,Res Solution group,Res-NPs group and Lf-Res-NPs group with 10 rats in each group.Normal group was given saline intraperitoneal injection and distilled water intragastric administration.Other groups were established with D-galactose 150mg·kg-1·d-1 intraperitoneal injection and Al Cl3 10mg·kg-1·d-1 intragastric administration continuously for 65 days.Since the 31 st day,normal group and AD model group were treated with saline intraperitoneal injection,Res intervention groups were treated with intraperitoneal injection of different forms of Res.The model has beenachieved 65 days later.The learning and memory ability was tested by morris water maze test.The activity of Glutathione peroxidase(GSH-PX),and Superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in serum and brain were measured by colorimetric method.The expression of beta amyloid protein1-42(A?1-42)and Cysteine-containing aspartate-specific protease 3(Caspase-3)in rats hippocampus were observed by immunohistochemistry staining method.The expression of A?1-42 in rats hippocampus were tested by ELISA.The expression of beta-amyloid precursor protein(APP),Casepase-3 and nuclear factor-?B(NF-?B)were measured by western blot.Results:(1)The best prescription and preparation process of Res-NPs were as follows that the oil-water ratio was 4m L:8m L,p H value of aqueous was 2.0,the amount of dextran-70 was 50 mg,the dosage of Res was 48 mg,the amount of BCA was 52?L.The particle size was(229.02±4.30)nm,drug loading was(10.13±0.96)%,and the encapsulation rate was(80.36±2.70)%.In the release of p H 7.4 PBS medium,The cumulative release rate of Res Solution was(80.26±1.61)% in the 2 hours and(86.16±0.10)% in the 8 hours.The cumulative release rate of Res-NPs was(56.77±2.47)% in 2 hours,(73.11±7.14)% in 8 hours and(80.24±2.78)% in 72 hours.,The cumulative release rate of Res-NPs adding plasma was(56.62±0.30)% in 2 hours,(82.22±3.22)% in 8 hours and(87.96±0.72)% in 72 hours.The Lf-Res-NPs particle size was(240±4.652)nm.Transmission electron microscope was used to approving the lactoferrin to connect with Res-NPs.(2)(1)The pharmacokinetic results showed that in Res-NPs group and Lf-ResNPs group,compared with Res Solution group the area under the curve(AUC(0-?)),biological half life(t1/2z),max time(Tmax),max concentration(Cmax)were increased and clearance(CLz/F)were decreased(P<0.05).In Lf-Res-NPs group,compared with the ResNPs group the AUC(0-?)and Cmax were increased(P<0.05).(2)The drug contents of Res Solution group and Res-NPs group were distributied in the kidney and liver while that of Lf-Res-NPs group was distributied in the liver and spleen.2 hours after intraperitoneal injection,the maximum value of brain-targeted index of Res Solution group was 14.263%,Res-NPs group was 20.980% and Lf-Res-NPs group was 26.879%.The maximum value of brain-targeted index of Lf-Res-NPs group was increased 1.3 times than that of Res-NPs group and it was increased 1.9 times than that of Res Solution group.10,12 and 24 hours after intraperitoneal injection,the brain-targeted index of Lf-Res-NPs group was higher than other groups.(3)(1)In spatial navigation of the water-maze test results showed that in model group,compared with normal group the escape latency on the first day to the fourth day was prolonged(P<0.05).In different forms of Res intervention groups,compared with model group the escape latency on the second day to the fourth day were shortened(P<0.05).In Lf-Res-NPs group,compared with Res Solution group and Res-NPs group the escape latency on the fourth day were shortened(P<0.05).Space exploration results showed that in model group,compared with normal group the quadrant entries times and retention time were decreased(P<0.05).In different forms of Res intervention groups,compared with model group the quadrant entries times and retention time were increased(P<0.05).Comparison between different forms of Res intervention groups,Lf-Res-NPs group the quadrant entries times and retention time were increased(P<0.05).(2)In model group,compared with normal group the activity of GXH-PX and SOD were decreased and the content of MDA was increased in serum and brain tissue(P<0.05).In Res-NPs group and Lf-Res-NPs group,compared with model group the activity of SOD and GSH-PX were increased and the content of MDA was decreased in serum and brain tissue(P<0.05).Comparison between different forms of Res intervention groups,Lf-Res-NPs group the activity of SOD and GSH-PX were increased and the contents of MDA were decreased in serum and brain tissue(P<0.05).(3)Immunohistochemistry results showed that in model group,compared with normal group the expression of Casepase-3 and A?1-42 in rats hippocampus were increased(P<0.05).In different forms of Res intervention groups,compared with model group the expression of Casepase-3 and A?1-42 were decreased(P<0.05).Comparison between different forms of Res intervention groups,Lf-Res-NPs group the expression of Casepase-3 and A?1-42 of Lf-Res-NPs group were decreased(P<0.05).(4)The ELISA results showed that in model group,compared with normal group the expression of A?1-42 in rats hippocampusl was increased(P<0.05).In different forms of Res intervention groups,compared with model group the expression of A?1-42 in rats hippocampus were decreased(P<0.05).Comparison between different forms of Res intervention groups,Lf-Res-NPs group the expression of A?1-42 in rats hippocampus of LfRes-NPs group was decreased(P<0.05).(5)The Western bolt results showed that in model group,compared with normal group the expression of Casepase-3,NF-?B and APP in rats hippocampus were increased(P<0.05).In different forms of Res intervention groups,compared with model group the expression of Casepase-3,NF-?B and APP in rats hippocampus were decreased(P<0.05).Comparison between different forms of Res intervention groups,Lf-Res-NPs group the expression of Casepase-3,NF-?B and APP in rats hippocampus of Lf-Res-NPs was decreased(P<0.05).Conclusion:(1)We have prepared Lf-Res-NPs,which size is(240±4.652)nm.(2)LfRes-NPs can prolong the efficacy of Res in vivo,be sustained release,increase the bioavailability of Res,improve the brain targeting of Res,reduce the intake of other organizations of Res.(3)Lf-Res-NPs can deliver more Res into the brain,increase the concentration of Res in the brain,improve antioxidant capacity,reduce the expression of APP and A?1-42 protein in rats hippocampus,reduce neuronal apoptosis and inflammation,the treatment effect be better than Res Solution and Res-NPs. |
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