Study On Lactoferrin Modified Diosmetin Brain Targeting Long Circulating Liposomes

Objective:To prepare a novel brain targeting long-circulating liposomes delivery system of diosmetin modified with lactoferrin,in order to improve the solubility and bioavailability of geranium lignin and achieve brain targeting,and provide reference for the development of Alzheimer’s disease（AD）drug.Methods:The Diosmetin Liposomes（Dios-Lip）were prepared by the thin film dispersion method,the Box-Behnken response surface method was used to optimize the formulation process,DSPE-PEG2000 and DSPE-PEG2000-COOH were modified on the surface of Dios-Lip by water bath constant temperature oscillation method to prepare Diosmetin Long circulating liposomes（Dios-LCL）.Finally,lactoferrin（Lf）was attached to prepare Lactoferrin Modified Diosmetin Long Circulating Liposomes（Lf-Dios-LCL）.The quality of liposomes was evaluated in vitro,and the pharmacokinetic characteristics of liposomes in rats and tissue distribution in mice were preliminarily investigated.Results:（1）The best prescription process for Dios-Lip:Dios 4 mg,phospholipids 21.04mg,cholesterol 6.31 mg were added to a 100 m L eggplant-shaped flask,dissolved in 20 m L methanol ultrasonically,placed on a rotary evaporator,and rotary steamed at 45℃for 30 min to remove the organic solvent and a uniformly dispersed film is formed at the bottom of the flask.17.68 m L UP water was added into the bottle,ultrasonically hydrated,the hydration solution was broken by ultrasonic for 2 min under the condition of 360 W ultrasonic cell breaker,and then a translucent Dios-Lip solution was obtained.（2）The best formulation process of Lf-Dios-LCL:DSPE-PEG₂₀₀₀and DSPE-PEG₂₀₀₀-COOH(DSPE-PEG₂₀₀₀-COOH:DSPE-PEG₂₀₀₀:phospholipid=1:6:100,mol/mol/mol)were added to Dios-Lip solution,It was shaken in a constant temperature water bath at 50℃and 100 r/min for 1 h to obtain Dios-LCL.The catalyst EDC and NHS(EDC:NHS:DSPE-PEG₂₀₀₀-COOH=10:10:1,mol/mol/mol)was added to Dios-LCL,shaken at room temperature for 15 min,and then Lf was added(Lf:DSPE-PEG₂₀₀₀-COOH=1:40,mol/mol),incubated in a 37℃water bath for 3 h,and dialyzed for 24 h to obtain Lf-Dios-LCL.（3）The best formulation process of Lf-Dios-LCL freeze-dried powder:8%of mannitol was used as a freeze-dried protective agent,stirred to dissolve,and dispensed into vials.Pre-freeze at-70℃for 12 h.At-50℃and 30mm Hg vacuum,the temperature was raised to-25℃at a rate of 5℃/h,and maintained for 24 h.Subsequently,the temperature continued to increase to 25℃at 5℃/h and maintained for 24 h to obtain Lf-Dios-LCL freeze-dried powder,which was sealed and stored at 4℃and protected from light.The obtained freeze-dried powder had uniform color,smooth surface and good redispersibility.（4）In vitro quality evaluation:Dios-Lip,Dios-LCL and Lf-Dios-LCL were spherical or quasi-spherical,with uniform particle size,complete and round surface,and clear membrane structure visible on the outer layer;the particle sizes respectively were（146±3.06）nm,（164±2.25）nm,（173±1.53）nm,showing a single peak distribution;Zeta potentials respectively are（-38.63±0.13）m V,（-33.64±0.10）m V,（-35.40±0.18）m V.The encapsulation rates of the three batches of Lf-Dios-LCL were（90.46±1.17）%,（88.11±0.72）%,（89.34±1.30）%,and the drug loading rates were（9.27±0.12）%,（9.03±0.07）%,（9.15±0.13）%.The connection rates of the three batches of Lf-Dios-LCL lactoferrin were 43.46%,41.59%,40.33%,and the average connection rate was（41.79±1.58）%.In the in vitro release experiment,Lf-Dios-LCL released about 30%in 12 h,about 55%in 24 h,and about 80%in 48 h.The release is almost complete.The in vitro release curve was best fitted by Weibull equation.（5）Pharmacokinetic study of rats:The main pharmacokinetic parameters of Dios and Lf-Dios-LCL at a dose of 1 mg/kg:T_1/2respectively were（3.767±1.121）h、（5.823±0.82）h;AUC_0-trespectively were（8.13±0.151）μg/m L*h、（13.899±0.305）μg/m L*h;AUC_0-∞respectively were（8.224±0.105）μg/m L*h、（14.743±0.536）μg/m L*h、C_maxrespectively were（1.508±0.061）μg/m L、（1.5±0.102）μg/m L.The T_1/2,AUC_0-t,AUC_0-∞and C_maxof Lf-Dios-LCL respectively were 1.55,1.71,1.79 and 0.99 times that of the Dios group.In the Lf-Dios-LCL group,the Dios in vivo action time was significantly prolonged,and the bioavailability was improved.（6）The results of the study on mouse tissue distribution showed that Dios and Lf-Dios-LCL were distributed in all tissues of the mice by intravenous injection of 1mg/kg,and the Cmax and r_evalue of the Lf-Dios-LCL group were 1.65 times and 2.02 times of that of the Dios group.Lf-Dios-LCL showed obvious brain targeting.Conclusion:A lactoferrin-modified diosmetin long-circulating liposome was successfully prepared,which had good drug release in vitro,prolonged in vivo action time,significantly improved bioavailability.It had brain targeting functions.The results of the subject research had laid a good theoretical foundation for the development of Dios preparations and the treatment of AD with active ingredients of Chinese medicine.