Study On Lactoferrin Modified Osthole Long Circulating Liposomes

Osthole(Ost)is an important active monomer extracted from Chinese traditional medicine Cnidium monnieri.It has many pharmacological effects,such as anti-inflammatory,anti apoptosis and so on.Some researchers have reported that osthole had neuroprotective effect,and the potential for treatment of neurodegenerative diseases such as Alzheimer's disease(AD)and Parkinson's disease(PD).In spite of the wide spectra of pharmacologic properties,the application of osthole in clinic has been hampered due to it's low solubility in aqueous solution.Liposomes can encapsulate poorly water-soluble drugs to increase the solubility and bioavailability of the drug.Lactoferrin receptors(Lf R)are ovexexpressed on neural cells and cerebral microvascular endothelial cells in neurodegenerative diseases.Liposome surface modified lactoferrin(Lf)can produce receptor mediated endocytosis or uptake.In this study,DSPE-PEG2000 was used to modify the surface of liposomes,in order to prolong the circulation time of the liposomes in vivo and improve the target efficiency.The brain targeting drug delivery system contained Ost was constructed,based on the covalent binding of the active carboxyl group at the terminal of DSPE-PEG2000-COOH and the amino group on the Lf,thereafter the physical and chemical properties,such as encapsulation efficiency,particle size,potential,release in vitro,and the protective effect of AD cell model were investigated.The main research methods and results are as follows:1)Preparation of osthole liposomes(Ost-Lips): By screening two methods of ethanol injection and thin film dispersion,the liposomes were prepared by ethanol injection method.After optimizing the prescription by orthogonal test,the encapsulation efficiency of the liposomes was 83±0.56%,the particle size was 101.9±2.7 nm,and the Zeta potential was-15.3±2.3 m V.2)Preparation of lactoferrin modified osthole long circulating liposomes(Lf-Ost-LCL):Osthole long circulating liposomes(Ost-LCL)was prepared by determining the total amount of PEG lipid based on investigating the stability of liposomes,thereafter the Lf-Ost-LCL was prepared by covalent binding of carboxyl group on DSPE-PEG2000-COOH and amino group on Lf.Purifying the liposomes by dialysis method.The concentration of Lf in liposomes was measured by BCA method with186.73±3.61 ?g/m L,and the coupling rate was 68.15±1.32%.The particle size and Zeta potential of Ost-LCL were 124.3±3.7 nm and-27.6±0.8 m V,and particle size and Zeta potential of Lf-Ost-LCL were 154.6±2.4 nm and-24.3±1.5 m V.3)In vitro release experiment: After screening the release medium,the HPLC analysis method of Ost in the release medium 1%Tween 80 normal saline was established.The cumulative release rates of Ost and three kinds of liposomal formulations in vitro were investigated.The results showed that the liposomes had longer retention time than the bulk drug,and the two kinds of long circulating liposomes showed obvious slow release effect compared with Ost-Lips.4)Protective effect of AD model in vitro: We established an AD model in vitro with A?25-35 induced PC12 cell to injury.The results of MTT showed that the protective effect of Lf-Ost-LCL on cells was stronger than that of Ost and Ost-Lips.The addition of free Lf could inhibit the binding of Lf-Ost-LCL to cells.