Studies On Transferrin Mediated Huperzine A Loaded Nanostructured Lipid Carriers For Brain Targeting Delivery Systems

The aim of the present study was to exploit the possibility of combination of transferrin mediated Huperzine A loaded nanostructured lipid carriers (Tf-NLC-Hup A) for brain-targeting delivery system. An effective treatment of Alzheimer's disease (AD) was limited due to the presence blood-brain barrier (BBB), so drug research and development have become the focus of attention. Transferrin was selected as a brain-targeting ligand, active targeting function of transferrin by transferrin receptor-mediated endocytosis to promote drug delivery to brain following intravenous administration with Hup A as model drug.Firstly, a quantitative RP-HPLC method was developed to determine the concentration of Hup A in vitro. The linear calibration curve was obtained in a concentration range of 5?100?g·mL-1. The precision, recovery, limit of detection and limit of quantitation accorded with the requirements of methodology. HupA is an alkaloid with slightly soluble in water. The drug entrapment efficiency (EE) was determined by measuring the concentration of free drug in the dispersion medium with ultrafiltration technique.Using a method of melt ultrasonication-high pressure homogenization, NLC-Hup A were prepared and the best formulation were optimized by Box-Behnken design. As a result, the designed nanoparticles showed nearly spherical particles with a mean particle size of 121.67±3.21 nm and-22.93±0.91 mV. The EE (%) and DL (%) could reach up to 89.18±0.28% and 1.46±0.05%, respectively. Differential scanning calorimetry (DSC) of Hup A loaded NLC indicated no tendency of recrystallisation. In vitro release studies showed a burst release at the initial stage and followed by a prolonged release of Hup A from NLC.Bicinchonic acid (BCA) protein assay was established to determine the content of Tf. The main goal for this study is to synthesized Tf-NLC-Hup A conjugates by binding Tf to the NLC-Hup A surface, using functional DSPE-PEG2000-COOH as linker lipid. The coupling efficiency of Tf was determined with a BCA protein assay. The Tf-NLC-Hup A conjugates may both maintain the advantages of PEGylation and achieve the function of active targeting brain cells. Compared to conventional NLC-Hup A, the average diameter, zeta potential of Tf-NLC-Hup A were found to be slightly increased. In vitro release studies showed a burst release at the initial stage and followed by a prolonged release of Tf-NLC-Hup A. The HPLC/Q-TOF MS methods were developed for the determination of Hup A in rat plasma, which was simple and precise. The results of Hup A solution, NLC-Hup A and Tf-NLC-Hup A after tail iv administration were compared. The pharmacokinetic parameters were studied according to non-compartment model. The results showed that the t1/2 of Tf-NLC-Hup A was 5.31-fold to Hup A solution and 2.00-fold to NLC-Hup A. MRT of Tf-NLC-Hup A were 4.92-fold to Hup A solution,1.99-fold to NLC-Hup A. These results suggested Tf-NLC-Hup A prolonged circulation time and increased bioavailability of Hup A.The tissues distribution was investigated by HPLC/Q-TOF MS, the results showed that Tf-NLC-Hup A could successfully target the brain and correspondingly improve the efficacy of Hup A to treat AD. The re values with Tf-NLC-Hup A were higher in brain and lung than NLC-Hup A. The re values with Tf-NLC-Hup A were lower in heart, liver, spleen and kidney. The results of distribution in rats suggested Hup A solution distributed rapidly and extensively, could somehow penetrate the blood brain barrier. Compared to Hup A solution and NLC-Hup A, Tf-NLC-Hup A could enhance the time in blood and increase the brain targeting efficiency of Hup A and decrease the kidney distribution in some extend.With AB25-35 induces the PC12 cell to injury establishes the AD cell model is the ideal model of the AD research in vitro. A?25-35 at concentration of 20.00?mol·L-1 inducing PC12 cells for 24 h could develop an ideal AD cell model by MTT assay. The various concentration of Tf-NLC-Hup A could significantly increase the cell viability than NLC-Hup A and free Hup A. Tf-NLC-Hup A in a concentration of 10.00?mol·L-1 had the strangest effect for protecting the AD cells. The cell apoptotic rate was measured by flow cytometry (FCM). The number of apoptosis cell in Tf-NLC-Hup A group reduced greatly compared with that of NLC-Hup A group and free Hup A group. Tf-NLC-Hup A have significantly protective effect on the PC12 cells injury induced by A?25-35.