Preparation, Exosyndrome And Application Of Nano Drug Carrier Targeted To Brain Based On Macromolecular Polymer

It is important for the basic research and clinical treatment of brain diseases thatdrugs transfect neurons and enter brain through the blood-brain-barrier (BBB). Thegoal of the project is to prepare nano carriers loading drugs which are able to transfectneurons and enter brain through the BBB.Lf-PEG-PLGA/DOPE nano carriers were made from Poly (lactic acid-glycolicacid) copolymer (PLGA), polyethylene glycol (PEG), lactoferrin (Lf) anddioleoylphosphatidyl ethanolamine (DOPE) by water/oil/water (W/O/W) doubleemulsion method. The Lf-PEG-PLGA/DOPE nano carriers were identificated andexosyndromed by means of FTIR,¹H-NMR, XRD, TGA, UV-vis, DLLS and TEM.We studied the nano carriers' ability of loading drugs, transfecting hippocampalneurons and carring them into brain through the BBB using the model gene drugsHeme oxygenase-1(HO-1) plasmid. The results showed that Lf was successfullylinked to PLGA through covalent bond, the prepared nano carriers had amorphousstructure and good thermal stability with an average particle size of142.2nm,uniform particle size distribution and Zeta potential of+16.4mV. The nano carrierscould bind plasmid with negatively charge through electrostatic adsorption andtransfect the hippocampal neurons, but they had temperate ability to enter brainthrough the BBB with the probalble reason that the binding between nano carriers andDOPE with positive charge was too slender because of the only imbedded form ofDOPE in the nano carriers.To stabilize the positively charged groups in the nano carriers, QMC wereprepared by grafting Methacryloxyethyltrimethyl ammonium chloride (DMC) andmyristic acid (MA) to chitosan (CS) through covalent bond. Lf was grafted to QMCthrough PEG-aldehyde and then Lf-PEG-QMC nano carriers were prepared by meansof self-assembly. The nano carriers were identificated, exosyndromed and functionevaluated. Their abilities of transfecting neurons and entering brain through BBBwere studied. The results showed that DMC with positive charge was grafted to CS,MA and Lf were also grafted to CS. Lf-PEG-QMC nano carriers had the crystalstructure, good thermal stability with an average diameter of244.7nm, more evenlydistributed, and the Zeta potential of+27.9mV. The nano carriers could load gene andpackage insoluble paclitaxel with high encapsulation efficiency of more than80%andgood slow-releasing effect. They could efficiently transfect the hippocampal neurons and enter brain through the BBB without significant toxicity for the isolatedhippocampal neurons and heart, liver, spleen, lung and kidney of mice except forQMC. But brief convulsions were found in the majority of mice and approximately10%of them died after the tail vein injection with Lf-PEG-QMC nano carriers.To improve the security of the intravenous injection of nano carriers,8poly-arginine (R8) with positive charge and MA were grafted to CS to prepare RMC.Lf was grafted to RMC through PEG-aldehyde and then Lf-PEG-RMC nano carrierswere prepared by means of self-assembly. The nano carriers were identificated,exosyndromed and function evaluated. Their abilities of transfecting neurons andentering brain through the BBB were studied. The results showed that R8and MAwas successfully grafted to CS, Lf was grafted to the RMC and Lf-PEG-RMC nanocarriers were prepared with the crystal structure, good thermal stability, averageparticle size of226.7nm, uniform distribution and Zeta potential of+18.7mV. Thenano carriers could load gene and package paclitaxel with high encapsulationefficiency of more than80%and good slow-releasing effect. They could efficientlytransfect the hippocampal neurons and enter brain through the BBB withoutsignificant toxicity for the isolated hippocampal neurons, heart, liver, spleen, lung andkidney and abnormity after the tail vein injection with the nano carriers in mice.To enhance the potential applications of nano carriers, oleic acid modified Fe₃O₄magnetic materials were used to modify QMC, RMC together with their derivativesand prepared corresponding series of magnetic nano carriers (MNC). They wereexosyndromed and performed by VSM analysis for their magnetic properties. Theresults showed that Fe₃O₄were successfully packed into QMC, RMC together withtheir derivatives. MNC had good thermal stability and magnetic properties withcrystal form consistent with that of the corresponding nano carriers without Fe₃O₄,and can easily enter the neurons.Lf-PEG-QMC, Lf-PEG-RMC, magnetic Lf-PEG-QMC and magneticLf-PEG-RMC nano carriers loading HO-1gene were initially applied to the braindiseases based on the ischemic stroke model of rats. The results showed that the4kinds of nano carriers loading HO-1especially the latter two kinds could significantlyinhibit the injury of hippocampal neurons induced by ischemic stroke.