| Objective:The incidence of brain diseases increases year by year with the aging of society.BBB hinders drug into the brain and limits the therapeutic effect.In this project,a novel brain-targeting drug delivery system will be established based on synergistic effect of receptor mediation and permeation enhancement.Searching"target" capacity of Aprotinin and LRP receptor-mediated endocytosis will be used to make nanoparticles(NPs)cross BBB into brain.And the effect of permeation enhancement of borneol will further increase the efficiency of drug through BBB.Methods:An ELSD-HPLC method and a LC-MS/MS method were established to simultaneously determine ginkgolide A,B,C and bilobalide content in Ginkgo terpenes in vitro and in vivo,respectively.To increase the entrapment efficiency and enhance the stability of nanoparticles(NPs),PEG-PLGA-NPs loaded with Ginkgo terpenes-phytosome were prepared by double emulsion solvent diffusion(DES-D).Aprotinin modified nanoparticles(Apr-NPs)was prepared through the reaction of maleimide groups and sulfhydryl groups.Brain targeting effect of Aprotinin or bornel was studied by in vitro uptake in BCECs.nude mice in vivo imaging and tissue distribution in mice.Results:The methodological study of the determination method verify its feasibility,as the specificity,linearity,limit of detection,limit of quantification,recovery,precision,repeatability and stability all meet the requirement.The complex rate of Ginkgo terpenes-phytosome reached 99.01%.Differential scanning calorimetry(DSC),Infrared(IR)and X-ray diffraction analysis proved the complex formation.The NPs had an average diameter of 75.9nm.Zeta potential was-9.86 mV.TEM photograph showed the NPs were spherical and of regular size,and the encapsulation efficiency was 65.92%.The in vitro release study during a 24 h incubation period showed that the delivery system had controlled release characteristics.After Aprotinin conjugation,the diameter of NPs was 82.6 nm.the encapsulation efficiency slightly was 61.32%,the zeta potential was-11.27 mv.There was no significant difference in morphology and release profile.In vitro uptake in BCECs and nude mice in vivo imaging showed that Aprotinin modification could significantly improve the transportation of NPs into brain(P<0.05).And it was demonstrated that Aprotinin could promote the NPs into the brain through LRP receptor-mediated endocytosis with the manner of energy dependency.The pathway included clathrin,caveolin.In vitro uptake inBCECs showed that adding borneol had little effect on the NPs uptake(p>0.05):nude mice in vivo imaging indicated that adding boraeol could promote the NPs into the brain significantly(p<0.05),and concentration-dependent,considering the stomach irritation of large doses,choosing dose of bomeol was 30 mg/mL.The optimum time delay of the borneol administration was 15 min,and the promote action was more obvious to the NPs with 80 nm than 150 nm.Borneol administration through gavage was recommended.The mechanism investigation showed that borneol could induce a raise of BCECs membrane fluidity.The results indicated that borneol decreased the obstacle to permeate through BBB by the loss of tight junction function,while it had no obvious effect on the endocytosis or NPs(p>0.05).In vitro uptake in BCECs,nude mice in vivo imaging and tissue distribution in mice showed that Aprotinin modification could significantly improve the transportation of NPs into brain(p<0.05);adding borneol also had the effect on the NPs into brain(p<0.05);and combination of the two could further promote the brain targeting efficiency of NPs.Conclusion:Apr-NPs/bomeol delivery system was successfully built and characterized.The action and mechanism of borneol and Aprotinin were investigated.The novel drug delivery system established in this study based on the synergistic effect of borneol resuscitation and Aprotinin modification was demonstrated to be effective to improve the brain targeting of Ginkgo terpenes. |
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