Aprotinin-Conjugated Nanoparticles Drug Delivery System Unite With Promote Penetration-Action Of Borneol For Brain Targeting

The blood-brain barrier (BBB), formed by brain vessel endothelial cells linked together by tight junctions, restricts the transfer of most drug substances from the bloodstream into the brain, impeding the brain drug delivery in patients who suffer from central nervous system (CNS) disorders. Although some drugs exhibit high therapeutic efficacy in vitro, their safe and effective delivery into the CNS remains a challenge.In the past few years, nanoparticle delivery has become an effective delivery method to pass through the BBB and target drugs to the CNS. However, because of the relatively low efficiency of the passive targeting delivery, active targeting delivery has become an alternative ways for the high efficiency by receptor mediated transport through the BBB. Some commonly used ligand such as insulin and transferrin has a shortcoming and side effect. insulin often cause hypoglycemia while brain targeting ability mediated by transferrin often descend remarkably by high concentration of endogenous transferrin saturation action in plasma. Thus, there is an increasing need of novel drug carriers to overcome the problems and enhancement transfer efficiency to brain.Low-density lipoprotein receptor-related protein (LRP) is a multi-ligand cell surface receptor overexpressed in brain. the ability of mediate drug into the brain has been validated. LRP ligands mainly contains lipoproteins E(ApoE), β-amyloid precursor protein(APP) and Aprotinin. Those ligands possess the Kunitz protease inhibitor (KPI) domain which can be recognized by LRP.Aprotinin(Apr), with a molecular weight of6512Da. is a ligand of LRP and also possesses a KPI domain and has high affinity with LRP. Apr in transcytosis across bovine brain capillary endothelial cell (BBCEC) mono layers is at least10-fold higher than that of transferrin. Study clearly showed that Apr crosses the BBB much more efficiently than other proteins, and high concentration of Apr does not affect the integrality of BBB. The brain accumulation and the high transcytosis rate of Apr suggested that Apr might be advantageously used in new delivery system.Borneol, molecular weight154.24, is a monoterpenoid component in medicinal plants. It is widely used in traditional Chinese medicine and has freshening, heat-removing, pain-relieving and eye-rightening effects. There are two types of borneol-natural and synthetic. Recent studies have shown that borneol can accelerate the open of BBB and enhance the distribution of drugs in brain tissue, therefore, borneol is a promising promoter for oral brain-targeting drug delivery. But till now, all the researches in this area are limited to the improvement effect on specific drug by oral administered. The influence of borneol on the permeating through BBB of drug-loaded nanoparticulate system is necessary to be explored, because this system, which can protect drugs against enzyme inactivation, allow access across the BBB by masking their physicochemical characteristics, ensure a sustained release of the drug and increase drug tissue selectivity, if properly functionalized, is more suitable for drug delivery to CNS than pure drug. And if borneol could increase the brain distribution of nanoparticles as well as pure drugs, it will provide a simple, non-toxicity and non-immunogenicity way for brain targeting. To expound the enhancement mechanism systemically makes sense for the further application of borneol in the CNS delivery.Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by increased deposition of beta-amyloid (AP) peptides and progressive cholinergic dysfunction in regions of the brain involved in learning and memory processing. It is a common cause of dementia in the elder, affecting approximately10%of people aged older than65years worldwide. AD takes a long course and may last for decades, severely reducing the quality of life. Thus, a long-term therapy is required to eliminate redundant Aβ and repair degenerated nerve cells. In this regard, there is an urgent need to develop a safe and effective drug delivery system for the treatment of AD used Apr-conjugated PEG-PLGA nanoparticles with borneol ig.Hup A, a novel alkaloid isolated from the Chinese herb Huperzia serrata, is a potent, highly selective and reversible inhibitor of acetylcholinesterase (AChE). It has been widely used to improve the memory deficits in elderly people and AD patients. Many clinical trials have proved that Hup A could significantly improve cognitive ability. Therefore, Hup A was chosen as a model drug in this study.Based on the above, we, for the first time, developed the Apr-conjugated PEG-PLGA nanoparticles with borneol ig.to enhance the delivery of Hup A into brain. To achieve this goal, Apr-conjugated PEG-PLGA was synthesized to prepare Apr-modified nanoparticles by emulsion solvent evaporation method. DiR, a fluorescence tracer, was encapsulated into nanoparticles to investigate the distribution of Apr-NP in vivo, to evaluate the effect of borneol on the distribution of PEG-PLGA nanoparticles (NPs) to the brain in mice and investigate the mechanism of borneol as a promoter for brain targeting drug delivery. The memory improvement effect of the Apr-NP loaded with Hup A was also tested on AD model rats following caudal vein injection.The present work includes four parts:1. Preparation and characterization of NP and Apr-NPhi this part, the nanopartkles prepared with blends of mPEG-PLGA and maleimide■PEG-PLGA had an average diameter bebw lOOnm after Apr conjugation. TEM photograph showed the NP and Apr桸P were both generally spherical and of regular size, and2mg Hup A added with20mg polymer to assure a higher L.C.The results of the in vitro release study conducted in pH7.4PBS at37癈showed that50.01%and56.56%of Hup A was released from NP and Apr-NP after a96h incubation period, the delivery system has controlled release characteristics. Apr conjugated with polymer did not influence die release profile.2. Evaluation the brain targeting effect of Apr-NP/borneol delivery systemhi this part, brain targeting effect of Apr-NP/borneol delivery system were evaluated in vitro and in vivo.In vitro uptake test of coumarin-6loaded NP and Apr-NP by BCEC cells indicate The fluorescent intensity of Apr-NP was higher than that of NP in15min observed through fluorescence microscope in BCEC, the same effect of borneoL There was a significantly accumulated amount of dye of Apr桸P in the cells compared with that of NP for15,30,45and60min at37癈respectively, The time-dependent uptake of the nanoparticles suggested a process of endocytosis.The distribution images of DiR-loaded NP and Apr-NP in nude mouse at different time points and the quantitative results in brain region showed that the fluorescence intensity in the brain region of Apr-NP group and NP increased within1h, DiR intensity of Apr-NP group in brain region is statistically stronger than that ofNP group. The ex vivo results demonstrated that the DiR intensity of Apr-NP group in the brain was stronger than that of NP group in30min after administration, which is consistent with the results of the in vivo. And the same effect of borneol Promote penetration-action to delivery more DiR-loaded NP and Apr-NP to the brain, which indicate borneol is a promising promoter for brain-targeting delivering drug-loaded nanoparticles.Pharmacokinetic experiments show that nanoparticles containing Hup A in rats than Hup A solution area under the curve(AUC)was significantly longer, with a significant sbw-release effect, but aprotinin modified of NP-Hup A did not affect the pharmacokinetic behavior; borneol also did not affect the pharmacokinetic behavior of NP/Hup A. Tissue distribution of mice study found Apr-NP-Hup A than NP-Hup A brain targeting index(DTI) was1.97; borneol promote NP-Hup A into the brain was significantly increased compared with group not fed with borneol,the DTI was1.86; both borneol ig. and aprotinin modification of nanoparticles for brain targeting index was significantly increased for2.71. The results show that aprotinin-modified nanoparticles have properties of brain drug delivery, and also borneol can increase NP-Hup A into the brain, both aprotinin and borneol play the role of additive effects, with much more amount of nanoparticles into the brain.3. Pharmacodynamics study of Huperzin A loaded Apr-NP/borneol delivery system on AD ratsIn pharmacodynamics study, results of Morris water maze showed that escape latency, the pass times through the platform and percent of time spent in quadrant SW were improved in the group of APR-NP-Hup A, the APR-NP-Hup A nanoparticles and with borneol ig.group had stronger pharmacological efficacy than that of both NP and Hup A solutions, indicate that Huperzin A loaded Apr-NP/borneol delivery system can improve AD rats’memory ability significantly.4. Promote penetration-action mechanism of borneolThe mechanism of borneol to enhance the distribution of NPs to the brain was investigated. The experiment of DPH fluorescence polarization illustrated that borneol could induce a raise of BCEC cell membrane fluidity with a manner of concentration dependency. BCEC are recognized to represent the structural basis of BBB and have tight circumferential junctions which effectively abolish any aqueous paracellular diffusion pathways between the cells. ZO-1protein is located on a cytoplasmic membrane surface of intercellular tight junctions as a marker for tight junctions. The present study showed that borneol-could lead to a significant decrease of the ZO-1expression in BCEC especially at the relatively high concentration and the inhibition effect of ZO-1protein had an obvious concentration dependency, which meant that borneol could cause the loss of tight junction function, thus decrease the obstacle to permeate through BBB. The determination of ATP level with borneol was also carried out. It was verified by the significant decrease of P-gp expression and the significant ATP depletion in BCEC caused by borneol.In summary, A novel brain delivery system, APR conjugated poly(ethylene glycol)-poly(lactic-co-glycolic acid)(PEG-PLGA) nanoparticle, was established in this study.APR-NP were proven to be able to target BBB, mediate endocytosis and transcytosis through the BBB and into the brain, In addition, borneol can promote the the distribution of NP and APR-NP into the brain. Our results suggest that APR-NP/borneol delivery system is an effective drug delivery system for targeted therapy of CNS disorders.