Preparation And Evaluation Of Trimethylated Chitosan Surface Modified Nanoparticle As A Novel Drug Carrier For Brain Delivery

A large number of drugs to the central nervous system are impeded by the tight restriction of the blood-brain barrier (BBB). Therefore, the research of enhancement of brain drug delivery, especially development of novel drug delivery system for brain drug targeting, is on focusing in pharmaceutics. Nanocarriers capable of overcoming the BBB have shown great potential in delivering drug to the brain. In this study, trimethylated chitosan(TMC) surface modified poly(D,L-lactide-co-glycolide) nanoparticle (TMC/PLGA-NP) was synthesized as a novel drug carrier for brain delivery and its brain-targeting effects were investigated. The surface modified TMC can improve the nanoparticle’s brain entrance via absorptive-mediated transcytosis and the hydrophilic surface of the nanoparticles can avoid the uptake by mononuclear phagocytic system, thus enhance the opportunities to beyond the BBB.The optimization of preparation and prescription of coenzyme Q10-loaded nanoparticles (PLGA-NP) by Doehlert design was described. PLGA-NP was prepared by nanoprecipitation method. The effects of influencing factors such as the concentration of TPGS, the concentration of PLGA in organic phase, the ratio of PLGA to drug and the ratio of water phase to organic phase, on mean diameter, drug loading and entrapment efficiency were investigated. The optimal conditions, the concentration of TPGS is 0.015%(w/w), the concentration of PLGA in organic phase is 10 mg/ml, the ratio of PLGA to drug is 9:1 and the ratio of water phase to organic phase is 7:1. The multi-objective simultaneous optimization of the formulation of PLGA-NP is obtained. The deviations of the measured values from the predicted ones were less than 10% (n=3) and the stimation of the established model is good.Trimethylated chitosan was synthesized by reductive methylation of chitosan according to a two-step method and characterized by FTIR and 1H-NMR. Then TMC was covalently coupled to the surface of PLGA-NP via carbodiimide-mediated coupling. Physicochemical characteristics of the obtained TMC surface modified PLGA-NP(TMC/PLGA-NP) were investigated. Transmission electron micrograph and dynamic light scattering results showed that TMC/PLGA-NP loaded with Co-Q10 had round and regular shape with mean diameter less than 150 nm. The zeta potential of TMC/PLGA-NP was about 20 mV with the loading capacity around 8%. The security of preparation was evaluated and there was no hemolysis side-effect and blood vessels stimulation observed. The in vitro cytotoxicity of TMC/PLGA-NP was investigated by MTT assay. There was no difference on cell viability between TMC/PLGA-NP and PLGA-NP at the concentration of 0.025-8.0mg/ml and the low cytotoxicity of the TMC/PLGA-NP was thus confirmed. A lipophilic fluorescent dye,6-coumarin was incorporated into the nanoparticles as the fluorescent probe to evaluate the effects of brain delivery. Thirty minutes after PLGA-NP and TMC/PLGA-NP injected into mice caudal vein, fluorescent microscopy of brain sections showed there was a higher accumulation of TMC/PLGA-NP in the cortex, paracoele, hippocampal, the third ventricle and choroid plexus epithelial, while no brain uptake of PLGA-NP was observed. The results demonstrated that TMC/PLGA-NP passed through the BBB and reached the brain parenchyma. And the most likely mechanism is absorptive mediated transcytosis pathway.The pharmacological effects of different formulations in the APP/PS1 transgenic mice were evaluated. The behavioral results showed that the administration of Co-Q1o solution had little influence on memory deficits improvement. However, the injection of Co-Q10 loaded TMC/PLGA-NP greatly improved memory impairments to a normal level, but the efficacy was slight for the loaded PLGA-NP, without conjugation of TMC, indicating that TMC/PLGA-NP was a potential brain delivery carrier. The senile plaque staining results showed that there was a great accumulation of senile plaque in AD mice. After TMC/PLGA-NP treated, the senile plaque became less and smaller, and pale. The cells containingβ-amyloid around the senile plaque were obviously less than other groups. This study for the first time revealed that Co-Q10 could solve senile plaque in vivo. The biochemical parameters were in good agreement with the behavioral investigation and the brain-targeted effects of TMC/PLGA-NP were thus better proved.Our investigations for the first time demonstrated these TMC surface modified nanoparticles to be able to across the BBB, and clearly indicated that it might serve a promising brain drug delivery carrier with low toxicity. Besides, our results also helped to confirm the effects of oxidative stress in the process of AD and the important role of Co-Q10 in the development of therapeutics for AD.