Mechanism Of Wheat Germ Agglutinin Conjugated PEG-PLA Nanoparticles For Direct Nose-to-brain Delivery

Many kinds of peptides and proteins are active to treat the central nervous system (CNS) diseases due to the swift development of biotechnology and neuroscience. However, because of the blood-brain barrier (BBB), those peptides and proteins are hardly delivered into the brain for treating brain diseases.The direct pathways from nose to brain make it possible to delivery peptides and proteins into brain. Despite the attractiveness of the nasal route, a variety of factors may compromise nasal drug absorption and accessing the brain. They are mainly related to the enzymatic degradation and rapid mucociliary clearance in nasal mucosa. Furthermore, poor capability of these drugs to penetrate the mucosa also attributes to low amount of drugs in CNS. A novel WGA-modified PEG-PLA nanoparticle was constructed by PhD. Gao Xiaoling. WGA specifically binds to N-acetylglucosamine, which was more abundantly observed in the nasal cavity especially in the olfactory mucosa than in the respiratory mucosa. Although previous pharmacokinetics and pharmacodynamics tests demonstrated that WGA-NP effectively delivered peptides from nose to brain. However, the mechanism of nanoparticles for direct nose-to-brain delivery of drugs has not yet been devised. This research carries on thorough and systematical analysis of the Calu-3 celluar uptake and pathway of nanoparticles from nasal epithelium into brain and its cytotoxcity for safe and effective therapies with WGA-NP.In the first part, WGA-conjugated nanoparticles were prepared by incorporating maleimide into one end of the PLA-PEG copolymer and taking advantage of its thiol group-binding reactivity to conjugate with the lectins thiolated with 2-iminothialane. The ratio of WGA:maleimide was 1:25,1:10,1:3,1:1,2:1. The mean size of the resulted nanoparticles was about 100 nm and the zeta potential was-20mV. The coupling of WGA with PEG-PLA nanoparticles (NP) was confirmed by the existence of gold-labeled WGA-NP under TEM. The retention of biorecognitive activity of WGA after the covalent coupling procedure was confirmed by haemagglutination tests. The agglutinating activites are enhanced by increasing WGA density at the particle surface.To qualitively and quantitatively evaluate the celluar uptake of WGA-NP/NP and study its mechanism, a Calu-3 cell model was used to stimulate nasal epithelium cell and coumarin-6 was used as fluorescent probe. After cultivation for 11-14 days, Calu-3 cells showed a distinct monolayer structure, microvilli, tight junction, mucus secretion and membrane expression of N-acetylglucosamine also could be observed. TEER is between 300 and 400Ω·cm2, and Papp values for fluorescein sodium and dexamethasone were 2.46×10-7 cm/sec and 5.99×10-6 cm/sec, respectively. Coumarin-6-loaded WGANP/NP were prepared to trace celluar uptake. The uptake results confirmed (1) the endocytosis extent of WGA-NP was significantly higher than NP and in 2 h, WGA-NP uptaken more rapidly as well. (2) Calu-3 cells had the highest amount of binding and uptake with the raito of WGA:maleimide 1:10. (3) The uptake of WGA-NP by Calu-3 cells was concentration dependent within certain concentration range. The saturation concentration was 4 mg/mL. (4) Endocytosis of WGA-NP was obviously influenced by temperature, showing energy-dependent internalization. (5) Colocalization of WGA-NP with Golgi apparatus and lysosome could be observed and inhibition experiments were performed to elucidate the transport mechanism. The results indicated that interactions between Calu-3 cells and WGA-NP were mainly due to the immobilization of carbohydrate-binding pockets on the surface of NP. Reduce of cellular uptake of WGA-NP following adding Chlorpromazine, Filipin and Cytochalasin D suggested that WGA-NP was absorbed via both clathrin, caveolae-dependent endocytosis pathways and pinocytosis. Both BFA and Monensin reduced the uptake confirming the involvement of both Golgi apparatus and lysosome in intracellular transport of WGA-NP. Nocodazole had no effects on cellular uptake of WGA-NP, implying that the transport is independent from microtubules. The uptake and transport process were energy needed.In the third part, the transport pathways of WGA-NP from nose to brain were investigated. DiR, a near infrared dye serving as a fluorescence probe to evaluate evaluate the capacity of the WGA-NP for drug delivery into the CNS. WGA-NP resided longer in the nasal cavity of nude mice and had more brain delivery than NP. Nasal cavity and olfactory bulb slices of rats following intranasal administration were obtained and distributions of WGA-NP in different sections were observed by fluorescent microscope. The results indicated that the penetration of WGA-NP penetrated into nasal submucosa rapidly; Distribution profiles of WGA-NP in the nasal cavity indicated their higher affinity to the olfactory mucosa than to the respiratory one, which also indicated that WGA-NP in the submucosa might be transported into the CNS through the nerves or the connective tissues around the nerves in the lamina propria via intracellular pathway. Besides,125I-WGA-NP was used to investigate nose-to-brain routes and distribution in the brain. It was revealed that trigeminal pathway predominated in transporting WGA-NP into the cerebellum, brainstem and spinal cord after intranasal administration. Another part of WGA-NP accessed into olfactory bulb via olfactory pathway. Nevertheless, the cerebrospinal fluid rarely involved in facilitating WGA-NP into CNS.To achieve the assessment of WGA-NP cytotoxicity, we using CCK-8 assay, LDH, GSH and ROS detection. The results illustrated that the cytotoxicity of WGA-NP was concentration, time and WGA conjugated density dependent. Dosages used in pharmacokinetics and pharmacodynamics tests previously showed no obvious cytotoxicity. According to uptake results in the second part and cytotoxicity,1:10 was the optimaized raito of WGA:maleimide.