Lectin-conjugated Nanoparticles For Drugs Delivery: Pathway From Olfactory Epithelium To Brain

With development of the society and increase of the population of the aged person,many brain diseases have become a serious problem to people's health.Many peptides and proteins are active to treat the central nervous system(CNS) diseases. However,a significant challenge to their clinical administration is their ideal delivery to the CNS crossing the BBB.In fact,due to poor stability and the presence of the blood-brain barrier(BBB),those peptides and proteins are hardly delivery into the brain.BBB is a barrier system which is formed by tight junctions within the capillary endothelium of the vertebrate brain.It is a guard of brain,but a barrier for drug delivery into brain.There is a direct pathway between the olfactory epithelium and the brain.Intranasal drug delivery system offers a non-invasive alternative for the delivery of therapeutics,effectively bypassing the BBB.The encapsulation of peptides and proteins into nanoparticles(NP) might be a promising approach to increase the amount of drugs accessing the brain,since the formulations have been shown to protect the drugs from the degrading milieu in the nasal cavity and facilitate their transport across the mucosal barriers.However,the unmodified NP showed limited effect in increasing the amount of drugs into brain because of the following reasons:the size of NP is too big to cross through the tight junctions between cells;the resident time of NP in nasal cavity is short because of mucociliary clearance,which is not available for the complete absorption of the formulation;NP distributed in the nasal cavity without selectivity,resulting in poor brain targeting efficiency of the formulation.To address these problems,Ph.D Gao Xiaoling has constructed a novel lectin-modified nanoparticles.Wheat germ agglutinin(WGA),specifically binding to N-acetyl-D-glucosamine,which was abundantly observed in the nasal cavity especially in the olfactory mucosa,was selected as promising targeting ligands.The incorporation of peptides in the NP might improve their stability in vivo while the conjugation of lectin at the NP surface might induce strong mucoadhesion for a longer duration,or a close contact of the NP with the mucosal cells especially in the olfactory mucosa so as to produce a stronger penetration.Such WGA-NP might serve as potential carriers for brain delivery of peptides and proteins.To investigate the pathway of WGA-NP from olfactory epithelium into brain,the amount of uptake and transport of WGA-NP by Caco-2 cells has been inspected;also the photos of nasal tissue slices following intranasal administration.In the first part,WGA-NP 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.Coumarin-6 (Cou-6) and quantum dots(QDs) were used as fluorescent prod.The preparation protocol was optimized based on the following endpoints:lectin density at the particle surface,particle size and conjugation efficiency,through which the optimal ratio of MAL-PEG-PLA to MPEG-PLA around 1:9,WGA:2-iminothiolane 1:60,thiolated WGA:maleimide 1:3 and the conjugation time of 9 h were obtained.The mean size of the resulted NP was about 100 nm and the Zeta potential was -20mV.The coupling of WGA with NP was confirmed by the existence of gold-labeled WGA-NP under TEM. The retention ofbiorecognitive activity of WGA after the covalent coupling procedure was confirmed by haemagglutination tests.The encapsulation of QDs was confirmed by freeze-fracture electron microscopy.In the second part,the bioavailabilities of methadone via three different administrations in rats were investigated,and the results showed that it is more effective and safe to take drugs via nasal administration.WGA-NP was used to deliver vasoactive intestinal peptide(VIP) into the CNS via intranasal administration. VIP was efficiently incorporated into NP followed by surface modification with WGA. AF64A was used to induce Alzheimer's disease(AD) rats.Neuroprotection effects of intranasal administration of VIP solution,NP-VIP and WGA-NP-VIP by means of Morris water maze experiment were investigated.Subcutaneous administration and intranasal administration of VIP solution showed no effect on improvements in spatial memory;intranasal administration of NP-VIP showed an obvious effect on neuroprotection and intranasal administration of WGA-NP-VIP were more effective even with half dose,indicating that WGA-NP might serve as a promising carrier especially for biotech drugs such as peptides and proteins.In the third part,a Caco-2 cell model was used to stimulate olfactory epithelium cell in investigating the mechanism of cellular uptake of WGA-NP in Caco-2 cells. After caltivation for 21 days,Caco-2 cells showed a tight monolayer structure, microvilli and tight junction also could be observed.TEER is more than 400Ω·cm², and penetration amount of ¹⁴C-sucrose is less than 0.5%/h,which proved the tight structure of cell monolayer.Colocalization of WGA-NP with Golgi apparatus and lysosome could be observed,indicating that Golgi apparatus and lysosome were of use in cellular uptake of WGA-NP.Inhibition experiments were performed to elucidate the transport mechanism.High Content Cell Analysis System and HPLC were used to quantify cellular uptake and transport of WGA-NP.The results suggested that interactions between Caco-2 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 and Filipin suggested that WGA-NP was absorbed via both clathrin and caveolae-dependent endocytosis pathways.Both BFA and Monensin reduced the uptake confirming the involvement of both Golgi apparatus and lysosome in intracellular transport of WGA-NP. Nocodazole and Cytochalasin D also significantly reduced cellular uptake of WGA-NP,implying that the transport is both microtubules and actin filaments dependent.The uptake and transport process were energy needed.In the fourth part,nasal cavity slices of rats following intranasal administration were obtained and distribution of WGA-NP in different sections were observed by fluorescent microscope.It was observed that the penetration of WGA-NP into submucosa was faster than NP;More fluorescent signals representing WGA-NP were observed in the sieve plate,which was anatomically near the olfactory bulb, suggesting that the absorption of WGA-NP into the olfactory bulb was faster than that of NP.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.Such investigations on mechanism might provide useful information for the design of novel drug delivery systems.