Lectin-conjugated Nanoparticles For Drugs Delivery Into Brain Following Intranasal Administration

Drug delivery into the brain is made difficult by the presence of the blood-brain barrier(BBB), which is formed by tight junctions within the capillary endothelium of thevertebrate brain. The development of neuroscience has facilitated the discoveries ofpeptides and proteins with considerable potential in the treatment of central nervoussystem (CNS) diseases such as Alzheimer's disease and Parkinson's disease. However, asignificant challenge to their clinical administration is their ideal delivery to the CNScrossing the BBB.Intranasal drug delivery system offers a non-invasive alternative for the delivery oftherapeutics, effectively bypassing the blood-brain barrier. Indeed, the past few yearshave witnessed a sharp increase in the amount of research on the nasal pathway for theCNS drug delivery. However, the total amount of drugs accessing the brain has beenreported to be low, especially in the form of peptides and proteins, which were highlysusceptible to the unfavorable environment of the nasal cavity. The encapsulation ofthese drugs into nanoparticles might be a promising approach, since the colloidalformulations have been shown to protect the drugs from the degrading milieu in the nasalcavity and facilitate their transport across the mucosal barriers. Nevertheless, the amountof nanoparticles accessing the brain is still limited because of the following reasons: firstof all, the penetration of the nanometer-size particles through tight junctions betweencells was negligible and the amount of unmodified nanoparticles endocytosed by thenasal epithelium was limited; secondly, the resident time of nanoparticles in nasal cavityis short because of mucociliary clearance (particles cleared within the nose every 15 to 20min), which is not available for the complete absorption of the formulation; finally,unmodified nanoparticles distributed in the nasal cavity without selectivity, resulting inpoor brain targeting efficiency of the formulation.To address these problems, novel lectin-modified nanoparticles were constructed.Wheat germ agglutinin (WGA), specifically binding to N-acetyl-D-glucosamine and sialic acid, both of which were abundantly observed in the nasal cavity especially in theolfactory mucosa, and ulex europeus agglutinin I (UEA I), specifically binding toL-fucose, which was largely located in the olfactory epithelium, were selected aspromising targeting ligands. The incorporation of peptides in the nanoparticles mightimprove their stability in vivo while the conjugation of lectin at the nanoparticles surfacemight induce strong mucoadhesion for a longer duration, or a close contact of thenanoparticles with the mucosal cells especially in the olfactory mucosa so as to produce astronger penetration. Such lectin-modified nanoparticles might serve as potential carriersfor brain delivery of peptides and proteins with enhanced brain-targeting efficiency andminimized adverse effects in the peripheral tissues.In the first part, lectin-conjugated nanoparticles were prepared by incorporatingmaleimide into one end of the PLA-PEG copolymer and taking advantage of its thiolgroup-binding reactivity to conjugate with the lectins thiolated with 2-iminothialane. Themean 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 theexistence of gold-labeled WGA-NP under TEM. The retention of biorecognitive activityof WGA after the covalent coupling procedure was confirmed by haemagglutination tests.The preparation protocol was optimized based on the following endpoints: lectin densityat the particle surface, particle size and conjugation efficiency, through which the optimalratio of maleimide-PEG-PLA to MePEG-PLA around 1: 9, lectin: 2-iminothiolane 1: 60,thiolated lectin: maleimide 1: 3 and the conjugation time of 8-10 h were obtained.In order to evaluate the capacity of the lectin-conjugated nanoparticles for drugdelivery into the CNS, in the second part, a lipophilic fluorescent probe with highsensitivity, coumarin-6, was incorporated into the nanoparticles, and the concentrationsof the fluorescent marker in blood and brain tissues following intranasal administration ofWGA-NP were determined and compared with those after nasal delivery of NP. It wassuggested by the in vitro release study and the haemagglutination test that coumarin-6was an appropriate fluorescent probe and the fluorescence signal detected or observedwas mainly attributed to the coumarin-6 encapsulated into the nanoparticles. Strongergreen fluorescent signals were observed on both olfactory bulb and cerebrum slicesfifteen minutes after intranasal administration of WGA-NP than that of NP. In vivopharmacokinetie results in rats suggested that the WGA and UEA I modification at the nanoparticles surface facilitated the absorption of the associated coumarin-6 into thebrain following intranasal administration with significant increase in the peakconcentration (about 2.26 and 1.33 times, respectively) and the area under theconcentration-time curve (about 2 and 1.7 times, respectively) in different brain tissuescompared with that of coumarin-6 incorporated in NP. The brain drug-targetingefficiency of the nanoparticles was in the following order: UEA-NP＞WGA-NP＞NP.In the third part, WGA-NP was used to deliver vasoactive intestinal peptide (VIP) intothe CNS via nasal administration. VIP was efficiently incorporated into PEG-PLAnanoparticles followed by surface modification with WGA, producing nanoparticles withparticle size of 100 nm, zeta potential of-20 mV, encapsulation efficiency of more than70ï¼…and drug loading capacity of 1.4ï¼…. In vitro release of VIP from VIP-NP showed aslight burst at the beginning and a long sustained release of about 35ï¼…aider incubationin plasma for 12 h and 17ï¼…in nasal wash for 8 h. It was also showed that the stability ofentrapped VIP in the plasma and nasal wash was significantly improved compared withthat of VIP. In the pharmacokinetic study, VIP was radio-labeled with Na ¹²⁵I and intact¹²⁵I-VIP in the CNS was determined with HPLC coupled with aγ-counter. The amount ofintact VIP detected in the brain tissues suggested that the incorporation of VIP into thenanoparticles significantly increased the stability of VIP in vivo and the area under theconcentration-time curve of intact ¹²⁵I-VIP in mice brain was significantly enlarged by3.5～4.7 folds and 5.6～7.7 folds following intranasal administration of ¹²⁵I-VIP carried byNP and WGA-NP, respectively, compared with that after intranasal application of¹²⁵I-VIP solution. The same improvements in spatial memory and hippocampusconcentration of acetylcholinesterase in ethylcholine aziridium-treated rats were observedfollowing intranasal administration of 25μg/kg and 12.5μg/kg of VIP loaded by NP andWGA-NP, respectively, indicating that WGA-NP might serve as a promising carrierespecially for biotech drugs such as peptides and proteins.In the fourth part, the transport pathways of WGA-NP from nose to brain wereinvestigated. It was observed that the penetration of WGA-NP into submucosa was fasterthan NP; More fluorescent signals representing WGA-NP were observed in the sieveplate, which was anatomically near the olfactory bulb, suggesting that the absorption ofWGA-NP into the olfactory bulb was faster than that of NP. Distribution profiles ofWGA-NP and UEA-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 submucosamight be transported into the CNS through the nerves or the connective tissues around thenerves in the lamina propria. Besides, a novel fluorescent probe with better opticalstability, CdSe quantum dots (QDs), was applied as a fluorescent probe and the transportmechanism of WGA-QDs-NP through the nasal mucosa was investigated using an in situnasal perfusion technique. Inhibition experiment of specific sugar suggested that theinteractions between the nasal mucosa and WGA-NP were due to the immobilization ofcarbohydrate-binding pockets at the surface of the nanoparticles. The addition of ametabolic inhibitor, NAN3, significantly reduced the amount of WGA-NP associated withthe nasal mucosa, indicating that WGA-NP was absorbed into the nasal mucosa throughan active transport pathway. Both PhAsO and chlorpromazine inhibited the association ofWGA-NP with the nasal mucosa while filipin failed to affect the association, suggestingthat clathrin pathway might play an important role in the endocytosis of WGA-NP whilethe caveolae one might not be the main pathway. The addition of both BFA andmonensin also decreased the association of WGA-NP with the nasal mucosa, indicatingthat both Golgi apparatus and lysosome participated in the uptake of WGA-NP. Suchinvestigations on mechanism might provide useful information for the design of noveldrug delivery systems.In order to evaluate the safety of WGA-NP following intranasal administration, theirinfluences on the nasal cilia, morphology and cells differentiation of the nasal mucosawere investigated. Nasal ciliotoxicity studies were conducted on both toad palate and ratnasal mucosa. The duration of ciliary movement on toad palate was 12 hours and 11.75hours for the WGA-NP-treated and NP-treated mucosa, respectively, which wascomparable with that of the negative control (12.25 hours). The data were consistent withthe results shown by SEM in the rat nasal mucosa, which showed that after intranasaladministration of WGA-NP and NP, no visible change in the morphology and integrity ofthe cilia was observed, suggesting that the nasal ciliotoxicity of WGA-NP and NP werenegligible. Furthermore, it was showed that the morphology and cells differentiation ofthe nasal mucosa were not affected with intact olfactory mucosa of about 40μm, intactand dense cilia on the mucosa and glands, blood vessels and nerve bundles in the laminapropria comparable with those of negative control observed following nasaladministration of WGA-NP for 7 days. These data indicated that WGA-NP was safe following intranasal administration.