| With the population aging, the increasing prevalence of central nervous system (CNS) disease is seen worldwide. Alzheimer’s disease (AD), the commonly known senile dementia is one of the four common neurodegenerative diseases. About 10% of people over 65 have AD. The harm of this disease is increasingly prominent with the aging of the human society. Neuropeptides have showed neuroprotective effects on neurodegenerative diseases, and representing promising therapeutics to the treatment of ADIntranasal administration provides a noninvasive alternative to the brain delivery of bioactive agents which could bypass the BBB and allow direct access of the therapeutic substances to the brain. It has been demonstrated that transport via the trigeminal nerve pathway facilitated nose-to-brain delivery to caudal brain areas. In contrast, drug directly entered the rostral brain regions via the olfactory pathway. However, the nasal absorption of bioactive agents is still limited by its rapid enzymatic degradation by nasal cytochrome P450/peptidases/proteases, low permeability via the nasal mucosa and rapid mucociliary clearance. Over the last decade, PEG-coated polyester nanoparticles have attracted increasing attention as a drug delivery system (DDS) due to their favorable biological properties--their biocompatible/biodegradable properties and long-circulating behavior However,the system still has its drawbacks; the surface PEG chains could probably inhibit its interaction with cell surfaces. Lactoferrin (Lf) is a natural iron binding cationic glycoprotein of the transferrin family, expressed in various tissues and involved in various physiological processes. Extensive histological studies showed that Lf receptor (LfR) is highly expressed on the apical surface of respiratory epithelial cells, and also in the brain cells such as brain endothelial cells and neurons. Besides, LfR has also been demonstrated to be overexpressed in the CNS associate with age-related neurodegenerative diseases including AD, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis. Based on this information, we speculated that Lf might serve as a suitable ligand for mediating enhanced nose-to-brain delivery of nanoparticles following intranasal administration. Therefore, the aim of this study was to determine the potential of Lf-conjugated poly(ethyleneglycol)-poly (ε-caprolactone) nanoparticle (Lf-NP) for delivering neuroprotective agents to the treatment of Alzheimer’s disease.The first part is about construction and characterization of Lf-NP. Unmodified nanoparticles (NP) were prepared using the emulsion/solvent evaporation technique, while Lf-NP were prepared via a maleimide-thiol coupling reaction in HEPES buffer (pH 7.0) at room temperature for 9 h with the purified thiolated Lf. The nanoparticles exhibited an average diameters 70-90 nm under transmission electron microscopy. The nanoparticles with or without Lf modification showed similar acceptably zeta potentials (around -24 mV). Under our experimental conditions (molar ratio of 2-IT to Lf 40:1, incubation time for conjugation reaction 9 h), Lf conjugation efficiency was 22.6%, and Lf density on the nanoparticle surface was around 46. XPS analysis showed 0.32% nitrogen on the Lf-nanoparticle surface while none on the unmodified nanoparticles.In the second chapter, using 16HBE14o-cells as the cell model, a fluorescent dye, coumarin-6, serving as a nanoparticle probe, was incorporated in Lf-NP to investigate its cellular accumulation characteristics in vitro cell model with unconjugated nanoparticle as control. In vitro release study conducted in pH 4.0 and pH 7.4 showed that almost all the coumarin-6 remained in nanoparticles during the experimental period, indicating that the fluorescent signal of coumarin-6 could represent the fate of nanoparticles both in vitro and in vivo. The uptake of Lf-NP by 16HBE14o-cells was much higher than that of NP. And both the uptake of Lf-NP and the uptake of NP showed a time, concentration and temperature-dependent cellular uptake pattern indicated an active endocytotic process. Lf-NP was found to exhibit significantly enhanced cellular uptake than that of unmodified NP via both clathrin-mediated endocytosis and caveolae-mediated endocytosis. Preincubated free lactoferrin competitively reduced the epithelial cellular uptake of Lf-NP, suggesting that the internalization of Lf-NP involved lactoferrin receptor-mediated.In the third chapter, Lf was labeled with Cy5.5, a near-infrared fluorescent probe, serving as a protein probe, and its brain targeting ability was evaluated by a real-time brain distribution analysis under an in vivo imaging system with Cy5.5 labeled BSA as control. The results indicated that Lf possessed higher efficiency for brain accumulation. Systemic pharmacokinetics and biodistribution of coumarin-6 were determined following intranasal administration of coumarin-6-loaded Lf-NP and NP. The AUCo-8 h of the fluorescent probe detected in the rat olfactory bulb, olfactory tract, cerebrum with hippocampus removed, cerebellum and hippocampus was 2.57,2.70, 2.22,2.70 and 2.23 folds, respectively, compared to that of coumarin-6 carried by NP. The AUCo-8h ratio of brain tissues to blood (AUCbrain/AUCblood) of coumarin-6 incorporated in Lf-NP was also much higher than that of coumarin-6 associated to the unmodified NP (2.69,2.27,3.80,3.57 and 3.51 folds for the olfactory bulb, olfactory tract, cerebrum with hippocampus removed, cerebellum and hippocampus, respectively. Brain distribution analysis suggested Lf-NP after intranasal administration could be delivered to the central nervous system along both the olfactory and trigeminal nerves pathways. The findings clearly indicated that the Lf-NP appears as an effective and safe carrier for nose-to-brain drug delivery.In the fourth chapter, In order to evaluate the neuroprotective effect of NAP encapsulated Lf-NP, we here use mice intracerebroventricularly co-injected with pre-aggregated Aβ1-4o and a small amount of ibotenic acid as the animal model. Lf-NP was used to deliver a protein drug NAP into the CNS. The Morris water maze (MWM) experiment, one of the most frequently laboratory tools to study the neurocognitive disorders, was subsequently conducted to evaluate the effect of different NAP preparation on the mice spatial memory and learning dysfunction. The NAP-related neuroprotection was also studied by determining the activity of biochemical indicators--acetylcholinesterase and cholineacetyltransferase in the mice hippocampus. According to the behavioral research and biochemical indicators results, intracerebroventricularly co-injected with preaggregated Aβ-40 and a small amount of ibotenic acid could lead to neurocognitive disorders. While direct intranasal administration of NAP solutions or NAP encapsulated NP had less improvement action on AD model mice with the nasal absorption of NAP formulations limited by its rapid enzymatic degradation by nasal cytochrome P450/peptidases/proteases, as well as low permeability via the nasal mucosa and rapid mucociliary clearance. In both behavioral studies and in histology study, administration of NAP-loaded Lf-NP had shown better improvement compared to other NAP formulations even in a lower drug dose (0.05ug/mouse). When the dose of NAP-loaded Lf-NP was increased to 0.1 μg/mouse, the latency was shortened significantly in behavioral tests and the acetylcholinesterase and cholineacetyltransferase activity was recovered to normal level compared to sham mice. These results definitely indicated that the Lf-NP drug delivery system could mediate NAP transport into the brain for the treatment of Alzheimer’s disease, and may offer an effective non-invasive approach to facilitate the access of neuropeptides to the CNS. |
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