Nanoparticles For Brain Delivery Of NR2B9c Peptide To Treat Ischemic Brain Damage Following Intranasal Administration

Stroke is a major public health problem leading to high rates of death and disability in adults.Although excessive stimulation of N-methyl-D-aspartate receptors(NMDARs)mediate ischemic brain injury,directly inhibiting NMDARs can cause severe side effects because they also mediate essential neuronal excitation.All attempts at treating strokes by pharmacologically reducing the human brain's vulnerability to ischaemia have failed.NR2B9c(KLSSIESDV),which comprise the nine COOH-terminal residues of NR2 B,was constructed to disrupt the interaction of NMDARs with the postsynaptic density protein PSD-95.This procedure dissociated NMDARs from downstream neurotoxic signaling without blocking synaptic activity or calcium influx.NR2B9 c can prevented NMDA-induced excitotoxicity and cerebral ischemic damage in rodents.Not only that,that stroke damage can be prevented in non-human primates in which NR2B9 c is administered after stroke onset in clinically relevant situations.Currently,the study of NR2B9 c is focused on the therapeutic effect of HIV-Tat-mediated NR2B9 c penetrating through the blood-brain barrier and cell membrane.However,a lot of people were concerned about the safety of HIV-Tat vector.Therefore,the development of effective strategies to enhance NR2B9 c delivery to the brain is of great interest in both clinical and pharmaceutical fields.However,the drug delivery system development for NR2B9 c needs the systemically understanding of its physicochemical properties,which have not yet been studied.This study systematically evaluated the impact of different factors that commonly used in the pharmaceutical formulation development,such as p H,temperature and specific oxidizing agents,on the stability of the NR2B9 c.As wheat germ agglutinin will selectively bind to N-acetyl-D-glucosamine highly expressed in olfactory mucosa and neuron,wheat germ agglutinin-modified PEG-PLGA nanoparticles were developed for NR2B9 c delivery to brain by nasal route efficiently and safely.This nanoparticles are prepared to investigate the physicochemical characteristics,the safety,the nose-to-brain delivery property and its neuroprotective effect in vitro and in vivo.Chapter 1 Preformulation study of NR2B9cIn this study,a reversed-phase high-performance liquid chromatography(HPLC)was applied to study the forced degradation behavior and stability of NR2B9 c.HPLC studies were performed with an C8 column using a mobile phase consisting of acetonitrile(14.5:85.5,v/v)and aqueous solution(0.1% trifluoroacetic acid(TFA)and 0.05 mol/l KH2PO4).The flow rate and the wavelength set during HPLC detection were 1.0 ml/min and 205 nm,respectively.The octanol/water partition coefficient of NR2B9 c in water(0.026±0.003)showed its poor permeability.The degradation pattern of NR2B9 c aqueous solution followed pseudo first-order kinetics.The degradation rate at p H 7.5 was the slowest according to the plotting V-shaped p H-rate profile.The influence of temperature on the rate of reactions was interpreted in terms of Arrhenius equation(r2>0.98).Thermodynamic parameters were calculated based on Eyring equation(r2>0.98).The result of this study showed that p H,temperature,and oxidation were the most significant factors affecting the stability of NR2B9 c.Solution with low concentration of NR2B9 c,neutral p H,low buffer concentration,high ionic strength,low dielectric constant,and without oxidation as well as low storage temperature and low ultrasound power showed beneficial effect on the stability of NR2B9 c.In comparison,the stirring rate and light investigated in the study did not show any impact on its degradation.In a word,the elucidation of the effects of different factors on NR2B9 c can help us to choose suitable conditions for preparation of NR2B9 c formulation in future.Chapter 2 Preparation and Characterization of NR2B9 c Loaded Wheat germ agglutinin-modified PEG-PLGA nanoparticlesThe WGA modified PEG-PLGA nanoparticles loaded with NR2B9 c were prepared using the double emulsion(W/O/W)method.A three factor,three-level Box–Behnken design was used for the optimization procedure,choosing the amount of NR2B9 c,the amount of Mal-PEG-PLGA,the weight ratio of Mal-PEG-PLGA and WGA-SH as the independent variables.The chosen dependent variables were particle size,dosage loading and conjugation efficiency of WGA.The optimized NR2B9c-WGA-NPs were almost spherical with good dispersity,the mean particle size was 140 nm and the zeta potential was-23 m V.The entrapment efficiency and drug loading were about 50% and 11%,respectively.The WGA conjugation efficiency and surface density on nanoparticles were 60% and 18 ?g WGA/mg NP,respectively.Haemagglutination test showed that WGA,conjugated to the surface of nanoparticles,still remain their carbohydrates binding bioactivity.The optimized formulation showed a sustained release following the Weibull equation.Furthermore,the NR2B9c-WGA-NPs were suitable to be stored at low temperature(4 °C).The stability of NR2B9 c was increased when it was entrapped into WGA-NPs in plasma and nasal wash.Chapter 3 WGA-modified PEG-PLGA nanoparticles for drug delivery to the brain after intranasal administrationUsing Calu-3 cells and neurons as the cell model,WGA-NPs were found to exhibit significantly enhanced cellular accumulation than that of unmodified NP via both clathrin-independent and caveolae-mediated endocytosis.Part of the uptaked WGA-NPs were co-localized with the endosomes,which were marked by Lyso Tracker Red,indicating that the cellular uptake was associated with the endosomes.Following intranasal administration of coumarin-6-loaded WGA-NPs,the AUC0-8h of the fluorescent probe detected in the rat cerebrum,cerebellum,hippocampus,olfactory tract and olfactory bulb was found to be 1.81,1.86,2.01,1.56 and 1.62 folds(p < 0.05),respectively,compared with that of coumarin-6 carried by NPs.WGA-NPs demonstrated higher brain targeting efficiency in different brain tissues than unmodified NPs(p < 0.05).The findings clearly indicated that the brain delivery of nanoparticles could be greatly facilitated by WGA and the WGA modified PEG-PLGA nanoparticles appears as an effective and safe carrier for nose-to-brain drug delivery in potential diagnostic and therapeutic applications.NR2B9c was entrapped in nanoparticles conjugated with WGA,which selectively binds to N-acetyl-D-glucosamine on the nasal epithelial membrane and plasma membranes of neurons.The brain distribution of the NR2B9 c formulations following intranasal administration was assessed using liquid chromatography-tandem mass spectrometry method.The areas under the concentration-time curve of NR2B9 c in the olfactory bulb,olfactory tract,hippocampus,and cerebrum,cerebellum of rats following nasal administration of NR2B9c-WGA-NPs were 1.99-3.84 folds of that of NR2B9 c via tail vein injection of NR2B9 c solution(p < 0.01),and 1.96-2.67 and 1.14-1.46 folds higher compared with intranasal solution(p < 0.01)and unmodified nanoparticles,respectively(p < 0.05).WGA-NPs also delivered more NR2B9 c to the brain in the rats subjected to middle cerebral artery occlusion(MCAO)and reperfusion.The results show that WGA-NPs may serve as a promising nose-to-brain NR2B9 c delivery carrier.Chapter 4 Neuroprotection and preliminary safety evaluation of NR2B9c-WGA-NPsTo evaluate whether NR2B9c-WGA-NPs could be neuroprotective in vitro,cortical neurons were exposed to an NMDA challenge,NR2B9c-WGA-NPs as well as Tat-HA-NR2B9 c inhibited lactate dehydrogenase release and reduced the number of PI-positive cells,it also ameliorated morphological changes,suggesting a neuroprotective effect against NMDAR-mediated excitotoxicity.To examine whether NR2B9c-WGA-NPs has benefits in cerebral ischemia,MCAO rats were treated with NR2B9c-WGA-NPs(i.n.)immediately after reperfusion and Infarction volume and neurological outcome measurements were performed at 24 h after reperfusion.NR2B9c-WGA-NPs as well as Tat-HA-NR2B9 c reduced infarct size and neurological deficit.CCK-8 method was used to evaluate cytotoxicity of NPs and WGA-NPs with and without NR2B9 c on Calu-3 cells and neurons.The result exhibited that under our experimental condition,the cell viability was more than 90%.The rats were intranasally given WGA-NP for 7 continuous days,tumor necrosis factor ?(TNF-?)concentrations in rat olfactory bulb and olfactory tract,brain and peripheral organs were measured to estimate the toxicity of WGA-NPs.There were no significant changes in TNF-? levels.The safety of NR2B9 c formulations was evaluated using histopathology assays of HE staining.No obvious pathological lesions were detected in nasal mucosa,heart,liver,spleen and lung of all groups of rats following nasal administration of NR2B9 c formulations.Accordingly,WGA-NPs were considered as a promising drug carrier without observable toxic effects.Conclusion:WGA modified PEG-PLGA nanoparticles following intranasal administration offer a safe and effective non-invasive delivery system for NR2B9 c to enter the brain and play the therapeutic role.This application will help us to discover other potential therapeutic peptide and protein drugs for central nervous system diseases treatment.