The Design Of Lactoferrin Loaded Hupa Nanoemulsion And Research Of Brain Targeting Mechanism Via Intranasal Route

Alzheimer’s disease（AD）is a common disease which results in the degeneration of the central nervous system.The number of cases of AD disease is increasing year by year,making it an increasingly common CNS disease.However,the development of new drugs for the treatment of AD disease is restricted by many factors,among which the most important one is the problem of how to cross the blood brain barrier（BBB）.After oral or injection of systemic drugs,almost all large molecule drugs and water-soluble small molecule drugs cannot enter into CNS,which becomes the bottleneck of CNS disease treatment.The traditional trans-BBB methods,such as intracranial drug transport or lipidation of small water-soluble molecules,have many disadvantages and defects.The nasal-brain pathway is considered a promising brain targeting strategy to treat different CNS diseases.In addition,with the development of pharmacy,the advantages of nano-technology-based pharmacy methods in nasal delivery across BBB have aroused people’s interest.For example,the drug delivery system of Nanoemulsion（NE）has a high efficiency of nasal-brain transport.However,the mechanism of nasal-brain transport across BBB in nano-drug-delivery systems is still controversial.Huperzine A（HupA）is a reversible cholinesterase inhibitor,which can be used to treat benign memory disorders and senile dementia caused by AD.At present,tablets,capsule and injection of HupA are widely used in clinic.Oral administration of the drug could cause gastrointestinal symptoms such as nausea,vomiting,diarrhea and other peripheral cholinergic side effects like sweating,insomnia and blurred vision.It is also easy to produce first pass effect and affect the efficacy.Furthermore,injection administration causes great pain and inconvenience to the patient.In order to break through the bottleneck of brain disease treatment,achieve the nasal brain targeted therapy and improve the effect of clinical treatment of AD,this paper used AD healing Chinese medicine HupA as the model drug to design and optimize the nano nasal drug delivery system.Furthermore,NE was modified by targeting functional molecular Lf to obtain the brain targeting Lf-HupA-NE.In this paper the nanomedicine delivery system was well studied in vitro and in vivo.This paper is mainly divided into the following seven parts:The first part is to establish the methodology of measuring the HupA in Vitro and in Vivo.It is conducted by the high performance liquid chromatography（HPLC）.This method is accurate and reliable because of the high specificity,high precision,high recovery and good stability.The second part is about screening and optimization of HupA-NE prescription.According to the characteristics of HupA as lipid soluble,the oil phase that can dissolve HupA,the surfactant with appropriate HLB value and the cosurfactant were first selected to determine the solubility of HupA in various components seperately.HupA has good solubility in surfactant Cremophor EL,cosurfactant Labrasol/PEG400and oil phase Capryol90/L.A.S.IPM,as a small molecule oil phase,has strong permeability to hydrophilic surfactant to form NE easily.Therefore,the mixture of highly soluble oil phase and IPM was selected as the mixed oil phase.The composition systems with phase conversion and blue fluorescence were selected as nanoemulsions by mechanical agitation.The prescription of nanoemulsion was mixed Capryol 90 and IPM as the oil phase,Cremophor EL and Labrasol as surfactant and cosurfactant.The mixed surfactant（Smix）,oil phase and water phase were taken as three vertices,and the pseudo-ternary phase diagram was drawn according to the different proportions of oil phase and Smix as well as the water content of critical points.The larger the area of pseudo-ternary phase diagram indicates the larger the area of nanoemulsion.The area of pseudo-ternary phase diagram is the largest when the ratio of surfactant to cosurfactant Km is 2:1.The proportion of each component was further optimized by using the Surface Response Method.Finally,the prescription of nanoemulsion was determined to be 3.00%IPM,3.81%Capryol 90,40%Cremophor EL+Labrasol and 53.19%water.The third part is about the preparation,quality evaluation,in vitro release and stability evaluation of LF-HupA-NE delivery system.Based on the formulation design of ternary phase diagram and response surface method optimization,the preparation of NE delivery system（HupA-NE）was conducted.Lf-HupA-NE was obtained by electrostatic adsorption of positively charged targeting molecule lactoferrin（Lf）onto negatively charged NE surface.The prepared nanoemulsion was determined to be O/W type by staining.The particle size,PDI and Zeta potential of HupA-NE measured by Marvin particle size detector were 15.24±0.67nm,0.128±0.074 and-8.06±0.53mV,respectively.The particle size,PDI and Zeta potential of LF-HupA-NE were 16.78±0.4nm,0.038±0.028 and+5.67±0.39mv,respectively.The morphology of the NE,HupA-NE and LF-HupA-NE,were spherical and evenly distributed,and their size was basically consistent with that of the particle size measured by the analyzer.The drug loading capacity of HupA-NE was 0.49±0.024%which is 4.94±0.127 mg·mL^-1.The content of HupA in LF-HupA-NE was about4.87±0.239 mg·mL^-1.The results of in vitro release tests showed that both nanoemulsions could increase drug release.The pH values of HupA-NE and LF-HupA-NE were 5.75 and 6.15 which are in the range of nasal pH（5.5-6.5）reported in literature,which could reduce the irritation or damage to nasal mucosa.The HupA-NE and LF-HupA-NE delivery systems prepared in this study have stable quality and meet the requirements of nanoemulsion delivery system.The fourth part investigated the mechanism of nanoemulsion into BBB in vitro.In vitro BBB model was established by Human cerebral microvascular endothelial cells,D3 clone（hCMEC/D3）cells.The BBB model was successfully established by leakage test and monitoring TEER.The toxicity of HupA-NE,LF-HupA-NE and HupA solutions on hCMEC/D3 cells was investigated by MTT method.There was no significant toxicity to hCMEC/D3 cells within 24h between 0.1 and 10μg·mL^-1.In this chapter,Western Blot method was used to prove that p-gp,MRP1 and BCRP efflux proteins were present in hCMEC/D3 cells,and the intracellular HupA content could be increased by adding corresponding efflux protein inhibitors.The improvement of HupA in the cells after the treatment of HupA-NE and LF-HupA-NE was lower than that in the HupA solution.On the one hand,it indicates that HupA is transported through the three efflux proteins.On the other hand,combined with immunohistochemical analysis,it was concluded that the nanoemulsion had inhibiting effect on efflux.The results of uptake inhibition experiments confirmed that the uptake of HupA-NE and LF-HupA-NE was mediated by low-density lipoprotein receptor-related protein（LRP）receptor,and clathrin and macropinocytosis.The NE modified with Lf also enters the cell mediated the Lf related receptor（LfR）across the BBB.The fifth part study the nasal-brain pathways.After nasal administration of HupA-NE and LF-HupA-NE in SD rats,the two NEs showed no irritation to nasal mucosa by HE staining of nasal mucosa.The P₂ probes provide extremely significant tools to track the in vivo fate of nanoparticle.The probes are kept illuminated in the nanoparticles,but quench whenever they leave the integral matrix of the nanoparticles.The olfactory nerve was blocked in the rat model and the direct pathway of the nasal and brain olfactory nerve was cut off.The nanoemulsion could still be detected in the brain,but the distribution range and intensity in the brain were lower than that in the non-operation group.So the olfactory nerve pathway was the main pathway.After intranasal administration of the complete nanoemulsion containing HupA,it can bypass the BBB through the olfactory nerve or cross the BBB after systemic circulation through the nasal mucosa absorption.The sixth part investigated the distribution of NE in rat brain.Rhodamine B（RhB）was used as a fluorescent probe.The experiment prepared the compound RhB-HupA by chemical synthesis.IR method demonstrated that the RhB and HupA were linked by the reaction of to the amino group and carboxylic acid.RhB-HupA-NE and Lf-RhB-HupA-NE,which were connected with fluorescent probes,were given to the nasal cavity of the rats.The frozen sections of brain tissue showed that the red fluorescence distribution in the brain of the NE preparation increased significantly compared with that of the drug solution.And the distribution of Lf-RhB-HupA-NE in the brain was higher than that of RhB-HupA-NE.The results showed that the drug dosage in the brain could be increased by transnasal administration of NE,and Lf mediated NE greatly increased drug transport into the brain,which was consistent with the expected results.The Seventh part is about the pharmacokinetics and targeting evaluation of NEs in rats.Compared with the conventional oral administration,the pharmacokinetic method was used to investigate the drug delivery characteristics of HupA-NE and Lf-HupA-NE in rat brain.After nasal administration,the concentration of HupA in rat plasma and tissues at each time point was quantitatively determined by HPLC,and the pharmacokinetic parameters were calculated by DAS 2.0 software.In brain tissue,the AUC（0-t）of the NEs were significantly higher than that of the oral drug solution and the Lf-HupA-NE>HupA-NE>HupA solution.The peak concentration of Lf-HupA-NE>HupA-NE>HupA solution;mean repair time of Lf-HupA-NE>HupA-NE>HupA solution;Half-life Lf-HupA-NE>HupA-NE>HupA solution;Clearance rate Lf-HupA-NE<HupA-NE<HupA solution.This indicated that nasal administration of NEs could increase the amount of HupA entering the brain,and achieve the purpose of sustained release to prolong the action time in the brain.In particular,Lf-HupA-NE was improved significantly.The Lf receptor-mediated trans-BBB transportation is more conducive to the concentration of Lf modified nano delivery system in brain tissue.In other tissues,NEs can reduce the Cmax of heart,liver and kidney,thereby reducing the toxicity of heart,liver and kidney.Liver and kidney clearance rate is fast,NEs may be metabolized by liver and kidney.Peak concentration ratio Ce and relative uptake rate Re were used to evaluate the targeting of nanoemulsion.Both Ce and Re were greater than 1,indicating that compared with the traditional oral intragastric solution group,the transnasal nanoemulsion could increase the distribution of HupA in the brain,with a brain targeting effect.The Lf-HupA-NE targeting index DTI constructed by the targeted functional molecule Lf was 2.61.It indicated that the brain targeting of Lf-HupA-NE was stronger than HupA-NE,and specific distribution in vivo.In summary,the paper successfully constructed a nanometer drug delivery system of Lf modified Lf-HupA-NE,which can effectively increase the drug concentration to the brain through nasal delivery,and preliminarily speculated the mechanism of cross-BBB.The results of this study provide a new idea for the research of drugs for the treatment of brain diseases such as AD.