Nose-to-Brain Targeting Delivery Of Lactoferrin-modified,Borneol And Dopamine Co-loaded Nanoparticles For Parkinson’s Disease

Parkinson’s disease（PD）is a neurodegenerative disorder characterized by progressive degeneration of nigrostriatal dopaminergic neurons.The key to PD treatment is to deliver an effective amount of drugs into the brain.Delivering the dopamine to the brain is still the most effective treatment.At present,there are two problems in the delivery of drugs into the brain.Firstly,the amount of drug delivered into the brain is small,and the presence of the blood brain barrier（BBB）prevents most drugs from entering the brain,which only allows small lipophilic drugs to across.Secondly,drugs have a wide distribution into the brain that are not selective.This project aimed to design a dopamine nose-to-brain targeted drug delivery system,which used the osmotic effect of borneol to promote the drug into the brain,and the lactoferrin as a targeting molecule to modify the nanoparticles（NPs）to concentrate in the PD lesions,thus achieving effective treatment of PD and reducing side effects:borneol anddopaminewereco-encapsulatedinmethoxypoly（ethylene glycol）-b-poly（lactide-co-glycolide）（MPEG-PLGA）NPs,lactoferrin（Lf）was used as a targeted molecule to modify the NPs to achieve active targeting,and in vitro and in vivo evaluation was investigated.The main research contents are as follows:1.Preparation and characterization of NPsBorneol and dopamine co-loaded NPs（DA BNPs）were prepared by emulsion solvent evaporation method,and the formulation and preparation process were optimized by single-factor method.And the optimal formula was as follows:dopamine theoretical drug loading was 30%,borneol theoretical drug loading was 4%,and polymer concentration was 20 mg/ml.The oil phase was dichloromethane,the external aqueous phase was 1%PVA aqueous solution,and the ultrasonic particle power was 300W that the particle diameter,the borneol and dopamine encapsulation efficiency,the drug loading,and the Zeta potential were both ideal.Lf-modified,borneol and dopamine co-loaded NPs（DA Lf-BNPs）were then obtained by attachment of the thiolated Lf to the maleimide group on the surface of the NPs.The DA Lf-BNPs that prepared by the optimal formula had a particle size of（160.7±16.7）nm,a dopamine drug loading of（7.98±1.80）%,a borneol drug loading of（1.78±0.22）%,and a Zeta potential of（-21.70±3.39）mV.The results of in vitro release indicated that dopamine entrapment in the NPs had good sustained release properties in the release medium of PBS（pH 7.4）.2.Cytotoxicity and cellular uptake of NPsTo evaluate the toxicity and drug delivery properties of the drug delivery system to brain tissue and nasal mucosa,human neuroblastoma cell line（SH-SY5Y）and human bronchial epithelial cells（16HBE）were used to investigate the toxicity and uptake behavior of NPs.The cytotoxicity of the carrier materials and dopamine-loaded NPs was investigated by MTT method.The results showed that the carrier materials had good safety and biocompatibility.In contrast,compared with free dopamine,dopamine-loaded NPs showed significantly less toxic of SH-SY5Y cells and 16HBE cells.The fluorescent probe was encapsulated into the NPs.The uptake behavior of the Nile red NPs was qualitatively observed by fluorescence microscopy.The uptake of the coumarin-6 NPs was quantitatively analyzed by flow cytometry.The results showed that the modified NPs of Lf can significantly enhance uptake and have better brain targeting for SH-SY5Y cells,and the Lf modification and co-encapsulated borneol can synergistically promote cell uptake for 16HBE cells.3.Brain targeting evaluation of NPsDiR was encapsulated into NPs that were administered by intranasal administration to qualitatively evaluate the drug delivery characteristics of the NPs using in vivo real-time fluorescence imaging analysis.The experimental results showed that the fluorescence intensity of the Lf-BNPs group was stronger,indicating that Lf modification and borneol co-loaded can promote more drug delivery into the brain and achieve better brain targeting following intranasal administration.Moreover,the drug was mainly concentrated in brain tissue,and was rarely distributed in other organs,further indicating that intranasal administration can reduce the toxicity of drugs compared with conventional administration methods such as oral administration and intravenous administration.4.Pharmacokinetic study of NPsThe pharmacokinetic behavior of NPs in plasma and brain after intranasal administration was investigated.The results showed that NPs had similar pharmacokinetic behavior,indicating that Lf modification and borneol co-loaded of NPs had no significant effect into the blood post intranasal administration.But the AUC_0-12h value of the Lf-NPs group in the brain was 1341.8±109.8 ng·h/g,which was significantly different from the Lf-BNPs（（1696.4±119.3）ng·h/g）group and the NPs group（（830.6±108.9）ng·h/g）（p<0.05）.These results demonstrated that Lf modification and borneol co-encapsulated can significantly increase the amount of NPs delivered into the brain and achieved targeting.5.Pharmacodynamic evaluation of NPsIn order to evaluate the therapeutic effect of the drug delivery system on PD,PD rats’model was prepared by inducing dopaminergic neuron injury by unilateral striatum injection of 6-OHDA.The contralateral rotation behavior and the content of dopamine and its metabolites in the brain were evaluated after intranasal administered different NPs.The results showed that after 20 days administered of Lf-BNPs group,the number of contralateral rotations was significantly lower than that of NPs（p<0.05）.The levels of dopamine and dihydroxyphenylacetic acid in the lesioned striatum were significantly higher than those in the PBS group（p<0.05）.These demonstrated that the drug delivery system achieved a better therapeutic effect after intranasal administration for PD.