Experimental Study Of Focused Ultrasound Combined With EXO-RPM In The Treatment Of Parkinson's Disease

Parkinson’s disease(PD)is one of the common neurodegenerative diseases,its incidence is only lower than Alzheimer’s disease.Its main clinical symptoms are tremor,limb stiffness,and slow action and so on.With the gradual aging of our population,the Parkinson’s disease’s incidence rate is increasing year by year,which seriously threatens the health and quality of life of human beings.The main clinical treatments for PD are dopamine-like drugs that directly or indirectly supplement dopamine,and some invasive craniotomies,but these treatments’ effect is not ideal.Rapamycin(RPM)is an autophagy inducer that can activate autophagy through mTOR-dependent pathways to protect the homeostasis of nerve cells and improve the symptoms of PD.The blood-brain barrier(BBB)is one of the key factors restricting the treatment of all brain diseases.It blocks 98%of small-molecule drugs and almost 100%of large-molecule drugs from entering the brain tissue,resulting in a situation that brain diseases are difficult to treat and have poor results.Because of BBB,it is difficult to treat brain diseases and the treatment efficacy is bad.Therefore,how to overcome the BBB and achieve the targeted enrichment of drugs to improve therapeutic efficiency is the main scientific problem to be solved in the treatment of Parkinson’s disease.With the rise of nanotechnology,the preparation of nano-drug carriers has attracted the attention of researchers.Exosomes(EXO)is a natural endogenous drug carrier that can be secreted by a variety of cells.It has the advantages of good biocompatibility and the ability to overcome potential physiological barriers.In particular,the brain-targeting potential of blood exosomes has received widespread attention.However,the efficiency of drug-carrying drugs across the BBB and its targeting to lesion sites still has significant limitations.In order to improve its targeting performance,a large number of researchers have modified it by chemical synthesis or genetic engineering,but it is particularly time-consuming and labor-intensive.It still cannot avoid off-target effects.Studies have shown that focused ultrasound(FUS)combined with microbubbles can reversibly open BBB,which has the advantages of targeting by controlled operation,high safety,and low toxic and side effects.FUS has been approved by the FDA for the treatment and diagnosis of brain diseases.Based on the above research background,this study intends to develop a non-invasive and highly biocompatible natural drug delivery system:first,using FUS to safely and reversibly open the BBB,then injecting blood exosomes carrying RPM in an open window to promote accurate delivery and enrichment of RPM to brain lesions.Second,RPM can up-regulate autophagy to promote the degradation of α-synuclein(α-syn)and reduce the pathological accumulation of a-syn protein,thereby achieving the purpose to protect dopaminergic neurons.Finally,the effectiveness,feasibility,and biological mechanism of the research system for PD treatment were evaluated from behavioral indicators and tissue section observations,which may provide a foundation for exploring new approaches to PD diagnosis and treatment.This thesis mainly focuses on the preparation and characterization of EXO-RPM,the protective effects of EXO-RPM on MPTP-induced PD model of SH-SY5Y cells,ultrasound-targeted delivery of EXO to the brain,the alleviated effect of FUS combined with EXO-RPM on MPTP-induced PD model mice and so on,the main contents are summarized as follows:Part1 Preparation and Characteristic Analysis of EXO-RPMOptimizing the extraction steps and using ultracentrifugation to extract blood exosomes,then using the extracted exosomes as drug carrier for loading rapamycin(RPM).The rapamycin was encapsulated by a gentle incubation method,then we further optimized the EXO-RPM preparation process and drug loading ratio.The prepared EXO-RPM showing good stability,the properties and chemical physiological characteristics characterized were identified.Nanoparticle tracking analysis system(NTA)was used to measure the size and potential distribution of exosomes.Transmission electron microscopy(TEM)was used to observe the ultra-morphological structure.Western blotting were used to identify the expression of exosomal mark proteins such as CD63,CD9 and transferrin receptor(TfR).The results showed that the EXO-RPM has uniform dispersion,regular morphology,moderate particle size and potential distribution,good stability,and several important mark proteins are expressed,which was consistent with related literature reports.It has the excellent properties of nano-drug carriers and can be used for subsequent research.Part 2 The protective effect of EXO-RPM on MPTP-induced PD model of SH-SY5Y cellsLaser confocal microscopy and flow cytometry were used to detect the uptake of exosomes and its subcellular localization in the cells,using the CCK8 method to screen the drug concentration and incubation time,it can provide a basis for the mechanism analysis of cell autophagic response.The cell viability was measured after treatment to verify whether EXO-RPM had a protective effect on model cells.At the same time,observing the changes of cell autophagy protein and α-syn protein can provide a basis for subsequent treatment research of PD mice.The results indicate that SH-SY5Y cells presented a time-dependent uptake of EXO,and EXO was mainly distributed in the cytoplasmic matrix.EXO-RPM can up-regulate intracellular autophagy and degradeα-syn protein in vitro,suggested that the drug loading system may have a certain protective effect on PD neurological damage.Part 3 Ultrasound promoted targeting delivery of EXO to the brainAn in vitro BBB model was established to explore whether ultrasound can promote exosomes to pass through the BBB.The action parameters and reversibility extent of focused ultrasound combined with microbubble to open the BBB disorder was observed in mice to determine the optimal time window for later administration.The distribution of EXO in mice after different treatments was observed by using small animal in vivo imaging equipment,especially the enrichment of EXO in the brain.The results demonstrated that focused ultrasound combined with microbubble opening BBB was safe and reversibly,ultrasound can instantly improve the efficiency of EXO crossing the BBB,and can promote the targeted enrichment of EXO-RPM to PD lesions in the brain region.Indirectly,it suggested that the drug delivery system of ultrasound combined with EXO can increase the drug into the brain and improve the treatment efficiency of Parkinson’s disease.Part 4 Protective effect of FUS combined with EXO-RPM on MPTP-induced PD model miceTo evaluate the therapeutic effect of PD mice,the behavioral changes of mice was examined by open field experiments and rotarod tests,and the dopamine(DA)content in the striatum of mice was detected by HPLC.Immunohistochemical staining was employed to investigate the activity of tyrosine hydroxylase(TH)and the expression ofα-syn protein.Transmission electron microscope was used to observe the autophagy level.The safety of this treatment mode was evaluated by monitoring the changes in body weight of mice,serum biochemical indicators,as well as the histopathological differences in brain,heart,liver,spleen,lung,kidney and other treatment groups.The results showed that the behavioral disorders of PD model mice were largely recovered after treatment,DA and TH levels in the brain were reversed,autophagy levels were upregulated,and α-syn protein degradation was enhanced.The findings suggested that FUS combined with EXO-RPM protected the damaged neurons in the brain of PD model mice induced by MPTP and improved the motor ability of the mice.This treatment mode was relatively safe and effective.ConclusionThis study successfully designed and developed an endogenous nano-delivery system that combing focused ultrasound with rapamycin loaded blood exosomes.The therapeutic effects of FUS+EXO-RPM on PD models were evaluated by analyzing the cellular responses and animal behaviors both in vivo and in vitro.The results suggested that the treatment mode of FUS combined with EXO-RPM has a good therapeutic effect both in vivo and in vitro.This treatment strategy of FUS combined with EXO-RPM has a good therapeutic effect on Parkinson’s disease,showing great potentials in future application.