| Background&ObjectiveTranscranial drug delivery has always been a research hotspot.Parkinson’s disease is caused by selective apoptosis of dopaminergic neurons in the nigra striatum.The key to the treatment of PD lies in effective transcranial administration,but the existence of blood-brain barrier shuts out macromolecular drugs.At present,the microbubbles or nanobubbles combined with low-intensity focused ultrasound targeting brain drug delivery system has great potential after several years of research.However,due to the large particle size and poor compression resistance of traditional microbubbles,it is easy to "burst" due to transient cavitation effect after focused ultrasound irradiation,emitting extreme physical events such as microjets and shock waves,and damaging fragile brain tissue.In this study,curcumin-loaded Lipid-PLGA nanobubbles(CLPNBs)with good compression resistance,long action time and safe opening of blood-brain barrier through stable cavitation was prepared by changing materials and improved methods.Curcumin has been demonstrated to be effective in relieving PD.It is mainly through anti-inflammation;anti-oxidation;activating Wnt/β-catenin pathway to increase cell vitality;activating autophagy system to promote the elimination of α-Syn,which has a neuroprotective effect.However,curcumin has strong hydrophobicity and poor bioavailability,so this study intends to improve the hydrophilicity of curcuminMethods&Materials1.PLGA and Lipid were mixed and dissolved into the first phase by double emulsion solvent evaporation,and they will be used as the coating structure of nanobubbles.The nanobubbles are coated with PEG-6000-modified curcumin,which can increase the amount of curcumin dissolved in water.The morphology of nanobubbles was observed by scanning electron microscope(SEM)and transmission electron microscope(TEM)The particle size,fluorescence wavelength change,drug loading and ultrasonic imaging effect in vivo and in vitro were measured by related instruments2.To explore the range of ultrasound Mechanical Index(MI)parameters of low intensity focused ultrasound(LIFU)combined with CLPNBs to open the blood-brain barrier(BBB).The best MI value was screened by Evans blue staining,HE staining,fluorescence microscope observation,ultrasonic cavitation detection system and so on The depth and distribution of drug delivery in the brain were observed by the fluorescence of curcumin.3.16-week-old C57BL/6J male mice were divided into 5 groups:① healthy control group;② ultrasound combined with nanobubbles group;③ simple nano-bubble group;④ simple ultrasound group;⑤ animal model group.Among them,①②③④⑤groups were given corresponding intervention measures(once a day)on the 0th,2nd,4th,6th,8th and 10th day of the experiment respectively.Group①②③④⑤was injected intraperitoneally with mptp solution at the dose of 30mg/kg once a day on the 1st-7th day of the experiment(6 hours after the implementation of the intervention or at the same time).After the establishment and intervention of mptp subacute Parkinson’s mouse model,the behavior experiment(rotating rod test,pole climbing test)was used to detect the passive movement intention of Parkinson’s mice,and to evaluate whether the treatment of target group ② was effective or not.HE staining was used to observe whether there was brain injury after the use of the best MI.Hemolysis test was used to observe whether different doses of CLPNBs would have adverse reactions to blood circulation.Results1.Curcumin-loaded Lipid-PLGA nanobubbles with a particle size of(357±18.53)nm were successfully prepared.There were many pores on the surface of the shell and large cavities inside.Under electron microscope,it was observed that the dispersion was good,there was no polymerization,and the effect of contrast-enhanced ultrasound in vivo and in vitro was good.2.Through exploration,the method of CLPNBs combined with LIFU can open the blood-brain barrier.By screening parameters,the nanobubbles still maintained stable cavitation during MI=1.75,and no obvious brain tissue damage was found.At the same time,the drug delivery depth in the brain could reach the substantia nigra nucleus.3.The corresponding intervention measures of each group were used to deal with Parkinson’s model mice(ultrasonic MI=1.75).The behavioral experiment showed that the exercise ability of group ② was significantly lower than that of group ①,but the exercise ability of group②was significantly higher than that of group③④⑤(P<0.05).ConclusionIn this study,curcumin-loaded Lipid-PLGA nanobubbles have been successfully developed.The morphology of curcumin-loaded nanobubbles is regular and round,and the internal cavity can carry drugs,and the imaging performance is good.CLPNBs combined with LIFU can open the blood-brain barrier through the skull and maintain the state of stable cavitation at the best mechanical parameter MI=1.75.At the same time,the drug can be delivered to the deep brain nucleus through the skull.CLPNBs+LIFU is effective in the treatment of Parkinson’s C57 mice,and behavioral experiments show that it is effective,and there is no additional brain tissue damage.Different doses of CLPNBs had no hemolysis. |
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