Study Of Magnetic Nanobubbles Applied For The Tracking And Ultrasound Regulation Of Neural Stem Cells

Cells and regenerative medicine are the newly emerging biomedical field with important and valuable clinical applications,which aims to promote wound repair and disease treatment through stem cell transplantation,regulation of differentiation and tissue regeneration.In recent years,neuronal dysfunction caused by neuron cells damage is a difficult point for the neurological disease treatment.Neural stem cells(NSCs),as the pluripotent cells with self-renewing ability and multidirectional differentiation potential,have attracted much attention in recent years on neuron repair and the treatment of neuron-related diseases.However,the migration,survival and differentiation in vivo cannot be accurately evaluated and its clinical application remain challenging.With the development of nanotechnology,novel bio-nanomaterials have played an important role in stem cell labeling and its controllable differentiation regulation.In this thesis,superparamagnetic iron oxide nanoparticles(SPIONPs)are used to construct magnetic nanobubbles(MNBs)based on systematically optimizing of physicochemical properties.The interaction between magnetic nanobubbles and NSCs,as well as the mechanism of NSCs differentiation have been explored.Furthermore,we have investigated the influence of NSCs differentiation efficiency with nanobubblemediated low intensity pulse ultrasound(LIPUS).The mouse photothrombotic ischemic stroke(PTS)model was prepared to track MNB-labeled NSCs as well as monitor their fate in vivo.The specific research content includes as the following four parts:(1)Synthesis of magnetic nanobubbles and NSCs labelingWe developed a new gas(SF6)-liquid interface self-assembly method to prepare magnetic nanobubbles with shell embedded with SPIONPs.The diameter of the MNBs was 227.40 ± 87.21 nm with good stability.The SEM and TEM images of MNBs clearly exhibited that the nanoparticles were assembled on the surface of interface of nanobubbles.Ultrasound(US)imaging and magnetic resonance imaging(MRI)experiments indicated that the MNBs showed good US and MR contrast capabilities.Moreover,the NSCs were labeled with MNBs by two approaches: under ultrasound exposure,and co-incubation.The labeling efficiency was measured through inductively coupled plasma mass spectrometry(ICP-MS),transmission electron microscopy(TEM)and synchrotron transmission X-ray microscopy(STXM).The results showed that more MNBs were uptake after 6 h co-incubation(9.64 ± 0.06 pg per cell),while after 40 s ultrasound exposure,the cellular uptake up to 2.84 ± 0.68 pg per cell,which indicated that ultrasound exposure was a non-destructive and rapid labeling method.(2)Mechanism of NSCs differentiation mediated by intracellular MNBsThe NSCs were labeled with MNBs under co-incubation conditions,and the differentiation fate of cells was studied after interaction with MNBs.Through analyzing the total iron mass including intracellular and extracellular,as well as the expression of iron metabolism-related genes at different time points,we have verified the MNBs can be non-destructive metabolism pass the ferritin heavy chain gene and the ferritin light chain gene in the cell that ensuring long-term biocompatibility between MNBs and NSCs.Furthermore,the expression of specific protein markers for astrocyte and neuron was analyzed by immunofluorescence staining and flow cytometry to investigate the NSCs differentiation after 7 days interaction.The results demonstrated that intracellular MNBs can activate the NSC differentiation pathway.The staining results for actin filaments(F-actin)in NSCs revealed that the distribution of F-actin changed and areas of increased actin density appeared on the cell surface after interaction,meanwhile the atomic force microscope(AFM)results verified cells became increasingly stiffer during interaction.All these mechanical property changes could activate Piezo1 channel,inducing the Ca2+ influx and subsequent the BMP2/Smad signaling pathway response.(3)Magnetic nanobubble response to acoustic stress accelerates NSCs differentiation to neuronBased on the acoustic responsiveness of intracellular MNBs,the LIPUS was used to study the viability of NSCs under different parameters,and the regulation of NSCs differentiation fate(especially the direction of neuron differentiation)under different LIPUS exposure.The results of immunofluorescence staining and flow cytometry showed that after two LIPUS parameters(45 m W / cm2 for 20 minutes and 60 m W /cm2 for 30 min)exposure,the number of differentiated neurons increased about 3-fold than 7 days interaction.While under the same LIPUS stimulation,the number of neurons and astrocytes differentiated from NSCs decreased with the decrease amount of intracellular MNBs.All these results further demonstrated that the increased neurons differentiation was caused by the acoustic response of intracellular MNBs.Further immunofluorescence staining and quantitative analysis results of the presynaptic marker synaptophysin and the postsynaptic marker postsynaptic density protein 95(PSD 95)indicated that the LIPUS exposure accelerated synapse formation and maturation.(4)MRI and US dual imaging surveillance and neural stem cells fate in vivoFinally,in the mouse ischemic stroke model,the distribution of MNB-labeled NSCs around the stroke lesion,and the proliferation and differentiation fate of transplanted NSCs with and without LIPUS were evaluated.The results of MRI and US imaging showed that the implanted MNB-NSCs could achieve enhanced US and MRI dual imaging in stroke lesions.The Prussian blue staining results verified the retention of MNBs around the lesion.The results of immunofluorescence staining and H&E staining of brain sections showed that orthotopically transplanted MNB-NSCs could effectively slow down the cell infiltration during the development of ischemic stroke and reduced the inflammatory response.Moreover,the implanted NSCs retained proliferation and differentiation capability,especially under the exposure of LIPUS,NSCs could differentiate towards to functional neurons type around the stroke lesion area,and promoted the synapse formation of differentiated neurons.