Experimental Study Of The Impact Of Focused Ultrasound Applied Lipid PLGA Hybrid Bubbles On Blood Brain Barrier

Chapter1 Preparation and characterization of lipid PLGA hybrid bubblesObjective:Lipid PLGA hybrid bubbles,A kind of ultrasound contrast agent with good acoustic response,was prepared and delivered to nervous system through ultrasound targeted microbubble destruction.Methods:In this paper,lipid PLGA hybrid bubbles,were prepared by the combination of solvent evaporation and freeze-drying.The morphology and structure were observed by TEM and SEM,the particle size distribution and dispersion were analyzed by Malvern particle size analyzer,and the imaging ability was detected by ultrasonic imaging device.Results:TEM and SEM revealed that of lipid PLGA hybrid bubbles were well-defined morphology.Malvern particle size analyzer showed that the average particle size was 852.8±153.6nm,and the dispersion coefficient was 0.32.Moreover,lipid PLGA hybrid bubbles had a stable and lasting ultrasound imaging ability of about 1h in vivo,with the characteristics of general contrast agents and unique advantages.Conclusion:In this study,lipid PLGA hybrid bubbles were successfully prepared,which has the characteristics of regular morphology,uniform particle size,good dispersion coefficient and good ultrasound imaging.Chapter2 Effect of focused ultrasound combined with microbubbles on the function of blood brain barrier model in vitroObjective:To establish a sustained drug delivery system for an in vitro blood brain barrier model disruption Induced by focused ultrasound combined with microbubbles.Methods:Mouse brain microvessel endothelial cells were co-cultured with brain microvessel pericytes and astrocytes through transwell chamber so as to establish an in vitro blood brain barrier model,and the barrier function was evaluated by transendothelial electrical resistance(TEER)and fluorescein sodium permeability.After the model was built,it was divided into 0.4,0.6,and 0.8 Mpa groups and the control group.The different parameters were used to focus the ultrasound combined with the microbubble to irradiate the transwell chamber,and the BBB was observed at 0.5,4,12,and 24 h after ultrasound combined with microbubble irradiation.TEER changes to screen for appropriate ultrasound parameters.After determining the appropriate parameters,the dynamic changes of the doxorubicin hydrochloride and FITC-Avidin permeability at 0.5,4,12 and 24 h after ultrasound irradiation of the BBB model were observed.Results:(1)The TEER of the three cells co-cultured for 6 days reached(253.1±10.86)Q/cm2,and the fluorescein sodium permeability coefficient was(3.490±0.4646×10-6)cm/min,and the model was established successfully.(2)Compared with the control group The TEER of the 0.4Mpa group changed little within 24h(P>0.05),while the TEER of the 0.6Mpa group and the 0.8Mpa group was the smallest at 0.5h(P<0.05),and the TEER of the BBB gradually became larger at 4,12 and 24h.The 0.6Mpa group returned back to normal at 24h(P>0.05),while the 0.8Mpa group failed to return back to normal at 24h(P<0.05).(3)Focused ultrasound at 0.4Mpa increased the permeability of DOX and FITC-Avidin(p<0.05),and basically recovered back to normal level at 24h(P>0.05).Conclusion:Focused ultrasound combined with microbubbles can safely and reversibly increase the permeability of BBB model of different molecular weight substances.Chapter3 Detection and imaging of ultrasound-induced apoptotic events during blood-brain barrier openingObjective:Lipid PLGA hybrid bubbles were prepared,which can open the blood-brain barrier and detect apoptosis and have the function of anti apoptosis simulta-neously.Methods:(1)Annexin v-avidin-icg-bsa(AAIB)and Avidin-icg-bsa(AIB)were designed and synthesized by biotin avidin system.The particle size,morphology and fluorescence intensity were detected by Malvern particle size analyzer,transmission electron microscope and fluorescence gradiometer respectively,and the apoptosis model established by hydrogen peroxide was also detected.(2)Preparation of AAIB and AIB by the combination of solvent evaporation and freeze-drying,The particle size,morphology and fluorescence imaging of loaded targeted AAIB and loaded non-targeted AIB lipid PLGA hybrid bubbles were detected by Malvern particle size analyzer,transmission electron microscope,scanning electron microscope and IVIS small animal imaging instrument respectively;the localization of fluorescence was detected by laser confocal;the composition and loading rate of AIB lipid PLGA hybrid bubbles were analyzed by sda-page electrophoresis with the change of the input;the ultrasound targeted microbubble damage was detected by small animal ultrasound imaging instrument.With the change of ultrasonic intensity,SEM was used to detect the change of morphology after blasting.(3)The apoptosis model was established by hydrogen peroxide,and the apoptosis was detected by laser confocal observation of loaded targeted AAIB PLGA hybrid vesicles and loaded non-targeted AIB PLGA hybrid vesicles.(4)The amount of Evans blue exudate was observed to observe the degree of opening blood-brain barrier by different ultrasound parameters combined with loaded targeted AAIB lipid PLGA hybrid bubbles.Meanwhile,HE and TUNEL staining were used to detect its bleeding and apoptosis.The fluorescence imaging effect and distribution of its main organs in vivo were measured on IVIS small animal imaging instrument.According to the selected ultrasound parameters,the time-varying trend of fluorescence imaging was observed with IVIS small animal imaging instrument after loaded AAIB and non-targeted AIB PLGA hybrid vesicles opened the blood-brain barrier,and the change rate of targeted and non-targeted groups was analyzed with mathematical model.The ultrasound parameters and anti apoptotic drugs of AAIB and AIB lipid PLGA hybridized bubbles were selected by cell activity experiment,and wrapped in the lipid PLGA hybridized bubbles with fluorescence probe.The morphology and particle size were detected by SEM and Malvern particle size meter,and the drug loading rate was detected by liquid chromatography.The apoptosis of drug loaded group and drug not loaded group was detected by confocal laser,and the general mechanism of inhibition of apoptosis was detected by reactive oxygen species and mitochondrial superoxide staining.(5)Using IVIS small animal imaging instrument to observe the time-varying trend of fluorescence imaging after opening the blood-brain barrier of loaded drug and AAIB lipid PLGA hybrid vesicles and loaded AAIB lipid PLGA liybrid vesicles,TUNEL staining to detect apoptosis,DHE staining to detect the level of active oxygen in vivo.HE staining and blood routine examination were used to detect the toxic and side effects of loaded AAIB and AIB loaded PLGA hybrid vesiclesResults:(1)The results of Malvern particle size analyzer showed that the average particle size of non-target avidin-ieg-bsa fluorescent probe was 31.2nm The average particle size of Annexin V-avidin-icge-bsa is 20.3nm;TEM shows that the shape of probe is regular,the particle size is more uniform and spherical,the dispersion is better,and there is no adhesion and aggregation phenomenon;the results of fluorescence indexer show that the emission wavelengths of ICG in fluorescence probe are 795-845nm,and there is no shift.(2)The moephology of annexin v-avidin-icg-bsa-plga hybrid bubble was spherical,with a cavity inside and good dispersion.The results of Malvern laser particle size analyzer showed that the microbubbles were wrapped in annexin The particle size of MBs-Annexin V-Avidin-ICG-BSA and MBs-Avidin-ICG-BSA was 844.3±114.5nm,and there was no significant change.The lipophilic surface of DiO was observed under confocal laser scanning microscope.The probe uniformly dispersed inside the microbubbles,and fluorescence co localization revealed that the green fluorescence of DiO covered the red fluorescence of the probe.SDS-PAGE gel electrophoresis showed that the target microbubbles contained Annexin.V-targeted protein,but not targeted microbubbles,only avidin and BSA,and with the increase of probe input,the amount of microbubble loaded probes also increased,when the input reached 100ug,it reached saturation;IVIS small animal imaging instrument detection showed that the annexin v-avidin-icg-bsa and avidin icg-bsa lipid PLGA hybrid vesicles had good fluorescence imaging effect,and with the increase of concentration,the fluorescence signal became stronger.(3)The results of confocal laser microscopy show that it has a target annexin The fluorescence probe of AAIB and propidium iodide stained the necrotic cells and the cells in the late stage of apoptosis at the same time,but the non-target group was only stained by propidium iodide;PLGA heterozygous vesicles loaded with the target fluorescence probe were simultaneously blasted with 1.0MPa ultrasound energy and incubated for 20min,and the fluorescence intensity of apoptotic cells in the ultrasound blasted group was stronger than that in the non ultrasound group.(4)With the increase of ultrasound energy,EB exudation in brain parenchyma increased.At the same time,he staining showed that when the ultrasound energy was 0.6 and 0.8MPa,there was no obvious hemorrhage,but when the ultrasound energy was 1MPa,there was obvious hemorrhage.TUNEL showed that green fluorescent apoptotic bodies increased with the increase of ultrasound energy.The mathematical model shows that the net entry rate of the fluorescent probe in the targeted group and the non targeted group is 0.76 and 0.7 respectively,there is no significant difference,but after 3 hours,the net entry rate of the fluorescent probe in the targeted group and the non targeted group is 0.32 and 0.56 respectively.(5)With the increase of ultrasound energy and irradiation time,the activity of cells decreased.Drug therapy showed that gas,GSH and DMSO could increase the activity of cells,but DMSO with high concentration had toxic effect on cells.The morphology,particle size and dispersion of PLGA hybrid vesicles with and without drug loading were not significantly different from those without drug loading.After incubation at 37? for 4h,the apoptosis rate of MGAAIB group decreased from 27.8%to 17.3%.IVIS imaging results showed that the increase of brain fluorescence intensity in MGAAIB group was consistent with that in MAAIB group in the first three hours,and then the decrease of brain fluorescence intensity in MGAAIB group in the 12th hour was faster than that in MAAIB group,and there was significant difference in 12h and 16h,.TUNEL staining results showed that the total fluorescence intensity of apoptosis was consistent with IVIS imaging results,indicating that the drug can reverse apoptosis and reduce the retention of AAIB in the brain.DHE staining showed that the level of ROS in MGAAIB group was lower than that in MAAIB group,and the fluorescence intensity of AAIB and TUNEL was also decreased.Conclusion:In this study,we have successfully prepared a fluorescence probe targeting apoptosis and a lipid PLGA hybrid bubble loaded-with fluorescence probe.The fluorescence probe can detect apoptosis well,and the lipid PLGA hybrid bubble loaded with fluorescence probe can be released to detect apoptosis in vivo and in vitro after ultrasound blasting,and the anti apoptosis drug gas is wrapped in it,so that it has the function of inhibiting apoptosis while detecting apoptosis It provides a new perspective for the integration of focused ultrasound and microbubble to open blood-brain barrier,and promotes the transformation of this technology to clinical practice.