Downregulation Of P2X3 Receptor In The Carotid Body For Reducing Blood Pressure By Acoustic Gene Target-delivery In Canines

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CATIONIC LIPID MICROBUBBLES PREPARATION AND CHARACTERIZATIONObjectives:We aimed to prepare the cationic microbubbles(CMBs)and detect its concentration,particle-size distributions and zeta potentials,estimate the acoustic activity of CMBs in vitro and in vivo.The optimum working parameters of low-intensity focused ultrasound(LIFU)was estimated to provide the experimental foundation for ultrasound-mediated gene delivery.Methods:DPPC,DSPE-PEG2000 and DC-Chol were mixed for preparation of CMBs using membrane hydration-mechanical oscillation method.C3F8 gas was used to fill the microbubbles.CMBs labeled with DIO were also prepared to better observe the surface physical characteristics of the microbubbles.The morphology and distribution of the microbubbles were observed by light microscope,transmission electron microscope(TEM)and laser confocal microscope,the concentration of the microbubbles was detected by a particle-counting device,and the size distributions and zeta potentials were detected by a dynamic light scattering-measurement device.The acoustic activity of CMBs and optimum working parameters of LIFU were estimated in the 3%agarose gel.The location of the carotid body and contrast-enhanced ultrasound imaging of the common carotid artery were detected using the high-frequency ultrasound probe.Results:CMBs are gas-filled particles consisting of a C3F8 gas core and a stabilized liposome shell.The CMBs were uniform in size and distribution observed by optical microscope and confocal microscope.The TEM images of CMBs clearly showed that the CMBs were spherical and well dispersed.The size distribution and zeta potentials were 926.6±24.5nm and 28.2±3.2mv,respectively.The average concentration of CMBs was(6.3±0.8)×10~8/ml,as determined with a hemocytometer.The average depth of the carotid body area was 2.95±0.17cm,the contrast-enhanced ultrasound imaging of the common carotid artery was clearly detected by high-frequency ultrasound probe.The optimum acoustic parameters of LIFU were:focal length 2.8±15%cm,6w/cm2,focal region 3.30×2.59×10.58mm~3,duty cycle 50%,irradiation time,2min,respectively.Conclusion:The zeta potentials of CMBs could create strong electrostatic interactions between microbubbles and nucleic acids leading to their protection against nucleases degradation and high concentration within the target tissue.CMBs was stable enough to withstand nucleic acids interaction.The good size distribution for in vivo administration and enough acoustic activity to be detected by echography could provide advantages for ultrasound-mediated microbubbles targeted gene delivery.PART ? LOW-INTENSITY FOCUSED ULTRASOUND MEDIATED TARGETED GENE DELIVERY FOR DOWNREGLATION THE P2X3 RECEPTOR OF CAROTID BODY IN CANINESObjectives: The purpose of this study was to explore a feasibility approach of targeted downregulating P2X3 receptor in CBs,providing a noninvasive and repeatable modulation strategy of CBs for hypertension therapy via ultrasound-mediated gene delivery technique.Methods: P2X3-g RNA plasmid was produced using the CRISPR/Cas9 system.P2X3-g RNA plasmid and cationic microbubbles were mixed to prepare plasmid-loaded microbubbles.Laser confocal microscopy was used to observe the Cy5-labeled-plasmid loaded microbubbles cationic,and flow cytometry was used to analyze the binding rate of plasmid and microbubbles.Canines with a baseline systolic BP(SBP)above 140 mm Hg and a diastolic BP(DBP)above 90 mm Hg were eligible for our study and were randomly divided into four groups,including canines(i)injected with cationic microbubbles(CMBs)loaded with a P2X3 guide RNA(g RNA)via LIFU(treatment group,n = 14),(ii)CMBs loaded with a negative-control plasmid via LIFU(negative-control group,n = 10),(iii)CMBs-only via LIFU(LIFU + CMBs group,n = 4),and(iv)a LIFU-only treatment group(LIFU group,n = 4).The CB area was located with a 4–13 MHz probe with B-mode ultrasonic imaging.15 ml of treatment mixture was injected intravenously into each CB,and contrast-agent images were monitored in contrast-enhanced mode.The LIFU transducer was placed on the target area and used to deliver exposures with a transmission frequency of 650 k Hz,a pulsed ultrasonic power of 6.0 W/cm2,and a 50% duty cycle,with a focal distance of 2.80 ± 15% cm,until the microbubbles were invisible in ultrasonic images.The transfection efficiency was analyzed by GFP positive cells using a flow cytometer,insertion and deletion(indel)rate was detected by T7 Endonuclease I(T7EI)assay,gene mutation site was assessed by Sanger sequencing.m RNA expression level of P2X3 receptor was determined by RT-q PCR,protein expression level of P2X3 receptor was detected by Western Blot.Results: Confocal microscopy showed that cationic microbubbles could intake of P2X3-g RNA plasmid,and flow cytometric analysis showed that the binding rate of plasmid and microbubbles was 74.99%.High-frequency ultrasound probe could accurately locate the CB area and assist in LIFU-mediated gene delivery.The 4ml microbubbles at a concentration of 1*108/ml were continuously enhanced in the common carotid artery for about 2min.There were significant differences in m RNA and protein expression levels of P2X3 receptor between high-blood group and non-hypertensive canines(P < 0.05).Flow cytometry showed that 33.15% of transfected cells expressed the green fluorescent protein reporter gene.T7 endonuclease I assays showed an insertion-deletion rate of 8.30%.The P2X3 receptor m RNA-and protein-expression levels in CBs decreased by 56.31% and 45.10%,respectively,in the treatment group.The gene mutation site was detected by Sanger sequencing.Compared with the treatment group,there was no statistically difference in LIFU group,LIFU+CMBs group and negative control group(P > 0.05).Conclusion: The expression level of P2X3 receptor in hypertensive canines CB is significantly higher than that in non-hypertensive canines,which is correlated with CB activity.P2X3 receptor is a promising target of CB modulation for hypertension therapy.LIFU-mediated gene delivery technique was a feasible strategy to targeted downregulation of the P2X3 receptor in CBs,providing a noninvasive and repeatable modulation strategy of CBs for hypertension therapy.PART ? EFFICACY AND SAFETY EVALUATION OF BLOOD PRESSION REDUCTION VIA LOW-INTENSITY FOCUSED ULTRASOUND-MEDIATED P2X3-GRNA DELIVERY TO CAROTID BODY IN CANINEObjectives: To investigate the effect and safety of target down-regulating P2X3 receptor expression in carotid body(CB)to reduce blood pressure via low-intensity focused ultrasound(LIFU)-mediated P2X3-g RNA plasmid delivery.Methods: Baseline blood pressure of canines was measured by femoral artery intubation after anesthesia,and canines with a systolic/diastolic blood pressure?140/90 mm Hg were included and randomly divided into 4 groups,including canines(i)injected with cationic microbubbles(CMBs)loaded with a P2X3 guide RNA(g RNA)via LIFU(treatment group,n = 14),(ii)CMBs loaded with a negative-control plasmid via LIFU(negative-control group,n = 10),(iii)CMBs-only via LIFU(LIFU + CMBs group,n = 4),and(iv)a LIFU-only treatment group(LIFU group,n = 4).Blood pressure and ECG were monitored by Multichannel Electrophysiology Management System.The mean systolic and diastolic blood pressures were analyzed based on the blood pressure waveforms.The LF,HF and LF/HF of heart rate variability were analyzed using frequency domain method.The protein expression level of P2X3 receptor was detected by Western Blot and immunohistochemistry.The safety of CMBs injection and LIFU irradiation was assessed using blood sample and histology.Results: There was no statistically difference in baseline blood pressure between LIFU group,LIFU + CMBs group,negative control group and treatment group(P > 0.05).After LIFU-mediated gene-delivery treatment,the m SBP in the treatment group decreased from the baseline to day 14(152.5 ± 3.0 versus 138.0 ± 2.9 mm Hg;P= 0.003).In addition,the m DBP also decreased on day 14(97.8 ± 1.5 versus 87.2 ± 2.3 mm Hg,P= 0.002).Compared with the basal level,during the 14-day experimental period,the LF/HF ratio significantly decreased from 3.3 ± 0.1 to 1.7 ± 0.1 in the treatment group(P<0.001),the LF decreased from 71.8 ± 1.4 nu to 57.0 ± 2.4 nu,but the HF increased from 22.4 ± 1.2 nu to 34.5 ± 1.9 nu in the treatment group(both P<0.001).Reduction in heart rate also was observed during the 14 days(P<0.05).Compared with the treatment group,the m SBP,m DBP,heart rate and heart rate variability values showed no significant changes in the other three groups.Immunohistochemistry and Western Blot showed that the P2X3 receptor expression was deceased.The results of biochemical measurements and routine blood examinations were all within the normal physiological ranges and showed no significant statistically significant changes during the 14 days after CMB injection.Histology of CB showed no tissue thermal damage.Conclusion: Targeted downregulation of P2X3 receptor expression in CBs could reduce BP without structural and functional impairment of the CB,indicating LIFU-mediated gene delivery was a non-invasive and effective technique for modulating CB function,proving a safe and feasible strategy for BP reduction in a canine model.