Long noncoding RNAs HAND2-AS1 ultrasound microbubbles suppress hepatocellular carcinoma progression by regulating the miR-873-5p/tissue inhibitor of matrix metalloproteinase-2 axis

BACKGROUND Increasing data indicated that long noncoding RNAs(lncRNAs)were directly or indirectly involved in the occurrence and development of tumors,including hepatocellular carcinoma(HCC).Recent studies had found that the expression of lncRNA HAND2-AS1 was downregulated in HCC tissues,but its role in HCC progression is unclear.Ultrasound targeted microbubble destruction mediated gene transfection is a new method to overexpress genes.AIM To study the role of ultrasound microbubbles(UTMBs)mediated HAND2-AS1 in the progression of HCC,in order to provide a new reference for the treatment of HCC.METHODS In vitro,we transfected HAND2-AS1 siRNA into HepG2 cells by UTMBs,and detected cell proliferation,apoptosis,invasion and epithelial-mesenchymal transition(EMT)by cell counting kit-8 assay,flow cytometry,Transwell invasion assay and Western blotting,respectively.In addition,we transfected miR-837-5p mimic into UTMBs treated cells and observed the changes of cell behavior.Next,the UTMBs treated HepG2 cells were transfected together with miR-837-5p mimic and tissue inhibitor of matrix metalloproteinase-2(TIMP2)overexpression vector,and we detected cell proliferation,apoptosis,invasion and EMT.In vivo,we established a mouse model of subcutaneous transplantation of HepG2 cells and observed the effect of HAND2-AS1 silencing on tumor formation ability.RESULTS We found that UTMBs carrying HAND2-AS1 restricted cell proliferation,invasion,and EMT,encouraged apoptosis,and HAND2-AS1 silencing eliminated the effect of UTMBs.Additionally,miR-873-5p targets the gene HAND2-AS1,which also targets the 3’UTR of TIMP2.And miR-873-5p mimic counteracted the impact of HAND2-AS1.Further,miR-873-5p mimic solely or in combination with pcDNA-TIMP2 had been transformed into HepG2 cells exposed to UTMBs.We discovered that TIMP2 reversed the effect of miR-873-5p mimic caused by the blocked signalling cascade for matrix metalloproteinase(MMP)2/MMP9.In vivo results showed that HAND2-AS1 silencing significantly inhibited tumor formation in mice.CONCLUSION LncRNA HAND2-AS1 promotes TIMP2 expression by targeting miR-873-5p to inhibit HepG2 cell growth and delay HCC progression.

Latest advances and progress in the microbubble flotation of fine minerals:Microbubble preparation,equipment,and applications

In the past few decades,microbubble flotation has been widely studied in the separation and beneficiation of fine minerals.Compared with conventional flotation,microbubble flotation has obvious advantages,such as high grade and recovery and low consumption of flotation reagents.This work systematically reviews the latest advances and research progress in the flotation of fine mineral particles by microbubbles.In general,microbubbles have small bubble size,large specific surface area,high surface energy,and good selectivity and can also easily be attached to the surface of hydrophobic particles or large bubbles,greatly reducing the detaching probability of particles from bubbles.Microbubbles can be prepared by pressurized aeration and dissolved air,electrolysis,ultrasonic cavitation,photocatalysis,solvent exchange,temperature difference method(TDM),and Venturi tube and membrane method.Correspondingly,equipment for fine-particle flotation is categorized as microbubble release flotation machine,centrifugal flotation column,packed flotation column,and magnetic flotation machine.In practice,microbubble flotation has been widely studied in the beneficiation of ultrafine coals,metallic minerals,and nonmetallic minerals and exhibited superiority over conventional flotation machines.Mechanisms underpinning the promotion of fine-particle flotation by nanobubbles include the agglomeration of fine particles,high stability of nanobubbles in aqueous solutions,and enhancement of particle hydrophobicity and flotation dynamics.

Effect of magnetic field on expansion of ferrofluid-encapsulated microbubble

Magnetic microbubbles(MMBs) have great potential applications in drug delivery and target therapy because they can be controlled by magnetic fields. In this paper, dynamic equations are derived by Lagrangian formalism and the behavior of MMBs subject to a combination field of magnetic and ultrasound field in an incompressible infinite fluid is analyzed numerically. The results show that the magnetic field can promote bubble expansion and hinder its translational motion,and both the enhancement and obstruction effects will weaken with the decrease of bubble size. The initial translational velocity has almost no effect on bubbles motion. Besides, the maximum expansion radius of MMBs increases with the ferrofluid shell thickness, while that of the common MBs is just the opposite. In addition, the periodic change of Levich viscous drag caused by the rebound leads to the step-like translational motion. Finally, the ferrofluid-shell model can be replaced by the model of non-magnetic microbubbles in magnetic liquid at high driving frequency.

Dynamics of magnetic microbubble transport in blood vessels

Magnetic microbubbles(MMBs)can be controlled and directed to the target site by a suitable external magnetic field,and thus have potential in therapeutic drug-delivery application.However,few studies focus on their dynamics in blood vessels under the action of magnetic and ultrasonic fields,giving little insight into the mechanism generated in diagnostic and therapeutic applications.In this study,equations of MMBs were established for simulating translation,radial pulsation and the coupled effect of both.Meanwhile,the acoustic streaming and shear stress on the vessel wall were also presented,which are associated with drug release.The results suggest that the magnetic pressure increases the bubble pulsation amplitude,and the translation coupled with pulsation is manipulated by the magnetic force,causing retention in the target area.As the bubbles approach the vessel wall,the acoustic streaming and shear stress increase with magnetic field enhancement.The responses of bubbles to a uniform and a gradient magnetic field were explored in this work.The mathematical models derived in this work could provide theoretical support for experimental phenomena in the literature and also agree with the reported models.

A new method for preparing a rat intracerebral hemorrhage model by combining focused ultrasound and microbubbles

Background:We aimed to prepare a non-invasive,reproducible,and controllable rat model of intracerebral hemorrhage with focused ultrasound(FUS).Methods:A rat intracerebral hemorrhage(ICH)model was established by combining FUS and microbubbles(μBs),and edaravone was used to verify whether the free radical scavenger had a protective effect on the model.The brain tissue of each group was sectioned to observe the gross histology,blood-brain barrier(BBB)permeability,cerebral infarction volume,and histopathological changes.Results:Compared with the FUS group,the BBB permeability was significantly increased in the FUS+μBs(F&B)group(p=0.0021).The second coronal slice in the F&B group had an obvious hemorrhage lesion,and the FUS+μBs+edaravone(F&B&E)group had smaller hemorrhage areas;however,ICH did not occur in the FUS group.The cerebral infarction volume in the F&B group was significantly larger than that in the FUS group(p=0.0030)and F&B&E group(p=0.0208).HE staining results showed that nerve fibrinolysis,neuronal necrosis,microglia production,and erythrocytes were found in both the F&B group and the F&B&E group,but the areas of the nerve fibrinolysis and neuronal necrosis in the F&B group were larger than the F&B&E group.Conclusions:A rat ICH model was successfully prepared using theμBs assisted FUS treatment,and edaravone had a therapeutic effect on this model.This model can be used to study the pathophysiological mechanism of ICH-related diseases and in preclinical research on related new drugs.

Antitumor effect of VEGFR2-targeted microbubble destruction with gemcitabine using an endoscopic ultrasound probe:In vivo mouse pancreatic ductal adenocarcinoma model

Background:Ultrasound-targeted microbubble destruction(UTMD)induces cellular inflow of drugs at low intensity,while high intensity eradicates tumor vessels.Since vascular endothelial growth factor receptor 2(VEGFR2)is highly expressed in pancreatic ductal adenocarcinoma(PDAC),VEGFR2-targeted microbubble(MB)might additionally increase the tissue specificity of drugs and thus improve antitumor effects.In addition,fixing the dual pulse intensity could maximize MB properties.This study evaluated the one-off(experiment 1)and cumulative(experiment 2)treatment effect of UTMD by regulating the dual pulse output applied to PDAC using VEGFR2-targeted MB.Methods:C57BL/6 mice inoculated with Pan-02 cells were allocated to five groups:VEGFR2-targeted MB+gemcitabine(GEM),VEGFR2-targeted MB,non-targeted MB+GEM,GEM,and control groups.After injection of GEM or GEM and either VEGFR2-targeted or non-targeted MB,UTMD was applied for several minutes at low intensity followed by high intensity application.In experiment 1,mice were treated by the protocol described above and then euthanized immediately or at the tumor diameter doubling time(TDT).In experiment 2,the same protocol was repeated weekly and mice were euthanized at TDT regardless of protocol completion.Histological analysis by CD31 and VEGFR2 staining provided microvascular density(MVD)and VEGFR2 expression along vessels(VEGFR2v)or intra/peripheral cells(VEGFR2c).Results:In experiment 1,TDT was significantly longer in the VEGFR2-targeted MB+GEM group compared to the non-targeted MB+GEM,GEM,and control groups,while the VEGFR2-targeted MB group showed no statistical significance.MVD and VEGFR2v in the immediate euthanasia was significantly lower in the VEGFR2-targeted MB+GEM and VEGFR2-targeted MB groups than other conditions.In experiment 2,the VEGFR2-targeted MB+GEM group produced significantly longer TDT than the GEM or control groups,whereas the VEGFR2-targeted MB group showed no significant difference.Histology revealed significantly reduced VEGFR2v and VEGFR2c in the VEGFR2-targeted and non-targeted MB+GEM groups,while only VEGFR2v was significantly less in the VEGFR2-targeted MB group.Conclusions:UTMD-mediated GEM therapy with the dual pulse application using VEGFR2-targeted MB substantially suppresses PDCA growth.

Effect of viscoelasticity on nonlinear vibration of single microbubble

Based on the Church-Hoff model, the nonlinear oscillations of a single encapsulated microbubble with a finite thickness shell are theoretically studied. The effects of viscoelasticity on radial oscillations and the fundamental and harmonic components are researched. The peaks of radial oscillations and magnitudes of power spectra of the fundamental and harmonic components all increase gradually with the shear modulus of shell varying from 0 to 10 MPa by an interval of 0. 1 MPa at the same shear viscosity, while they decrease as the shear viscosity increases from 0 to 1 Pa · s by an interval of 0. 01 Pa · s at the same shear modulus. The fluctuation ranges of subharmonic and ultraharmonic signals are much larger than both the fundamental and second harmonic components. It means that the effect of viscoelasticity on the subharmonic and ultraharmonic signals is greater than that on the fundamental and second harmonic components. So adjusting the viscoelasticity of the shell is a potential method to obtain a perfect microbubble contrast agent used for the subharmonic and ultraharmonic imaging. Four points with significant fundamental and harmonic components are chosen as an example： a shear viscosity of 0. 39 Pa · s with shear modulus of 3.9, 6. 6, and 8.6 MPa, respectively; a shear modulus of 6.6 MPa with a shear viscosity of 0.42 Pa · s.

Impact of the diameter of SonoVue microbubbles on binding characteristics of a new contrast agent targeted to choriocarcinoma cells in vitro

Objective:To evaluate the impact of the diameter of SonoVue microbubbles on binding characteristics, including the adhesion rate and stability, of a new contrast agent targeted to choriocarcinoma cells(JARs) in vitro, in order to establish a foundation to explore targeted ultrasound imaging for localization of tumor cell antigens and increase the early diagnostic rate for tumors.Methods:The objects were divided into three groups:the large microbubble group(n = 15), the middling microbubble group(n = 15) and the tiny microbubble group(n = 15).The rosette formation rate was counted.JARs were calculated by flow cytometry(FCM).The targeted contrast agent was prepared by mixing SonoVue microbubbles of different diam-eter with rabbit anti-human chorionic gonadotrophin(HCG) antibody.The binding rates of the targeted contrast agent to JARs before and after PBS rinse were analyzed.Results:The binding rate was significantly lower in the large microbubble group(61.7 ± 1.8)% than in the middling microbubble group(82.6 ± 4.5)% and the tiny microbubble group(91.3 ± 5.8)%(P < 0.05).The binding rates of different diameter microbubbles to JARs before and after PBS rinse were different.The middling microbubbles were the most stable ones, with the binding rate of(82.3 ± 4.5)% and(80.4 ± 3.9)% before and after PBS rinse(P > 0.05).The binding rates of the targeted microbubbles labeled with fluorescence to JARs were 68.6%, 81.3% and 89.3% in the large microbubble group, the middling microbubble group and the tiny microbubble group, respectively(P < 0.05).Conclu-sion:The binding capacity of the targeted SonoVue microbubbles to JARs is related to the diameter of the microbubble, which is determined by the shaking method before preparation.Modulating the diameter of SonoVue microbubbles may increase the binding rate and stability of targeted microbubbles to JARs, thus to improve the image of JARs.

Basic physical, acoustic properties, and in vitro ultrasound imaging enhancement of the lipid-coated microbubbles

Aim To research on a stable microbubble with good acoustic properties and excellent imaging enhancing effect, and to make it to be a promising agent for the enhancement of ultrasound imaging in the ultrasound diagnosis of cardiovascular system diseases. Methods The morphology, size and zeta potential of lipid-coated microbubbles （LCM）, the acoustic properties of backscatter, and the second harmonic scatter of LCM were determined. Furthermore, the relationship between the concentrations and the amplitude values of the second harmonic was investigated by testing the intensities of the second harmonic at different concentrations. The imaging effect of LCM was also studied in vitro. Results The mean diameter of LCM was 3.38 μm with 95% of the bubbles under 5 μm. The scatter signal generated by microbubbles was observed under different concentrations in the bistatic modes while the position of transmitting transducer and receiving transducer was orthogonal. The intensity of the second harmonic scatter fell with the decrease of microbubble concentration. The increase rate of the second harmonic amplitude values generated by the microbubbles versus that by physiological saline was linear with the natural logarithm of bubble concentrations. The LCM could enhance the ultrasound image of thrombus. Conclusion The LCM exhibited good physical state and acoustic properties, which could increase the imaging quality.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Nanobubble generation and its application in froth flotation (part I): nanobubble generation and its effects on properties of microbubble and millimeter scale bubble solutions

A special nanobubble generation system has been developed for evaluating the effect of nanobubble on froth flotation. In this study, an eight-factor five-level Central Composite Experimental Design was conducted for investigating eight important parameters governing the median size and the volume of nanobubbles. These process parameters included surfactant concentration, dissolved oxygen (O2) content, dissolved carbon dioxide gas (CO2) content, pressure drop in cavitation tube nozzle, <50 nm hydrophobic particle concentration, <50 nm hydrophilic particle concentration, slurry temperature and the time interval after nanobubble generation. The properties, stability and uniformity of nanobubbles were investigated. The study of the produced nanobubble’s effects on the characteristics of microbubble solutions and millimeter scale bubble solutions was performed in a 50.8 mm column.

Numerical simulation of a self-absorbing microbubble generator for a cyclonic-static microbubble flotation column

The Cyclonic-Static Microbubble Flotation Column (FCSMC) is currently a widely used, novel type of flotation device. The self-absorbing microbubble generator is the core component of this device. The structure of the microbubble generator directly influences flotation column performance by affecting bubble size and distribution as well as gas holdup in the column. However, the complicated flow inside the generator results in high R&D costs and difficulty in testing. Thus, the CFD software, FLUENT, was used to simulate the gas-liquid two-phase flow inside a self-absorbing microbubble generator. The effect of area ratio, a key structural parameter, was studied in detail. Critical flow-field parameters including velocity, turbulent kinetic energy, minimum static pressure and gas holdup were obtained. The simulation results demonstrate that the optimum area ratio is 3.

Clinical study of ultrasound and microbubbles for enhancing chemotherapeutic sensitivity of malignant tumors in digestive system

Objective： To explore the safety of ultrasound and microbubbles for enhancing the chemotherapeutic sensitivity of malignant tumors in the digestive system in a clinical trial, as well as its efficacy.Methods： From October 2014 to June 2016, twelve patients volunteered to participate in this study. Eleven patients had hepatic metastases from tumors of the digestive system, and one patient had pancreatic carcinoma. According to the mechanical index （MI） in the ultrasound field, patients were classified into four groups with MIs of 0.4, 0.6, 0.8 and 1.0. Within half an hour after chemotherapy, patients underwent ultrasound scanning with ultrasound microbubbles （SonoVue） to enhance the efficacy of chemotherapy. All adverse reactions were recorded and were classified in 4 grades according to the Common Terminology Criteria for Adverse Events version 4.03 （CTCAE V4.03）. Tumor responses were evaluated by the Response Evaluation Criteria in Solid Tumors version 1.1 criteria. All the patients were followed up until progression.Results： All the adverse reactions recorded were level 1 or level 2. No local pain occurred in any of the patients. Among all the adverse reactions, fever might be related to the treatment with ultrasound combined with microbubbles. Six patients had stable disease （SD）, and one patient had a partial response （PR） after the first cycle of treatment. At the end of follow-up, tumor progression was restricted to the original sites, and no new lesions had appeared.Conclusions： Our preliminary data showed the potential role of a combined treatment with ultrasound and microbubbles in enhancing the chemotherapeutic sensitivity of malignant tumors of the digestive system. This technique is safe when the MI is no greater than 1.0.

Ultrasound-triggered microbubble destruction enhances the radiosensitivity of glioblastoma by inhibiting PGRMC1-mediated autophagy in vitro and in vivo

Background:Ultrasound-triggered microbubble destruction(UTMD) is a widely used noninvasive technology in both military and civilian medicine,which could enhance radiosensitivity of various tumors.However,little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism.This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy.Methods:GL261,U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation(IR) or IR plus UTMD.Autophagy was observed by confocal microscopy and transmission electron microscopy.Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1(PGRMC1),light chain 3 beta 2(LC3B2) and sequestosome 1(SQSTM1/p62) levels.Lentiviral vectors or siRNAs transfection,and fluorescent probes staining were used to explore the underlying mechanism.Results:UTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo(P<0.01).UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells.Suppression of autophagy by 3-methyladenine,bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells(P<0.05).Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression(P>0.05).Furthermore,UTMD inhibited PGRMC1expression and binding with LC3B2 in IR-exposed glioblastoma cells(P<0.01).PGRMC1 inhibitor AG-205 or PGRMC1siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells,thereby promoting UTMD-mediated radiosensitization(P<0.05).Moreover,PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation,subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells.Finally,compared with IR plus UTMD group,PGRMC1 overexpression significantly increased tumor size [(3.8±1.1) mm^(2)vs.(8.0±1.9) mm^(2),P<0.05] and decreased survival time [(67.2±2.6) d vs.(40.0±1.2) d,P=0.0026] in glioblastoma-bearing mice.Conclusions:UTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.

Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent

Objective: To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122 a plasmids into nonsmall cell lung cancer A549 cells. Methods: Antisense miRNA-224 and miRNA-122 a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively. Results: The expression of mi RNA-224 decreased and the expression of miRNA-122 a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group(P<0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122 a were inhibited, and the differences were significant as compared with the control group(P < 0.05). Besides, the inhibition of miRNA-122 a group was the most significant and there was statistically significant difference as compared with miRNA-224 group(t =-4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group(P < 0.05). What's more, the apoptotic peak appeared in miRNA-122 a group. Its invasion ability decreased most obviously(40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups(P < 0.05). Conclusions: A549 cells transfected by ultrasound microbubble-mediated antisense miRNA-224 and mi RNA-122 a plasmids possessed good transfection efficiency. The cell growth, invasion and colony forming abilities of transfected A549 cells were suppressed, which laid a solid foundation for the gene therapy of non-small cell lung cancer.

Comparison of Microbubble and Air Layer Injection with Porous Media for Drag Reduction on a Self-propelled Barge Ship Model

Ship resistance issues are related to fuel economy,speed,and cost efficiency.Air lubrication is a promising technique for lowering hull frictional resistance as it is supposed to modify the energy in the turbulent boundary layer and thereby reduce hull friction.In this paper,the objective is to identify the optimum type of air lubrication using microbubble drag reduction(MBDR)and air layer drag reduction(ALDR)techniques to reduce the resistance of a 56-m Indonesian self-propelled barge(SPB).A model with the following dimensions was constructed:length L=2000 mm,breadth B=521.60 mm,and draft T=52.50 mm.The ship model was towed using standard towing tank experimental parameters.The speed was varied over the Froude number range 0.11–0.31.The air layer flow rate was varied at 80,85,and 90 standard liters per minute(SLPM)and the microbubble injection coefficient over the range 0.20–0.60.The results show that the ship model using the air layer had the highest drag reduction up to a maximum of 90%.Based on the characteristics of the SPB,which operates at low speed,the optimum air lubrication type to reduce resistance in this instance is ALDR.

In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

Overexpression of the neuroglobin gene delivered by ultrasound-targeted microbubble destruction protects SH-SY5Y cells against cobalt chloride induced hypoxia

In this study, we examined the effects of neuroglobin gene （Ngb） transfection into SH-SY5Y cells, using ultrasound-targeted microbubble destruction （UTMD）, on cobalt chloride-induced hypoxia. With an ultrasound intensity of 0.8 W/cm2, a 60-second exposure duration, 50% duty cycle, and 20% microbubble concentration, pAcGFP1-C1-Ngb-transfected cells exhibited the highest cell viability and transfection efficiency. The efficiency of plasmid delivery was significantly higher with UTMD than transfection with plasmid alone, transfection with plasmid using microbubbles, or transfection of plasmid by ultrasound. In addition, during cobalt chloride-induced hypoxia, caspase-3 activity in pAcGFP1-C1-Ngb-transfected cells was significantly lower than in untransfected cells. Ngb protein and mRNA expression were significantly higher in cells transfected by UTMD than in cells transfected with the other methods. These results demonstrate that UTMD can very efficiently mediate exogenous gene delivery, and that Ngb overexpression protects cells against cobalt chloride-induced hypoxia.

Efficient Gene Delivery to Myocardium with Ultrasound Targeted Microbubble Destruction and Polyethylenimine

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein （EGFP） gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction （UTMD） and polyethylenimine （PEI）. SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.

Microscopic study of ultrasound-mediated microbubble destruction effects on vascular smooth muscle cells

Objective:To observe vascular smooth muscle cell morphological changes induced by ultrasound combined with microbubbles by Atomic Force Acoustic Microscopy(AFAM).Methods:A7r5 rat aortic smooth muscle cells were divided into groups:control group(without ultrasonic irradiation,no micro bubbles)and US+MB group(45 kHz、0.4 W/cm^2 ultrasound irradiate for 20 seconds with a SonoVuc^(TM)concentration of[(56-140)×10~5/mL].Cell micromorphological changes(such as topographic and acoustic prognosis)were detected,before and after ultrasound destruction by AFAM.Results:In cell morphology,smooth muscle cells were spread o and connected to each another by fibers.At the center of the cell,the nuclear area had a rough surface and was significantly elevated from its surroundings.The cytoskeletal structure of the reticular nucleus and cytoplasm in the morphology of A7r5 cells(20μm×20μm)were clear before microbubble intervention.After acoustic exciting,the cell structure details of the acoustic image were improved with better resolution,showing the elasticity of different tissues.In the acoustic image,the nucleus was harder,more flexible and uneven compared with the cytoplasm.Many strong various-sized echo particles were stuck on the rough nuclear membrane's substrate surface.The nuclear membrane did not have a continuous smooth surface;there were many obstructions(pores).After ultrasound-intervention was combined with microbubbles,the dark areas of the A7r5 cell images was increased in various sizes and degrees.The dark areas showed the depth or low altitudes of the lower regions,suggesting regional depressions.However,the location and scope of the acoustic image dark areas were not similar to those found in the topographic images.Therefore,it was likely that the dark areas,both from the topographic and acoustic images,were sound-holes.In addition,some cell nuclei become round in different degrees after irradiation.Conclusions:Atomic force microscopy and acoustic excitation method can noninvasively and completely display a cell's structure,connections and elastic properties at a nano scale in just several minutes.The dark areas,both from the topographic and acoustic images,may be sound-holes;therefore,it would be helpful if these sound-holes were found.These findings provide a relationship between cell apoptosis after ultrasound and microbubble ultrasound irradiation,and the sound-hole effect.