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.

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.

Enhancing effect of ultrasound-mediated microbubble destruction on gene delivery into rat kidney via different administration routes

Objective:To investigate the efficiency ofβ-galactosidase gene transfer into rat kidney with ultrasound-mediated microbubble destruction via different injection routes.Methods:A total of 25 Wistar rats were randomly divided into 5 groups.Four groups received a mixture of optison microbubbles(0.2 mL) and lacz plasmids(25μg) injection via renal artery,tail vein,anterior tibial muscle and renal parenchyma,respectively.The control group received a mixture of PBS (xx mL) and lacz plasmids(25μg) via renal artery.Three days after the gene transfer,ultrasound with fixed frequency and power(1 MHz,xxW) was delivered to the kidneys for 3 min.The efficiency of the gene transfer and expression was evaluated on the basis ofβ-galactosidase expression.The side effects of this method were evaluated by immunohistological method. Results:β-galactosidase expression could be observed only in tubules but not in glomeruli and interstitial area.The efficiency of renal artery group was higher than that of tail vein,anterior tibial muscle and renal parenchyma group(P【0.05).Immunohistochemical analysis revealed co-expression ofβ-galactosidase with a roximal tubule marker,megalin,which suggested that ultrasound enhanced gene transfer into the proximal tubular epithelial cells.Noβ-galactosidase expression was observed in the extrarenal organs.There were no evident pathological and biochemical changes after gene transfer.Conclusions:Ultrasound-mediated microbubble destruction can transfer gene into kidney via renal artery,tail vein,anterior tibial muscle and renal parenchyma.Compared with renal artery,administrating microbubbles via tail vein and anterior tibial muscle are more convenient and less vulnerarious.

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.

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.

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.

Investigation on the relationship between overpressure and sub-harmonic response from encapsulated microbubbles

Sub-harmonic component generated from microbubbles is proven to be potentially used in noninvasive blood pressure measurement. Both theoretical and experimental studies are performed in the present work to investigate the dependence of the sub-harmonic generation on the overpressure with different excitation pressure amplitudes and pulse lengths. With 4-MHz ultrasound excitation at an applied acoustic pressure amplitude of 0.24 MPa, the measured sub-harmonic amplitude exhibits a decreasing change as overpressure increases; while non-monotonic change is observed for the applied acoustic pressures of 0.36 MPa and 0.48 MPa, and the peak position in the curve of the sub-harmonic response versus the overpres- sure shifts toward higher overpressure as the excitation pressure amplitude increases. Furthermore, the exciting pulse with long duration could lead to a better sensitivity of the sub-harmonic response to overpressure. The measured results are ex- plained by the numerical simulations based on the Marmottant model. The numerical simulations qualitatively accord with the measured results. This work might provide a preliminary proof for the optimization of the noninvasive blood pressure measurement through using sub-harmonic generation from microbubbles.

Effect of secondary radiation force on aggregation between encapsulated microbubbles

Secondary radiation force can be an attractive force causing aggregates of encapsulated microbubbles in ultrasonic molecular imaging. The influence of the secondary radiation force on aggregation between two coated bubbles is investigated in this study. Numerical calculations are performed based on four simultaneous differential equations of radial and translational motions. Results show that the secondary force can change from attraction to repulsion during approach, and stable microbubble pairs can be formed in the vicinity of resonant regions; the possibility of microbubble aggregations can be reduced by using low exciting amplitude, ultrasonic frequencies deviating from the resonant frequencies or microbubbles with small compressibility.

Effect of CXCR4 pretreated with ultrasound-exposed microbubbles on accelerating homing of bone marrow mesenchymal stem cells to ischemic myocardium in AMI rats

Objective: To investigate the role and potential mechanism of CXCR4 in promoting targeted homing of bone marrow mesenchymal stem cells(BMSCs) with ultrasound-exposed microbubbles(UM) pretreatment. Methods: Third generation BMSCs were divided into four groups control group, ultrasound(US) group, UM group and ultrasound-exposed microbubbles plus catalase group. RT-PCR and western blot were performed to determine the levels of CXCR4 m RNA transcription and protein expression, respectively. Third generation BMSCs were labeled with Fluo-α/AM and divided into three groups: control group, US group and UM group, and flurorescence intensities in the cells were observed immediately, 5 min and 15 min after intervention underflurorescence microscope. The calcium iron levels in the cells were analyzed. BMSCs were divided into i ve group: group A without calcium in the medium, group B, group C, group D and group E containing calcium chloride with concentration of l mol, 2 mol, 4mol, anti-calciurn-sensing receptor antibody, respectively. RT-PCR and western blot were performed to determine the levels of CXCR4 m RNA transcription and proteins expression of the third generation BMSCs of each group, respectively. Results: The levels of CXCR4 m RNA transcription and protein expression between US group and control group had no statistically signii cant dif erence(P>0.05) shown by RT-PCR and western blot; the transcription level in the UM group was signii cantly higher than that in US group and control group(P<0.05); and in the ultrasound-exposed microbubbles plus catalase group, the transcription level was much lower than that in UM group. Fluorescence intensify in the cells of US group had no signii cant dif erence compared with that in the cells of the control group(P>0.05), which in the cells of UM group was signii cantly higher than that in the cells of both US group and control group(P<0.05). Compared to group A, expressions of CXCR4 of group B to D were signii cantly increased in concentration-dependent manner showed by RT-PCR and western blot(P< 0.05). Compared to group C, expressions of CXCR4 of group E were signii cantly decreased(P< 0.05). Conclusions: UM can promote the inl ux of calcium in BMSCs and increase m RNA transcription and protein expression of CXCR4. The latter may partly be caused by influx of calcium.

Difference-frequency ultrasound generation from microbubbles under dual-frequency excitation

The difference-frequency （DF） ultrasound generated by using parametric effect promises to improve detection depth owing to its low attenuation, which is beneficial for deep tissue imaging. With ultrasound contrast agents infusion, the harmonic components scattered from the microbubbles, including DF, can be generated due to the nonlinear vibration. A theoretical study on the DF generation from microbubbles under the dual-frequency excitation is proposed in formula based on the solution of the RPNNP equation. The optimisation of the DF generation is discussed associated with the applied acoustic pressure, frequency, and the microbubble size. Experiments are performed to validate the theoretical predictions by using a dual-frequency signal to excite microbubbles. Both the numerical and experimental results demonstrate that the optimised DF ultrasound can be achieved as the difference frequency is close to the resonance frequency of the microbubble and improve the contrast-to-tissue ratio in imaging.

Effect of Pressure on Intracardiac Backscatter from Microbubbles

The backscatter from sonicated albumin microbubbles (Albunex) was analyzed using acoustic densitometry in an in vitro pulsatile heart model to evaluate the effects of pressure on the backscatter from Albunex, and the cardiac cyclic changes of intracardiac backscatter from sonicated albumin microbubbles in 16 healthy persons were analyzed. It was found that the Albunex microbubbles were compressed in systole and decompressed in diastole, causing corresponding changes of backscatter in cardiac cycle. Although the intensities of backscatter in diastole and systole were related to the concentration of microbubbles, the concentration of microbubbles had no effect on the difference of end-diastolic and end-systolic backscatter. The difference of the backscatter was highly correlated with end-systolic pressure (r=0.96, P=0.001). In human studies, we also observed same intracardiac cyclic changes of backscatter from sonicated albumin microbubbles. Our study indicates that it is possible to evaluate the intracardiac pressure non-invasively by analyzing the intracardiac backscatter from the microbubbles with acoustic densitometry.

Ultrasound-triggered Microbubble Destruction in Combination with Cationic Lipid Microbubbles Enhances Gene Delivery

This study aimed to examine the preparation of cationic lipid microbubble（CLM）,and evaluate its physical and chemical properties and toxicity,measure the gene transfection efficiency by ultrasound triggered microbobble destruction（UTMD） in combination with CLM.The CLM was prepared by the method of the thin film hydration,and its morphology was observed under the electron microscopy at 1st,3rd,7th,10th,and 14th day after preparation,respectively.The size,Zeta potential and stability of CLM were tested.The acute toxicity of CLM was assessed.The green fluorescent protein gene（EGFP） transfection efficiency was evaluated.The experiment grouping was as follows：naked plasmid group（P group）,ultrasonic irradiation plus naked plasmid group（P-US group）,naked plasmid plus CLM group（P-CLM group）,naked plasmid plus ultrasound and CLM group（UTMD group）.The expression of EGFP was detected by fluorescent microscopy and flow cytometry.The results showed that CLMs were spherical in shape,with the similar size and good distribution degree under the light and electron microscopies.The size of CLMs was varied from 250.4±88.3 to 399.0±99.8 nm and the Zeta potential of CLMs from 18.80±4.97 to 20.1±3.1 mV.The EGFP expression was the strongest in the UTMD group,followed by the P-CLM group,P-US group and P group.Flow cytometry results were consistent with those of fluorescent microscopy.The transfection efficiency was substantially increased in the P-US group,P-CLM group and UTMD group as compared with that in the P group,almost 7 times,10 times and 30 times higher than that in the P group respectively.It is suggested that CLMs prepared by the method of thin film hydration are uniform in diameter,and proved non-toxic.UTMD combined with CLM can significantly increase the transfection efficiency of EGFP to targeted cells.

Preparation of Thrombosis-targeted Lipid Microbubbles and Determination of Rabbit Carotid Artery Thrombosis by Microbubbles Ultrasonogaphy

A kind of thrombus-targeted lipid-coated microbubbles were prepared, and the target prop- erty of the microbubbles and the effects of different methods detecting thrombosis in vessels were ob- served. Phospholipid-coated microbubbles were prepared by membrane-hydration method. Throm- bus-targeted lipid-coated fluorocarbon microbubbles were labeled with specific fluorescence and then integrated to the thrombus in vivo and ex vivo through an avidin biotin system. The thrombus was im- mediately observed for the distribution and property of the thrombus-targeted microbubbles under the optical microscope, fluorescence microscope and transmission electron microscope. The carotid throm- bosis models were set up in rabbits, and the effects of different methods detecting thrombosis in vessels were observed. The diameter of the phospholipid-coated microbubbles was 0.8-2.5μm, and even reached nanoscale in some of them. The zeta electric potential was about -11 mV and the concentration was about 1.08×10μmL. Immunofluorescence of rapid frozen sections in vivo and ex vivo showed that massive targeted lipid-coated microbubbles flocked around fresh blood clots and some aggregated within them under the light and fluorescence microscope. The number of aggregated microbubbles ex vivo was greater than that observed in the experiment in vivo, and the fluorescence observed in the ex- periment ex vivo was stronger than that in the experiment in vivo. The same imaging was observed un- der the electron microscope. Models of carotid thrombosis in rabbits were established successfully. Ef- fects of detecting thrombosis by means of thrombosis-targeted microbubble ultrasonoraphy and Sono Vue ultrasonography in vessels were more satisfactory than those by Color Doplor Flow Imaging （CDFI）, ordinary microbubbles and Three Dimensions-time of flight MR angiography （3D-TOF-MRA） （P〈0.01）. Compared to ordinary microbubbles ultrasonography, thrombosis-targeted microbubbles ultrasonography had the advantages whenever in imaging quality or in imaging time. Thrombus-targeted phospholipid-coated microbubbles were prepared successfully by membrane-hydration method. They could aggregate rapidly in fresh blood clots and enter deep into the internal part of the thrombus both in vivo and ex vivo, and had the targeted property of strongly conjugating with the thrombus. Compared to other thrombosis detection methods, ultrasonography with thrombosis-targeted microbubbles has obvious advantages in detecting thrombosis in vessels, mainly in： non-invasiveness, safety, good image quality, accuracy, and longer imaging time.

Effects of RNA Interference Combined with Ultrasonic Irradiation and SonoV ue Microbubbles on Expression of STAT3 Gene in Keratinocytes of Psoriatic Lesions

The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.

Multi-Layer Microbubbles by Microfluidics

Multi-layer microbubble has great potential in enabling the corporation of medical imaging with tumor therapy such as drug and gene delivery of therapeutics or other functional materials in medical applications. Microfluidic technique has advanced over the last decade and showed great promise in replacing traditional microbubble generating method. In this paper, a multi-layer microbubble structure was produced with the aspect as potentially used for drug loaded microbubble contrast agents.

Producing single microbubbles with controlled size using microfiber

Microbubble-mediated pore formation in sonoporation includes a combination of complex processes such as collision or coalescence of translating or collapsing microbubbles with vascular cells. Although understanding the pore formation mechanisms may improve drug delivery efficiency, their details are still poorly understood. In the present study, we describe an experimental model that produces single air bubbles with controllable size. A carbon microfiber in liquids is illuminated by an infrared laser to produce individual bubbles having size comparable to that of the microfiber. The microbubbles can be physically isolated from the fiber for placing at arbitrary positions in the liquids. The lifetime of the bubbles is several tens of minutes depending on the intensity of the laser used. The preparation of the controllable air bubbles may be useful in future investigations of ultrasound-mediated microbubble–cell interactions.

Ultrasound and Microbubbles: Their Functions in Gene Transfer In Vitro

To examine the role of ultrasound in gene delivery in vitro, three cells lines were exposed to the low-frequency ultrasound of varying intensities and for different durations to evaluate their effect on gene transfection and cell viability of the cells. Microbubble （MB）, Optison （10%）, was also used to observe the role of the microbubbles in gene transfection. The results demonstrated that as the ultrasound intensity and the exposure time increased, the gene transfer rate increased and the cell viability decreased, but at high energy intensities, the cell viability decreased dramatically, which caused the transfer rate to decrease. The most efficient ultrasound intensity for inducing gene transfer was 1 W/cm^2 with duration being 20 s. At the same energy intensity, higher ultrasound intensity could achieve maximal gene transfer rate earlier. Microbubbles could increase ultrasound-induced cell gene transfer rate by about 2 to 3 times mainly at lower energy intensities. Moreover, microbubbles could raise the maximum gene transfer rate mediated by ultrasound. It is concluded that the low-frequency ultrasound can induce cell gene transfer and the cell gene transfer rate and viability are correlated with not only the ultrasound energy intensity but also the ultrasound intensity, the higher ultrasound intensity achieves its maximal transfer rate more quickly and the ultrasound intensity that can induce optimal gene transfer is 1 W/cm^2 with duration being 20 s, and microbubbles can significantly increase the maximal gene transfer rate in vitro.

Ultrasound microbubbles combined with liposome-mediated pNogo-R shRNA delivery into neural stem cells

In the present study, ultrasound-mediated microbubble destruction （UMMD） alone and combined with liposome technology was used as a novel nonviral technique to transfect a Nogo receptor （Nogo-R） shRNA plasmid （pNogo-R shRNA） into neural stem cells （NSCs）. Using green fluorescent protein as a reporter gene, transfection efficiency of NSCs was significantly higher in the group transfected with UMMD combined with liposomes compared with that of the group transfected with UMMD or liposomes alone, and did not affect cell vitality. In addition, Nogo-R mRNA and protein expression was dramatically decreased in the UMMD combined with liposome-mediated group compared with that of other groups after 24 hours of transfection. The UMMD technique combined with liposomes is a noninvasive gene transfer method, which showed minimal effects on cell viability and effectively increased transfer of Nogo-R shRNA into NSCs.

Effects of ultrasound-combined microbubbles on hippocampal AchE fibers in rats

Objective:To investigate the protective effect of ultrasound-combined microbubbles on hippocampal acetylcholinesterase(AchE) fibers in rats.Methods:According to random digits table,60 SD rats were divided into two groups,marrow stromal cells(MSCs) intracranial transplantation group and MSCs intracranial transplantation + ultrasonic microbubbles group.Marrow stromal cells were cultivated and isolated in vitro;12 weeks after transplantation,spatial learning and memorizing abilities of rats were assessed by Morris water maze;AchE staining method was used to observe changes in density and appearance of AchE staining positive fibers in hippocampal CA1 region.Results:There was asignificant increase in spatial learning and memorizing abilities of rats in MSCs intracranial transplantation + ultrasonic microbubbles group.Hippocampal AchE staining suggested an increase in the density of AchE staining positive fibers in MSCs intracranial transplantation group;the fibers were regular,intact and dense.Density of hippocampal AchE positive fibers was negatively correlated with the escape latent period and was positively correlated with percentage of the time needed to cross each platform quadrant.Conclusions:Better promotion of spatial learning and memorizing abilities of rats in MSCs intracranial transplantation + ultrasonic microbubbles group may be related with the protective effect of ultrasound-combined microbubbles on hippocampal acetylcholine fibers.

Germination and appressorium formation of Pyricularia oryzae Cavara can be inhibited by reduced concentration of Blasin~? Flowable with carbon dioxide microbubbles

We investigated the possibility to reduce the usage of Blasin^Flowable （BF）, a disinfectant inhibiting the germination and appressorium formation of Pyricularia oryzae Cavara conidia, by using carbon dioxide microbubbles （CO2MB）. Germination was significantly inhibited by 10 000-fold diluted BF solution containing CO2MB generated by the decompression-type generator compared to CO2 millibubbles （CO2MMB） and CO2MB generated by the gas-water circulating-type generator. Appressodum formation in the 10 000-fold diluted BF solution containing both CO2MBs was less than that in CO2MMB. Scanning electron microscopy showed wrinkles and dents on the surface of conidia treated with 5 000-fold diluted BF solution containing both CO2MBs. Via transmission electron microscopy, we observed the expansion of the vacuole and the intracellular space and bloated or absent lipid granules in the conidia treated with BF solution containing both CO2MBs. Our results show that inhibition of the conidium germination and appressorium formation of P. oryzae Cavara by 10 000-fold diluted BF solution could be achieved by using the decompression-type CO2MB.