Research progress in micro/nanobubbles for ultrasound diagnosis or treatment / 药学学报

In the past few decades, microbubbles were widely used as ultrasound contrast agents in the field of tumor imaging. With the development of research, ultrasound targeted microbubble destruction technology combined with drug-loaded microbubbles can achieve precise drug release and play a therapeutic role. As a micron-scale carrier, microbubbles are difficult to penetrate the endothelial cell space of tumors, and nano-scale drug delivery system—nanobubbles came into being. The structure of the two is similar, but the difference in size highlights the unique advantages of nanobubbles in drug delivery. Based on the classification principle of shell materials, this review summarized micro/nanobubbles used for ultrasound diagnosis or treatment and discussed the possible development directions, providing references for the subsequent development.

Advantages of contrast-enhanced ultrasound in the localization and diagnostics of sentinel lymph nodes in breast cancer / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Sentinel lymph nodes (SLNs) are the first station of lymph nodes that extend from the breast tumor to the axillary lymphatic drainage. The pathological status of these LNs can predict that of the entire axillary lymph node. Therefore, the accurate identification of SLNs is necessary for sentinel lymph node biopsy (SLNB) to replace axillary lymph node dissection (ALND). The quality of life and prognosis of breast cancer patients are related to proper surgical treatment after the precise identification of SLNs. Some of the SLN tracers that have been identified include radioisotope, nano-carbon, indocyanine green (ICG), and methylene blue (MB). However, these tracers have certain limitations, such as pigmentation, radiation dangers, and the requirement for costly detection equipment. Ultrasound contrast agents (UCAs) have good specificity and sensitivity, and thus can compensate for some shortcomings of the mentioned tracers. This technique is also being applied to SLNB in patients with breast cancer, and can even provide an initial judgment on SLN status. Contrast-enhanced ultrasound (CEUS) has the advantages of high distinguishability, simple operation, no radiation harm, low cost, and accurate localization; therefore, it is expected to replace the traditional biopsy methods. In addition, it can significantly enhance the accuracy of SLN localization and shorten the operation time.

Experimental study on high-frequency subharmonic scattering characteristics of ultrasound contrast agent microbubbles under low ambient pressure / 生物医学工程学杂志

Correlation between nonlinear subharmonic scattering of ultrasound contrast agent microbubbles and ambient pressure is expected to be used for local brain tissue pressure monitoring. Although high-frequency ultrasound has achieved high-resolution imaging of intracranial microvessels, the research on high-frequency subharmonic scattering characteristics of microbubbles is insufficient at present, which restricts the research progress of estimating local brain tissue pressure based on high-frequency subharmonic scattering of microbubbles. Therefore, under the excitation of 10 MHz high-frequency ultrasound, the effects of different acoustic pressures and ambient pressures on the high-frequency subharmonic scattering characteristics of three different ultrasound contrast agents including SonoVue, Sonazoid and Huashengxian were investigated in this in vitro study. Results showed that the subharmonic scattering amplitudes of the three microbubbles increased with the increase of ambient pressure at the peak negative acoustic pressures of 696, 766 and 817 kPa, and there was a favorable linear correlation between subharmonic amplitude and ambient pressure. Under the above three acoustic pressures, the highest correlation coefficient of SonoVue was 0.948 ( P = 0.03), the highest sensitivity of pressure measurement was 0.248 dB/mm Hg and the minimum root mean square error (RMSE) was 2.64 mm Hg. Sonazoid's highest correlation coefficient was 0.982 ( P < 0.01), the highest sensitivity of pressure measurement was 0.052 dB/mm Hg and the minimum RMSE was 1.51 mm Hg. The highest correlation coefficient of Huashengxian was 0.969 ( P = 0.02), the highest sensitivity of pressure measurement was 0.098 dB/mm Hg and the minimum RMSE was 2.00 mm Hg. The above in vitro experimental results indicate that by selecting ultrasound contrast agent microbubbles and optimizing acoustic pressure, the correlation between high-frequency subharmonic scattering of microbubbles and ambient pressure can be improved, the sensitivity of pressure measurement can be upgraded, and the measurement error can be reduced to meet the clinical demand for local brain tissue pressure measurement, which provided an important experimental basis for subsequent research in vivo.

Research Progress of Targeted Ultrasound Contrast Agent BR55 / 中国医学科学院学报

BR55 is an ultrasound contrast agent targeting vascular endothelial growth factor receptor 2,which can be used to detect tumor neovascularization and improve the diagnostic accuracy.Overseas researchers have used BR55 for human ultrasound molecular imaging,which showed good safety and tolerance.We reviewed the research progress on BR55 applied in the evaluation of tumor neovascularization from the composition,characteristics,animal experiments,and clinical studies of BR55.

A new method for isolating gas vesicles from Microcystis for ultrasound contrast / 生物工程学报

Gas vesicles are a unique class of gas-filled protein nanostructures which are commonly found in cyanobacteria and Halobacterium. The gas vesicles may scatter sound waves and generate harmonic signals, which enabled them to have the potential to become a novel ultrasound contrast agent. However, the current hypertonic cracking method for isolating gas vesicles contains tedious operational procedures and is of low yield, thus not suitable for large-scale application. To overcome these technical challenges, we developed a rapid and efficient method for isolating gas vesicles from Microcystis. The new H2O2-based method increased the yield by three times and shortened the operation time from 24 hours to 7 hours. The H2O2 method is not only suitable for isolation of gas vesicles from laboratory-cultured Microcystis, but also suitable for colonial Microcystis covered with gelatinous sheath. The gas vesicles isolated by H2O2 method showed good performance in ultrasound contrast imaging. In conclusion, this new method shows great potential for large-scale application due to its high efficiency and wide adaptability, and provides technical support for developing gas vesicles into a biosynthetic ultrasonic contrast agent.

Experimental studies for noninvasive assessment of portal vein pressure based on contrast enhanced subharmonic sonographic imaging / 生物医学工程学杂志

Portal hypertension (PHT) is a common complication of liver cirrhosis, which could be measured by the means of portal vein pressure (PVP). However, there is no report about an effective and reliable way to achieve noninvasive assessment of PVP so far. In this study, firstly, we collected ultrasound images and echo signals of different ultrasound contrast agent (UCA) concentrations and different pressure ranges in a low-pressure environment based on an

The clinical application of microbubble contrast agent in high intensity focused ultrasound treatment for uterine fibroids / 介入放射学杂志

Objective To investigate the clinical utility of SonoVue,a microbubble-enhancing contrast agent,in the treatment of uterine fibroids carrying different signal intensities on T2WI with high-intensity focused ultrasound (HIFU).Methods Based on the preoperative MRI signal intensity on T2WI,a total of 64 patients with uterine fibroids,who were scheduled to receive HIFU,were divided into low-intensity group (n =24),iso-intensity group (n =22) and high-intensity group (n =18).MRI check-up examination was performed one day after HIFU treatment to evaluate the ablation effect.The parameters related to HIFU,including therapeutic power,irradiation time and therapeutic dose,and the indexes related to therapeutic effect,including volume ablation rate (non-perfusion volume ratio,NPVR),energy-efficiency factor,treatment time,were recorded,and the results were compared between each other among the three groups.Results In the low-intensity group,iso-intensity group and high-intensity group,the volume ablation rates were (84.83±18.49)％,(8.72±17.76)％ and (71.11±23.87)％ respectively,the energy-efficiency factors were (6.87±7.77) J/mm3 (7.99±6.58) J/mm3 and (12.93±9.38) J/mm3 respectively,the treatment time were (102.12±54.45) min,(153.86±66.04) min and (141.50±69.56) min respectively.Single factor analysis indicated that statistically significant differences in volume ablation rate,energy-efficiency factor and treatment time existed between each other among the three groups.Among the total 64 patients,3 patients developed lower abdominal pain (6/64,9.4％),3 patients complained of general aches with numb (3/64,4.7％),and no severe complications,such as skin burn in treatment area,etc.,occurred in all patients.Conclusion The curative effect of SonoVue combined with HIFU for low-intensity and iso-intensity uterine fibroids is better than that for high-intensity uterine fibroids;the treatment time for low-intensity uterine fibroids is shorter than that for iso-intensity and high-intensity uterine fibroids.SonoVue is a safe and effective synergist for HIFU treatment.

An application study on assessment for endometrial receptivity by detecting endom- etrial and sub-endometrial perfusion with contrast-enhanced ultrasound / 中国医学装备

Objective:To discuss the endometrial and sub-endometrial perfusion by using contrast-enhanced ultrasound (CEUS) so as to assess the clinical value of endometrial receptivity.Methods: 86 cases, that were divided into healthy group (49 healthy females) and infertility group (37 patients with infertility), were enrolled in the research. All of cases received detection of color doppler flow imaging (CDFI) and CEUS per vaginam at later period of proliferation, ovulatory period and window phase of implantation, respectively. In these cases, 20 healthy females and 18 patients received endometrial biopsy. The microvessel density (MVD) and time intensity curve (TIC) were analyzed.Results: At the later period of proliferation, the endometrial and sub-endometrial perfusion of the healthy females were significantly more abundant than that of the infertility females as the results of CDFI (x2=4.575,P<0.05). As the results of biopsy for later period of proliferation and ovulatory period, the endometrial MVD of infertility females were significantly lower than that of healthy females (t=7.821, t=8.659,P<0.05). As the results of CEUS for later period of proliferation and ovulatory period, the endometrial and sub-endometrial perfusion peak intensity (Pi) value and area under the curve (AUC) value of healthy females were significantly higher than that of infertility females (t=8.004,t=1.269,t=6.581,t=6.759,P<0.05). Besides, the sub-endometrial Pi were positive correlation with MVD at healthy group and infertility group, respectively.Conclusion:The CEUS can assess the endometrial receptivity through accurately detected endometrial and sub-endometrial perfusion. Therefore, it is worthy to be popularized in clinical practice.

The formulation and characterization of near infrared-mediated size reversible ultrasound contrast agent / 药学学报

The drug-loaded ultrasound (US) contrast nanoparticles, which can effectively accumulate in the tumor to penetrate into its deep section, were prepared. After being heated or under the near infrared (NIR) light irradiation, the size of nanoparticles would transform from nanometer-scale to micrometer-scale in vitro, which can vastly enhance the effect of US imaging. We evaluated the size changes of the nanoparticles in vitro, investigating their effect in ultrasound imaging and distribution in vivo. Liposomes containing hydrophobic modified hollow gold nanospheres (HAuNS), doxorubicin (DOX) and perfluorohexane (PFH), which were referred to DOX and HAuNS loaded PFH liposome (DHPL), were prepared by thin film evaporation and ultrasonic technique. The morphology and size of DHPL were measured by transmission electron microscopy and particle size analyzer with dynamic light scattering (DLS) method. The agar gel pore model was used to investigate the enhanced effect of nanoparticles in vitro US imaging under the NIR light irradiation. The biodistribution of DHPL in 4T1 tumor-bearing mice after intravenous injection was measured by the in vivo imaging system. The DHPL were spherical at a particle size of 302 ±5 nm and polydispersity index of 0.195 ±0.018. The HAuNS loaded on phospholipid membrane was observed in transmission electron microscope (TEM) image. Under the NIR light irradiation (1 or 2 W·cm-2), the temperature of the solution containing the DHPL (0.2, 0.04, 0.02 g·L-1 in terms of HAuNS) rose rapidly. And a certain amount of micrometer-sized particles could be detected by the particle size analyzer when the temperature of the analyzer was raised to 52℃. The abundant microbubbles, which would enhance the effect of US imaging, were detected by ultrasonic diagnostic apparatus when the nanoparticles were irradiated by NIR light in the in vitro US imaging experiment. The in vivo distribution experiment showed that the DHPL could effectively accumulate in the tumor due to the enhanced permeability and retention effect (EPR effect) of the tumor. In this study, we successfully made a nanometer-micrometer reversible nanoparticles that can accumulate inside the tumor to provide a feasible scheme for US imaging in the tumor site and the combinational photothermal-chemotheraphy simultaneously.

Application of Sulfur Hexafluoride Micro-bubble in the Treatment of T2WI High Signal Uterine Fibroids with High Intensity Focused Ultrasound / 昆明医科大学学报

Objective To evaluate clinical value of ultrasound contrast agent the sulfur hexafluoride micro-bubble in the treatment of T2WI high signal uterine fibroids with high intensity focused ultrasound (HIFU).Methods A total of 36 patients with T2WI high signal uterine fibroids were randomly divided into experimental group and control group.Eighteen patients in experimental group received HIFU treatment with sulfur hexafluoride micro-bubble before,during and after the operation while another eighteen in control group received HIFU treatment with sulfur hexafluoride micro-bubble only after the operation.We recorded operation time (min),changing time of massive gray (s),irradiation time (s),therapeutic dose (J),incidence of complications and ablation rate (％).Results Operation time,changing time of massive gray,irradiation time,and therapeutic dose in experimental group were (101.51±43.02) min,(856.42±451.14) s,(1077.37±546.23) s,and (496581.81 ± 267192.31) J,respectively while those in control group were (261.34 ± 85.53) min,(1 833.32 ± 642.67) s,(1 890.21 ± 1 268.43) s,and (784 608.31 ± 357 621.45) J,respectively.Significant differences were found among those parameters between the two groups (P ＜0.05).Tumor ablation rate in experimental group was 83.3％ while that in control group was 55.5％,which was of significant difference between the two groups (P＜0.05).One case of muscular stiffness was found in experimental group while one case of second-degree burn and two cases of muscular stiffness were found in the control group.The incidence of complications was 1/18 (5.5％).No severe complication like bowel perforation or bladder perforation was found in the two groups.Conclusion The effect of ultrasound contrast agent sulfur hexafluoride micro-bubble can shorten operation time,improve tumor ablation rate and reduce complications and it can play a guiding role in clinical work.

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-122a plasmids into non-small cell lung cancer A549 cells.@*METHODS@#Antisense miRNA-224 and miRNA-122a 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 miRNA-224 decreased and the expression of miRNA-122a 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-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a 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-122a 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 miRNA-122a 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.

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-122a plasmids into non-small cell lung cancer A549 cells. Methods Antisense miRNA-224 and miRNA-122a 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 miRNA-224 decreased and the expression of miRNA-122a 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-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a 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-122a 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 miRNA-122a 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.

Study on the diagnosis of common diseases of the upper gastrointestinal tract by the instant gastrointestinal ultrasound contrast agent / 实用医学杂志

Objective To investigate the diagnostic value of the instant gastrointestinal ultrasound contrast agent in the digestive diseases. Methods Five hundred and seventy-nine patients received the examination of the color Doppler after they drinked the ultrasonic contrast agent. Then the results were analyzed by consistency analysis. Results There was high consistency between the two examinations in the normal control, gastritis, peptic ulcer, gastric cancer and gastric leiomyoma. The Kappa value was 0.768, 0.913, 0.925, 0.939 and 1.000, respectively. But the consistency in the gastric polyp was low , the Kappa value was 0.368. Conclusion The color Doppler through the instant gastrointestinal ultrasound contrast agent has high diagnostic value in the common diseases of the digestive system.

Preparation and evaluation of lipid microbubbles ultrasound contrast agent / 中国药学杂志

OBJECTIVE: To prepare lipid microbubbles as well as to evaluate their characteristics and efficacy. METHODS: Octafluoropropane-containing lipid microbubbles were prepared by high speed shearing method. Orthogonal experimental design was used to optimize the prescription. The resultant microbubbles were characterized, in terms of morphology, concentration, size, distribution and the octafluoropropane content. Moreover, the contrast effect of the microbubbles in New Zealand rabbit's kidney and liver was studied. RESULTS: The average concentration of microbubbles was (2.99±0.19)×109 their average size was (2.46±0.05) μm, and 97% of them were smaller than 7 μm. Octafluoropropane content was (387.81±35.28) μg·mL-1. With the help of the lipid microbubbles, satisfactory ultrasound contrast images of rabbit's kidney and liver were got. CONCLUSION: The microbubbles prepared by high speed shearing method have regular morphology, and their size distribution is small and even. It can be used as an excellent ultrasound contrast agent which can enhance significantly the yield of ultrasound examination and thus allow better imaging. Copyright 2012 by the Chinese Pharmaceutical Association.

Preparation and preliminary evaluation of KGDS-targeted ultrasound contrast agent / 中南大学学报(医学版)

Objective To prepare a thrombus-targeted ultrasonic contrast agent and to investigate its targeted ability to fresh blood clots. Methods We first synthesized FITC-KGDS-Palm compound, and then prepared thrombus-targeted microbubbles using "ultrasound & high speed shearing method".Fluorescence labeling thrombus-specific peptides and KGDS,directed at the activated glycoprotein(GP)Ⅱb/Ⅲa receptor of platelets were attached to the surface of lipid microbubbles. The concentration and size of TUCA were measured by Malvern Zeta Sizer Nano-ZS590 and Coulter counter.Immunofluorescence was applied to confirm the conjugation.The conjunct ratio was assessed by flow cytometer (FCM).Results The KGDS-TUCA was straw yellow turbid liquor,and the concentration was 1.5×10~9/mL,and the average size was 1.5 μm. The targeted microbubbles conjugated with the thrombus-specific peptides showed bright green rings by fluorescence microscope.FCM demonstrated that the wavelength of shell of KGDS-TUCA changed greatly,and the conjunct ratio was 90.04%.In vitro study showed KGDS-TUCA remained stable for 48 h at 4 ℃ and target-attached to blood clots and showed good stability.Conclusion The ultrasound & high speed shearing method to prepare TUCA is easy and in favor of purification.KGDS-TUCA has high specific biological activity.The conjunct ratio and stability of KGDS-TUCA are excellent.

Application of ultrasound contrast agents in high-intensity focused untrasound on clinical treatment of hepatocellular carcinoma / 国际生物医学工程杂志

High intensity focused ultrasound(HIFU) is a technique that ultrasound energy is focused on tumor target to function the treatment through the biological effects including heating effect, cavitation effect and mechanical effect of body to caused cell coagulation necrosis in vivo. Ultrasound contrast agent is one of the most important applicational agent for the treatment of hepatocellular carcinoma(HCC). We discussed in this paper the application of ultrasound contrast agent in the diagnose and focusing of target before the treatment and the assesement of treatment of HCC. Considering the enhancement of cavitation effect, better acoustic environment that enhance biological efficacy of HIFU during the treatment of HCC, ultrasound contrast agent is drawing more attention as an HIFU enhancement agent.

Assessment of renal trauma by ultrasound contrast via vein / 第三军医大学学报

0.05) before and after the contrast.However,for the renal parenchyma,it increased significantly after the contrast(P

Enhanced human MxA gene expression in mice liver by ultrasound and microbubble destruction technique / 重庆医科大学学报

Objective:To investigate whether expression of MxA gene could be enhanced by intravenous injection of gene and contrast agent with ultrasound wave irradiation in mice liver and to investigate the effects of different ultrasound parameters on gene expression.Methods:(1) Effect of different methods on transfection efficiency:24 Kunming mice were randomly divided into 4 groups as plasmid group,plasmid and microbubble group,ultrasound and plasmid group,and plasmid,ultrasound and microbubble group.After 7 days,the lungs,hearts,spleens,kidneys,lungs and skeletal muscles were harvested and half quantization of MxA protein was detected with immunofluorescence technique.(2) Effects of different parameters on gene expression:20 Kunming mice were randomly divided into 4 groups as 0.25 W/cm2 group,0.5 W/cm2 group,0.75 W/cm2 group and 1.0 W/cm2 group according to different ultrasound parameters.After 7 days,liver tissues were harvested for the half quantization of MxA by the same method described above.Results:(1) Number of liver cells with positive expression was the highest in plasmid,ultrasound and microbubble group,followed by ultrasound and plasmid group,plasmid and microbubble group,and plasmid group in order;There was significant statistical difference between either two groups(P