Yanli Guo

Construction and in vitro/in vivo targeting of PSMA-targeted nanoscale microbubbles in prostate cancer.

Prostate‐specific membrane antigen (PSMA) is a highly specific biological marker and treatment target for prostate cancer. So ultrasound molecular imaging using PSMA antibody‐loaded targeted nanoscale microbubbles (MBs) may contribute to the early diagnosis of prostate cancer.

Ultrasonic Nanobubbles Carrying Anti-PSMA Nanobody: Construction and Application in Prostate Cancer-Targeted Imaging

To facilitate prostate cancer imaging using targeted molecules, we constructed ultrasonic nanobubbles coupled with specific anti-PSMA (prostate specific membrane antigen) nanobodies, and evaluated their in vitro binding capacity and in vivo imaging efficacy. The “targeted” nanobubbles, which were constructed via a biotin-streptavidin system, had an average diameter of 487.60 ± 33.55 nm and carried the anti-PSMA nanobody as demonstrated by immunofluorescence. Microscopy revealed targeted binding of nanobubbles in vitro to PSMA-positive cells. Additionally, ultrasonography indicators of nanobubble imaging (including arrival time, peak time, peak intensity and enhanced duration) were evaluated for the ultrasound imaging in three kinds of animal xenografts (LNCaP, C4-2 and MKN45), and showed that these four indicators of targeted nanobubbles exhibited significant differences from blank nanobubbles. Therefore, this study not only presents a novel approach to target prostate cancer ultrasonography, but also provides the basis and methods for constructing small-sized and high-efficient targeted ultrasound nanobubbles.

Experimental investigation of the penetration of ultrasound nanobubbles in a gastric cancer xenograft.

Nanobubbles as a type of ultrasound contrast agent have attracted much interest in recent years due to their many advantages, such as strong penetrating power and high stability. However, there is still insufficient morphological evidence concerning gas-filled nanobubbles in tumor tissue spaces and tumor angiogenesis. We used a gastric cancer xenograft as an example to study this question. Nanobubbles with a particle size of 435.2 ± 60.53 nm were prepared and compared with SonoVue® microbubbles in vitro and in vivo, and they exhibited a superior contrast imaging effect. After excluding the impact of the nanobubbles in blood vessels through saline flush, we used an ultrasound burst and frozen sectioning to investigate the distribution of nanobubbles in the gastric cancer xenografts and confirmed this by transmission electron microscopy. Preliminary results showed that the nanobubbles were able to pass through the gaps between the endothelial cells in the tumor vascular system to enter the tissue space. These findings could provide morphological evidence for extravascular ultrasound imaging of tumors and serve as a foundation for the application of nanobubbles in extravascular tumor-targeted ultrasonic diagnostics and therapy.

Safety of Sulfur Hexafluoride Microbubbles in Sonography of Abdominal and Superficial Organs: Retrospective Analysis of 30,222 Cases: Safety of Sulfur Hexafluoride Microbubbles

The purpose of this study was to investigate the safety of the sulfur hexafluoride microbubble contrast agent SonoVue (Bracco SpA, Milan, Italy) and to implement precautions with the intent of further improving the safety of this contrast agent.

Preparation of Nanobubbles Carrying Androgen Receptor siRNA and Their Inhibitory Effects on Androgen-Independent Prostate Cancer when Combined with Ultrasonic Irradiation

Objective The objective of this study was to investigate nanobubbles carrying androgen receptor (AR) siRNA and their in vitro and in vivo anti-tumor effects, when combined with ultrasonic irradiation, on androgen-independent prostate cancer (AIPC). Materials and Methods Nanobubbles carrying AR siRNA were prepared using poly-L-lysine and electrostatic adsorption methods. Using C4-2 cell activity as a testing index, the optimal irradiation parameters (including the nanobubble number/cell number ratio, mechanical index [MI], and irradiation time) were determined and used for transfection of three human prostate cancer cell lines (C4-2, LNCaP, and PC-3 cells). The AR expression levels were investigated with RT-PCR and Western blot analysis. Additionally, the effects of the nanobubbles and control microbubbles named SonoVue were assessed via imaging in a C4-2 xenograft model. Finally, the growth and AR expression of seven groups of tumor tissues were assessed using the C4-2 xenograft mouse model. Results The nanobubbles had an average diameter of 609.5±15.6 nm and could effectively bind to AR siRNA. Under the optimized conditions of a nanobubble number/cell number ratio of 100∶1, an MI of 1.2, and an irradiation time of 2 min, the highest transfection rates in C4-2, LNCaP, and PC-3 cells were 67.4%, 74.0%, and 63.96%, respectively. In the C4-2 and LNCaP cells, treatment with these binding nanobubbles plus ultrasonic irradiation significantly inhibited cell growth and resulted in the suppression of AR mRNA and protein expression. Additionally, contrast-enhanced ultrasound showed that the nanobubbles achieved stronger signals than the SonoVue control in the central hypovascular area of the tumors. Finally, the anti-tumor effect of these nanobubbles plus ultrasonic irradiation was most significant in the xenograft tumor model compared with the other groups. Conclusion Nanobubbles carrying AR siRNA could be potentially used as gene vectors in combination with ultrasonic irradiation for the treatment of AIPC.

Inhibition of prostate cancer growth using doxorubicin assisted by ultrasound-targeted nanobubble destruction

Ultrasound (US)-targeted microbubble destruction has been widely used as an effective drug-delivery system. However, nanobubbles (NBs) have better stability and stronger penetration than microbubbles, and drug delivery assisted by US-targeted NB destruction (UTND) still needs to be investigated. Our aim was to investigate the effect of doxorubicin (DOX) on the inhibition of prostate cancer growth under UTND. Contrast-enhanced US imaging of transplanted PC3 prostate cancer in mice showed that under a combination of 1 W/cm2 US power and a 100 Hz intermittent pulse with a “5 seconds on, 5 seconds off” mode, NBs with an average size of (485.7±33) nm were effectively destroyed within 15 minutes in the tumor location. PC3 cells and 20 tumor-bearing mice were divided into four groups: a DOX group, a DOX + NB group, a DOX + US group, and a DOX + NB + US group. The cell growth-inhibition rate and DOX concentration of xenografts in the DOX + NB + US group were highest. Based on another control group and these four groups, another 25 tumor-bearing mice were used to observe the treatment effect of nine DOX injections under UTND. The xenografts in the DOX + NB + US group decreased more obviously and had more cellular apoptosis than other groups. Finally, electron microscopy was used to estimate the cavitation effect of NBs under US irradiation in the control group, NB group, US group, and NB + US group. The results of scanning electron microscopy showed that PC3 cells in the DOX + NB + US group had more holes and significantly increased cell-surface folds. Meanwhile, transmission electric microscopy confirmed that more lanthanum nitrate particles entered the parenchymal cells in xenografts in the NB + US group compared with the other groups. This study suggested that UTND technology could be an effective method to promote drugs to function in US-irradiated sites, and the underlying mechanism may be associated with a cavitation effect.

Diagnosis of prostate cancer using anti-PSMA aptamer A10-3.2-oriented lipid nanobubbles

In this study, the lipid targeted nanobubble carrying the A10-3.2 aptamer against prostate specific membrane antigen was fabricated, and its effect in the ultrasound imaging of prostate cancer was investigated. Materials including 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, carboxyl-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and polyethyleneglycol-2000 were for mechanical oscillation, and nanobubbles were obtained through the centrifugal flotation method. After mice were injected with nanobubbles, abdominal color Doppler blood flow imaging significantly improved. Through left ventricular perfusion with normal saline to empty the circulating nanobubbles, nanobubbles still existed in tumor tissue sections, which demonstrated that nanobubbles could enter tissue spaces via the permeability and retention effect. Fluorinated A10-3.2 aptamers obtained by chemical synthesis had good specificity for PSMA-positive cells, and were linked with carboxyl-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine lipid molecules from the outer shell of nanobubbles via amide reaction to construct targeted nanobubbles. Gel electrophoresis and immunofluorescence confirmed that targeted nanobubbles were fabricated successfully. Next, targeted nanobubbles could bind with PSMA-positive cells (C4-2 cells), while not with PSMA-negative cells (PC-3 cells), using in vitro binding experiments and flow cytometry at the cellular level. Finally, C4-2 and PC-3 xenografts in mice were used to observe changes in parameters of targeted and non-targeted nanobubbles in the contrast-enhanced ultrasound mode, and the distribution of Cy5.5-labeled targeted nanobubbles in fluorescent imaging of live small animals. Comparison of ultrasound indicators between targeted and non-targeted nanobubbles in C4-2 xenografts showed that they had similar peak times (P>0.05), while the peak intensity, half time of peak intensity, and area under the curve of ½ peak intensity were significantly different (P<0.05). In PC-3 xenografts, there were no differences in these four indicators. Fluorescent imaging indicated that targeted nanobubbles had an aggregation ability in C4-2 xenograft tumors. In conclusion, targeted nanobubbles carrying the anti-PSMA A10-3.2 aptamer have a targeted imaging effect in prostate cancer.

Inhibition Effects of High Mechanical Index Ultrasound Contrast on Hepatic Metastasis of Cancer in a Rat Model

RATIONAL AND OBJECTIVES The liver is the most common organ for tumor metastasis. The development of new methods to depress hepatic metastasis is of great importance in improving survival. The aim of this study was to observe the effects of high-mechanical index ultrasound contrast on hepatic metastasis of colorectal cancer. MATERIALS AND METHODS Hepatic metastasis models were established by injecting human colon carcinoma LoVo cells into the spleens of Sprague-Dawley rats. The rats were divided into a control group, a microbubble plus ultrasound group, a simple ultrasound group, and a simple microbubble group. The ultrasound contrast agent SonoVue (1 mL/kg) was injected via the tail vein, and high-mechanical index ultrasound contrast (frequency, 1.5 MHz; mechanical index, 1.7) was performed on the spleen intermittently for 2 minutes. The animals were sacrificed after 10 days, and the sizes and number of hepatic metastases were measured and compared. Histologic pathology and splenic ultrastructure were observed. RESULTS The number and sizes of hepatic metastases patently decreased in rats in the microbubble plus ultrasound group (P < .01). There were no obvious differences among the control group, simple ultrasound group, and simple microbubble group in hepatic metastases (P > .05). Histologic pathology showed that the number of tumor cells in the spleens decreased considerably, and massive necroses, hemorrhages, and thrombi were observed in the tumor and spleen tissues of rats in the microbubble plus ultrasound group. Transmission electron microscopy showed that the mitochondria of tumor cells and endothelial cells were clearly swelled, and there were gaps among endothelial cells and platelets aggregated in capillary vessels. CONCLUSION This research shows that intermittent high-mechanical index ultrasound contrast may inhibit the hepatic metastasis of cancer in a rat model.

CAIX aptamer-functionalized targeted nanobubbles for ultrasound molecular imaging of various tumors

Purpose Targeted nanobubbles can penetrate the tumor vasculature and achieve ultrasound molecular imaging (USMI) of tumor parenchymal cells. However, most targeted nanobubbles only achieve USMI of tumor parenchymal cells from one organ, and their distribution, loading ability, and binding ability in tumors are not clear. Therefore, targeted nanobubbles loaded with carbonic anhydrase IX (CAIX) aptamer were fabricated for USMI of various tumors, and the morphological basis of USMI with targeted nanobubbles was investigated. Materials and methods The specificity of CAIX aptamer at the cellular level was measured by immunofluorescence and flow cytometry. Targeted nanobubbles loaded with CAIX aptamer were prepared by a maleimidethiol coupling reaction, and their binding ability to CAIX-positive tumor cells was analyzed in vitro. USMI of targeted and non-targeted nanobubbles was performed in tumor-bearing nude mice. The distribution, loading ability, and binding ability of targeted nanobubbles in xenograft tumor tissues were demonstrated by immunofluorescence. Results CAIX aptamer could specifically bind to CAIX-positive 786-O and Hela cells, rather than CAIX-negative BxPC-3 cells. Targeted nanobubbles loaded with CAIX aptamer had the advantages of small size, uniform distribution, regular shape, and high safety, and they could specifically accumulate around 786-O and Hela cells, while not binding to BxPC-3 cells in vitro. Targeted nanobubbles had significantly higher peak intensity and larger area under the curve than non-targeted nanobubbles in 786-O and Hela xenograft tumor tissues, while there was no significant difference in the imaging effects of targeted and non-targeted nanobubbles in BxPC-3 xenograft tumor tissues. Immunofluorescence demonstrated targeted nanobubbles could still load CAIX aptamer after penetrating the tumor vasculature and specifically binding to CAIX-positive tumor cells in xenograft tumor tissues. Conclusion Targeted nanobubbles loaded with CAIX aptamer have a good imaging effect in USMI of tumor parenchymal cells, and can improve the accuracy of early diagnosis of malignant tumors from various organs.

OP09.10: The value of echocardiography in the diagnosis and postnatal outcome of fetal pure ventricular septal defect

by sequential ultrasonographic examinations during fetal life and following birth. Spontaneous closure of the VSD, need for intervention, additional abnormalities and chromosomal aberrations were collected and analysed. Results: From the original cohort of 86 isolated VSDs 11 cases (12.7%) were later excluded because of lost to follow up, termination of pregnancy and diagnosis of an extra-cardiac structural malformation before delivery. Seventy-five cases of isolated VSDs with complete data on outcome further analysed. Muscular VSDs found in 64 fetuses (85.3%) and perimembraneous VSDs in 11 fetuses (14.6%). Spontaneous closure of the VSD occurred in 59 (92.2%) cases with muscular VSDs, and in five (45.5%) cases with perimembraneous VSDs. Postnatal treatment was indicated in three (27.3%) patients with a perimembraneous defect and in none of the patients with a muscular defect. Only one child (9.1%) with a perimembraneous defect had a chromosomal anomaly detected. Conclusions: Isolated muscular VSDs usually close spontaneously during pregnancy or in the first two years of life and do not increase the risk for chromosomal aberrations. These cases have a good prognosis and probably need no further prenatal work-up. On the other hand, isolated perimembraneous VSDs may be associated with chromosomal anomalies and may need intervention following birth and further genetic prenatal investigation.