| Purpose: The objective of this research is the development of a new targeted ultrasound contrast agent for early detection of ovarian cancer using antiCA125 antibody(MUC16) conjugated nanobubbles(AB-NB). In vitro we evaluated their characterization and ability of cell targeting OVCAR-3 ovarian cancer cells line which is CA-125 positive. In vivo we observed ultrasound detection properties of AB-NB versus no targeted nanobubble(NB) were conducted in a mouse model. Methods: 1. Formulation of Nanobubbles: To formulate NB, the lipids DPPC, DPPA, DPPE and DSPE-PEG-COOH were dissolved in chloroform, Pluronic L10 solution and glycerol. After that, drying and shaking the mixture. To formulate AB-NB, EDC and NHS chemistry was employed to conjugate the NB to the anti-CA125 antibody(MUC16).2. Nanobubble Characterization: The mean diameter and polydispersity of NB and AB-NB were measured using dynamic light scattering(DLS), and the in vitro ultrasound contrast imaging effects were observed, separately. Detected the stability and efficiency of ultrasound contrast imaging at three different temperatures(4 ℃ group, 25 ℃ group, 37 ℃ group) of AB-NB within three days after preparation. Under simulated in vivo environment, the in vitro ultrasound stability of NB and AB-NB were measured.3. Conjugation NB With anti-CA125 Antibody(MUC16): A secondary antibody(SAB) tagged with green fluorescence was added to the AB-NB and NB separately and dialyzed for 30 min at 4℃. Twice centrifuge wash were performed at the end to purify the nanobubbles. Fluorescent microscopy was used to observe the presence of bright green dots from the SAB and evaluate the efficiency of AB-NB binding to the anti-CA125 antibody(MUC16).4. In Vitro Fixing Cell binding experiment of AB-NB:Human ovarian cancer cell lines OVCAR-3 cells, which are CA-125 positive, and SKOV-3 cells, which are CA-125 negative, were cultured. Then cells were fixed separately when they have passaged for 3-4 times. The fixing OVCAR-3 cells samples were divided into a group and b group. The fixing SKOV-3 cells samples were divided into c group and d group. Green fluorescence encapsulated NB was added to a group and c group separately. Green fluorescence encapsulated AB-NB was added to b group and d group. After incubating for 3 hours, all the cell-nanobubble solution samples were washed four separate times carefully with PBS. Fluorescent microscopy was used to check for the presence of green dots localized at the cells and the fluorescence intensity will be measured to evaluate the efficiency of AB-NB binding to the targeted cells.5. Ultrasound Biodistribution of AB-NB In Vivo: Human ovarian cancer cell lines OVCAR-3 cells and SKOV-3 cells were cultured and passaged for 3-4 times. Balb/c female nude mice were inoculated subcutaneously, respectively with OVCAR-3 cells and SKOV-3 cells. First, AB-NB were injected through tail vein in tumor-bearing mice that had been inoculated with either ovcar-3 cells or SKOV-3 cells. Second, two days after injecting AB-NB, the same amount with the same ultrasound intensity of NB were injected to the same mice. Fifteen seconds after raw data acquisition started, nanobubble solution was administrated and continuous image acquisition continued for 10 min. The time-intensity curve(TIC) was calculated for statistical analysis. The intensities ratio(AB-NB/NB normalized intensity in the same tumor) after peak 0, 1, 2, 5 min of contrast enhancement were compared with the same tumor between NB and AB-NB in each group.The decay slope of ultrasound contrast was also analyzed from the TIC. 6. Histological analysis: After undergoing ultrasound images for one week, OVCAR-3 and SKOV-3 tumor-bearing mice were all injected green fluorescence encapsulated AB-NB contrast material through the tail vein. Fifteen minutes after bubble injection, tumors were excised and mounted tissues in optimal cutting temperature compound(OCT) and frozen in-80°C. The tissues were cut into slices and stained with DAPI. Fluorescent microscopy was used to check for the presence of green dots localized at the tissues and the fluorescence intensity will be measured to evaluate the efficiency of AB-NB binding to the targets. Results: 1. DLS measurements showed NB and AB-NB diameters of 66.6 ± 22.0 nm and 74.6 ± 16.7 nm, polydispersity of 0.208±0.069 and 0.203±0.020, respectively. In vitro ultrasound contrast imaging displayed AB-NB as uniform punctate hyperechoic.2. Detected the stability of AB-NB at three different temperatures: At 4 ℃ group, AB-NB size was still small after the third day of preparation. At 25 ℃ group and 37 ℃ group, AB-NB size was increased significantly after the next day of preparation. At 4 ℃ group and 25 ℃ group temperatures set of samples within three days after the preparation could still maintain a good ultrasound contrast imaging effect. However, at 37℃ group, the effect of ultrasound contrast imaging was significantly decreased at the first day after the preparation.3. Under simulated in vivo environment, the in vitro ultrasound stability of NB and AB-NB: 0-4 hours The average decay rate of NB and AB-NB within 0-60min、0-4 hours were similar.4. NB Conjugation With Secondary antibody(SAB) Confirmation: Fluorescence microscopy showed that AB-NB group could present a lot of green fluorescence. However, there was no proteins of any sort were incubated with the NB control. The difference of green fluorescence was significant statistically between the two group(p <0.05) using Image J, which in turn suggests that Anti-MUC16 itself was bound to the AB-NB. 5. In Vitro Attachment of AB-NB to tumor cells: Under fluorescence microscope, there were most green fluorescence attach to the OVCAR-3 cells in b group(AB-NB) but there was no or only a very small amount green fluorescence around the OVCAR-3 cells in a group(NB). In the meantime, there was no or only a very small amount green fluorescence around the SKOV-3 cells in d group(AB-NB) and c group(NB). The nanobubble fluorescence ratio(total bubbles fluorescence/cells fluorescence per field) in b group was significantly different compared to the other three groups(p<0.05). In fact, the AB-NB adhered to OVCAR-3 cells ratio approximately 12 times better than the NB.6. In Vivo Distribution of NB and AB-NB Visualized with Ultrasound: In the transplanted OVCAR-3 tumors, the intensity ratio after peak 0, 1, 2, 5 min were significantly different between AB-NB and NB(p<0.05). Although the mean decay slopes of AB-NB in OVCAR-3 tumor was slower than that of NB in the initial phase(0.39±0.06 compared to 0.98±0.20), the difference was not statistically significant(p>0.05). In the transplanted SKOV-3 tumors, the intensity ratio after peak 0, 1, 2, 5 min of AB-NB and NB was not significantly different(p>0.05).There were also no significant difference observed in the decay slope comparing AB-NB with NB in SKOV-3 tumor during the initial phase(p=0.05). No significant differences were noted in the terminal phase comparing AB-NB with NB neither in OVCAR-3 nor SKOV-3 tumor(p>0.05).7. Histology analysis: In the OVCAR-3 tumors(CA-125 positive), a considerable number of green fluorescence was present in the intercellular space using fluorescence microscope. In contrast, there was very few green fluorescence in the SKOV-3 tumors(CA-125 negative). The nanobubble fluorescence ratio(total bubbles fluorescence/cells fluorescence per field) in OVCAR-3 tumor tissue was significantly different(Fig.8c, p<0.05) compared to in SKOV-3 tumor signal using Image J. Conclusions: 1.The size, stability and the ultrasound contrast imaging effect of this targeted nanobubbles ultrasound contrast agent are suitable for the development of intravascular contrast agent.2.This study successfully prepared a new targeted nanobubble contrast agent in ultrasound by employing EDC and NHS chemistry to conjugate the NB to the anti-CA125 antibody(MUC16). Importantly, the quite big molecular weight of CA-125 antibody doesn’t affect the bubble size and the stability in vitro study. The 4 ℃ is the best temperature for preservation of AB-NB. It could still maintain a good ultrasound contrast imaging effect within three days after preparation.3.The binding rate between CA-125 antibody and the nanobubbles was efficient. They were found to aggregate selectively around OVCAR-3 cells and SKOV-3 cells in an in vitro cell binding experiment. The CA-125 monoclonal antibodies that coupled with the surface of the targeted nanobubbles were able to specifically recognize and bind to ovarian cancer cells with high CA125 expression.4. We established OVCAR-3 cells tumor and SKOV-3 cells tumor model in nude mice and performed in vivo enhanced-contrast imaging experiments with NB and AB-NB. The results showed that, the contrast enhancement intensities were significantly higher in transplanted OVCAR-3 tumors. That suggested that AB-NB strongly and specially accumulated in ovarian cancer tissue. And the proposed nanobubble has the potential to significantly advance ovarian cancer early detection.5.Upon histological analysis using fluorescence microscope, AB-NB showed more extensive extravasation beyond and inside the CA-125 positive tumor area, while it showed limited extravasation in the CA-125 negative tumor. |
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