| PurposeTo prepared pure- sized nanobubbles. To connect Affibody loaded with HER2 ligand to nanobubbles and synthesis nanoscale targeted ultrasound contrast agents “nanobubbleAffibody” which can recognize HER2 in breast cancer, and through the in vitro and in vivo experiments, to verify the specific targeting of “nanobubble-Affibody” to HER2 molecule on breast cancer, further to assess noninvasively the expression level of HER2 molecule in breast cancer. In addition, the study attempts to explore the preparation of multifunctio na l nanobubbles loaded with IR780, provide a new way for tumor molecular targeted contrast-enhanced ultrasound in diagnosis and treatment.Methods1.14 mg DPPC and DSPE-PEG2000(biotin) were mixed according to a certain proportion in the 25 ml rotary evaporation bottle and produced film, combined with mechanical vibration method, finally prepared pure nanoscale bubbles. Using fluoresce nce microscope, scanning electron microscope, the morphology of nanobubbles was observed. Then, the size distribution and surface potential were further detected through Particle size and Zeta potential analyzer. The MTT method detected nanobubbles’ toxicity to cell.2. The avidin-biotin method was used to combine nanobubbles with biotinylated antiErb B2 Affibody® molecules. Then, the “nanobubble-Affibody” conjugates were incubated with a variety of HER2(+) and HER2(-) tumor cel s, the in vitro targeting was observed by inverted fluorescence microscope; the targeting rate between “nanobubbleAffibody” and tumor cels with HER2(+) was assessed by flow cytometry; the stability of “nanobubble-Affibody” in simulated body temperature was observed via size/potential analyzer; the ultrasound contrast imaging of “nanobubble-Affibody” was detected via rubber glove device.3. We established nude mouse model loading with tumor, injected the “nanobubbleAffibody” by tail vein, the effect of ultrasound imaging to the target molecule was detected using ultrasound diagnostic instrument. Small animal fluorescence imaging system observed the distribution and metabolic rate of “nanobubble-Affibody” in tumor and the major organ. Pathological sections of tumor were observed by confocal laser scanning microscopy to detect the targeting distribution of “nanobubble-Affibody” in tumor.4. In addition, in the process of making nanobubbles, a little of IR780 was added into the mixture of DPPC and DSPE-PEG2000(biotin) and made film, directly prepared of multifunctional nanobubbles. Confocal laser scanning electron microscope, transmiss io n electron microscope and particle size analyze r detected the characteristics. Then, mult ip le tumor cel s were cultured and taken rat myocardial cel s as negative control. The specific targeting to tumor cel s and ultrasound contrast imaging ability in vitro with multifunctio na l nanobubbles was observed.Results1. By controlling the thickness of lipid membranes, we can prepare the pure nanobubbles with diameter: 478.2 ± 29.7 nm, under the scanning electron microscope, the nanobubbles were taken on as cavities. Additionally, the nanobubbles had some characteristics such as good stability, non-toxic to cells and so on.2. Synthesis of "nanobubble-Affibody" as molecular targeted ultrasound contrast agent. In vitro experiments showed that the "nanobubble-Affibody" could specific target the HER2(+) tumor cel s but not the HER2(-) tumor cel s; at the same time, the nanobubbles had no specific target to HER2(+) tumor cells.3. The contrast enhanced ultrasound in vivo showed that although the imaging duration of "nanobubble-Affibody" was short, but the peak time, and the enhancement of strength was significantly higher than that of Sono Vue and nanobubbles(104.539 ± 6.42 d B vs. 79.451 ± 8.05 d B, P < 0.05; 104.539 ± 6.42 d B vs. 70.753 ± 10.33 d B, P < 0.05). The small animal fluorescence imaging system proved that the "nanobubble-Affibody" group of tumor fluorescence uptake rate(4.79e07 ± 210797.3) was significantly higher than that of nanobubble group(4.27e07 ± 78270.26,P < 0.05) at 3min. At 30 min, although the fluorescence uptake rate was relatively decreased slightl y in "nanobubble-Affibody" group of tumor, it was still higher than that of the nanobubble group(5.32e07 ± 347809.7 vs. 2.13e07 ± 122119.9,P < 0.05). The pathological sections showed, "nanobubble-Affibody" more through the tumor blood vessels into tissue space and tumor cel s.4. The particle size distribution of the new multifunctional nanobubbles is about: 552.7 + 55 nm, which had good biocompatibility and specific targeting to human breast cancer cel s BT-474 and MDA-MB-231, however, there was no targeting to rat myocardial cells. ConclusionThrough controlling the thickness of the membrane, we can prepare pure nanoscale bubbles directly, thus avoiding the tedious steps separation nanobubbles from the micro / nano bubbles suspension. The "nanobubble-Affibody", as novel molecular targeted ultrasound contrast agents, can specific target breast cancer with HER2(+) in vitro or in vivo. The molecular targeted ultrasound imaging resulted from "nanobubble-Affibody", has provide the basis for determining expression of HER2 molecules in breast cancer noninvasively. At the same time, we successfully prepared multifunctional nanobubbles with the ability of infrared fluorescence imaging, ultrasound contrast imaging and tumor targeting, which provide also a new way for molecular targeted contrast-enhanced ultrasound of tumor. |
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