Preparation Of Melanin-loaded Lipid Nanobubbles Contrast Agents And Its Imaging Study

PARTⅠ PREPARATION AND CHARACTERIZATION OF MELANIN-LOADED LIPID NANOBUBBLES CONTRAST AGENTSObjective To prepare a novel endogenous optical absorber of melanin-loaded lipid nanobubbles(NBs) contrast agents, and investigate their physical and chemical properties and observe the enhancement effect for ultrasound imaging and photoacoustic imaging.Methods Melanin-loaded lipid nanoparticles were prepared using ether injection method. After lyophilization, melanin-loaded lipid NBs contrast agents were formed by replacement of C3F8 gas. The surface morphology and structure, the size and Zeta potential distribution, the concentration of melanin loading in lipid nanoparticles, and the absorption spectrum were detected. Different concentrations of NBs in vitro agarose gel model were used to explore its ultrasound imaging and photoacoustic imaging performance. The melanin-loaded lipid NBs were incubated with MDA-MB-231 cells for different hours(6h,12 h,24h),and then the viability of cells and the phagotrophic effect of NBs by MDA-MB-231 cells were observed.Results The melanin-loaded lipid nanoparticles were prepared successfully using ether injection method. The nanoparticles exhibited a good spherical morphology with narrow diameter distribution under light microscopy. The mean size was(284.20±85.54) nm and the apparent Zeta potential was(-51.30±5.98) m V. Lots of melanin granules were distributed evenly in the lipid nanoparticles under transmission electron microscope. The concentration of melanin loading in lipid nanoparticles was 95.10 μg/mg measured by UV-Vis spectrophotometry. In the range of 300 nm to 800 nm wavelengths, the melanin-loaded lipid nanoparticles showed strong light absorption, and the absorbance gradually reduced with the increase of light wavelength. After lyophilization, the melanin-loaded lipid NBs became brown black suspension when dissolved in double-distilled water. The melanin-loaded lipid NBs could enhance the B-mode ultrasound imaging and second harmonic imaging. The echo intensity within region of interest(ROI) increased with the increase of concentration of NBs. In photoacoustic imaging experiments, the melanin-loaded lipid NBs had a good photoacoustic imaging performance. And as the NBs concentration increased, the photoacoustic value increased. The results of trypan blue dye exclusion showed the cells viability was not affected by melanin-loaded lipid NBs. After phagocytosis, the Di I labeled melanin-loaded lipid NBs in the cytoplasm were observed using laser scanning confocal microscope. The cells phagocytosed with melanin-loaded lipid NBs exhibited a good photoacoustic imaging performance, and the photoacoustic signal value showed a good correlation with incubation time and concentration of bubbles.Conclusion Melanin-loaded lipid NBs have been successfully prepared with a stable physical and chemical properties, small size, good dispersibility and high biosafety. They had perfect enhanced ultrasound imaging and photoacoustic imaging performance, which make them a novel multimodal molecular probes based on ultrasound imaging.PART Ⅱ MELANIN-LOADED LIPID NANOBUBBLES CONTRAST AGENTS FOR ENHANCED ULTRASOUND AND PHOTOACOUSTIC IMAGING IN TUMORObjective To established the model of tumor of human breast cancer in nude mice, and to observe the effect of melanin-loaded lipid NBs for enhanced ultrasound and photoacoustic imaging in vivo.Methods To prepare melanin-loaded lipid NBs using ether injection method and lyophilization. The model of tumor xenografts in the nude mice was established by subcutaneous injection of MDA-MB-231 cells. Melanin-loaded lipid NBs were injected from the tail vein and then ultrasound imaging and photoacoustic imaging at the center of the tumor were observed dynamically. The echo intensity on the harmonic ultrasound images and photoacoustic value within region of interest were quantitatively determined by DFY software and Vevo LAZR software, respectively. After 2h injection of contrast agents labeled with Di I, excised tumor tissues and performed quick frozen section, the retention of contrast agents in the tumor was observed by laser scanning confocal microscopy.Results The melanin-loaded lipid NBs contrast agent s were prepared successfully. After injection of contrast agents by tail vein, ultrasound imaging and photoacoustic imaging were enhanced dramatically. In contrast-enhanced ultrasound experiment, the echo signal reached peak after 15 s injection of contrast agents. And then the echo signal decreased gradually and disappeared about after 12 minutes. In the photoacoustic imaging experiment, the photoacoustic signal showed slight enhancement within 30 min after injection of contrast agents. The photoacoustic signal reached its peak about 3 hours. Under laser scanning confocal microscopy, a lot of red-labeled contrast agents were aggregated in the tumor site.Conclusion Melanin-loaded lipid NBs contrast agents could enhance ultrasound imaging and photoacoustic imaging in vivo. They can penetrate the tumor blood vessel pores into tumor tissue, which make them a potential for imaging and treatment of neoplastic parenchyma.