| Purpose In this work, we fabricate nanobubbles directly by controlling the thickness of the phospholipid film without amphiphilic surfactants and fabrication of the compound of nanosized and micro-sized Ultrasound Contrast Agents. And evaluated its particle size, zeta potential, stability, cytotoxicity and so on. At last, we compared the ability of contrast enhanced imaging in vitro and in vivo and the transmission of tumor blood vessel with Sono Vue.Material and methods The phospholipids that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]( DSPE-PEG(2000)) were used in the fabrication of the nanobubbles using the thinfilm hydration method. According to fixed ratio, 7mg, 14 mg, 21 mg, and 28 mg phospholipids mixture of DSPE-PEG(2000) and DPPC were respectively add in four 25 ml rotary evaporation bottles and separately dissolved with 2ml of chloroform. At this step, a small amount of the fluorescent membrane probe Di I(red fluorescence) would be added into each of the rotary evaporation bottle for some experiments. Rotary evaporation was performed for 10 min, 55℃, 120rpm/min in Rotary Evaporator for each rotary evaporation bottle. After chloroform evaporated, milky white phospholipid thin-films were rested on the rotary evaporation bottle walls. Then, the milky white phospholipid thin-films were then hydrated separately with 500μl,1 m L,1.5ml and 2ml of hydration liquid which consisted of 10% glycerol and 90% 1×PBS(V/V). Put all the four rotary evaporation bottles in an incubator-shaker(New Brunswick Scientific) at 37℃,130 rpm for 60 min. Then, milky white suspensions formed in the bottles. 500μl each of the suspension in the bottles was transferred respectively to four vials that was sealed with plastics cap. The air in the vials was replaced with C3F8 gas by using long, fine needle and a 50-ml syringe. Finally, every vial was oscillated for 45 s in Mechanical Oscillator to form the bubbles. The bubbles of each vial were separately diluted to 8ml with PBS. Meanwhile, Sono Vue that is commercial microbubbles was used as control. The particle size and zeta potential of nanobubbles and Sono Vue were tested by dynamic light scattering(DLS). To observe the structure of the nanobubbles(fabricated by 14 mg DPPC and DSPE-PEG(2000)) and compare with phospholipids and Sono Vue, scanning electron microscopy(SEM) and fluorescence microscope were performed. The stability of nanobubbles at 25℃ was evaluated by the particle size and concentration of nanobubbles at 1min, 15 min, 30 min, 45 min and 60 min after the fabrication. The centrifuge’s(20g, 50 g and 805g) influence on the compound of nano-sized and micro-sized ultrasound contrast agents(fabricated by 21 mg DPPC andDSPE-PEG(2000)) was tested by DLS. Nanobubble cytotoxicity assay was evaluated by MTT. Compared nanobubbles’ ability of contrast enhanced imaging in vitro and tumorcarrying mice in vivo with Sono Vue. Confocal laser scanning microscopy(CLSM) was used to observe the location of Di I-labeled nanobubbles in vivo. The nude mice bearing tumors were sacrificed after injection of Di I-labeled nanobubbles and Sono Vue. Tumor were separated immediately to perform frozen slice. The right thigh muscle of the mouse injected nanobubbles were performed as negative control.Result The average diameter of bubbles with 7mg, 14 mg, 21 mg, 28 mg gross weight and fixed ratio DPPC and DSPE-PEG(2000) were 565.2±201.5nm(n=3), 457.9±113.8nm(n=3), 960.8±59.5nm(n=3), 1121.1±57.0nm(n=3) respectively. The average diameter of the control Sono Vue was 1614.8±224.7nm(n=3). Zeta potential measurements showed that the nanobubbles(fabricated by 14 mg DPPC and DSPE-PEG(2000)) had negative charge of-21.48 ± 7.46 m V(n=3), and that of Sono Vue is-32.29 ± 13.13 m V(n=3). Statistical analysis indicated that there was no significant difference(P=0.283). The scanning electron microscope shown that phospholipid(negative control) was small ball, the nanobubbles and Sono Vue(positive control) were small and not aggregated hollows which matched with the results of DLS. The average diameters of the nanobubbles stored separately at 25°C for 1, 15, 30, 45 and 60 min were 457.9 ± 113.8 nm(n = 3), 504.3 ± 74.1 nm(n = 3), 519.5 ± 95.5 nm(n = 3), 625.9 ± 100.6 nm(n = 3) and 709.5 ± 272.0 nm(n = 3).The average diameters of the bubbles(fabricated by 21 mg DPPC and DSPEPEG(2000)) were 828.4 ± 425.7 nm(n = 3), 882.1 ± 417.6 nm(n = 3) and 977.2 ± 65.9 nm(n = 3) after centrifugation speeds of 20 g, 50 g and 805 g, respectively.Obvious cytotoxicity appeared when the concentration of phospholipids increased to 10 μg/ml. The results revealed that the nanobubbles had no obvious cytotoxicity to the cell line AU-565 for the phospholipid concentration(<5 μg/ml), which was the concentration used for the subsequent ultrasound imaging tests.No significant difference was observed between the signal enhancements of nanobubbles and Sono Vue(P = 0.691); the grey scale intensity ofnanobubbles was 58.482 ± 28.192 d B(n = 5), while that of Sono Vue was 52.861 ± 11.491 d B(n = 5). Upon caudal vein injection of the nanobubbles and Sono Vue in tumorcarrying mice, both of the enhancement in the tumor increased over time. The nanobubbles and Sono Vue reached the peak 30 s. No statistically significant difference(P = 0.404) was observed in the peak intensity(PI) of the grey scale enhancement between nanobubbles(212.547 ± 5.414n=9), and Sono Vue(224.44 ± 3.969 d B; n = 9). After 2min, the grey scale intensity for the nanobubbles and Sono Vue appeared significant difference. The image enhancement sustained time of nanobubbles is longer than Sono Vue. The frozen sections of the tumors and skeletal muscle were observed under CLSM. a number of Di I-labeled nanobubbles(red) were present in the extravascular and intercellular space of the tumor tissue, while Di I-labeled Sono Vue was hardly detected outside the tumor blood vessels. In the skeletal muscle sections, however, Di I-labeled nanobubbles were rare.Conclusion We prepared pure nanobubbles directly by modulating the thickness of the phospholipid film, which omitted the process of separating pure-sized nanobubbles from the mixture of microbubbles and nanobubbles or adding some amphiphilic surfactants during the phospholipid film fabricated. The nanobubbles exhibited optimal size, good stability, notoxicity, and other physical characteristics. The ability of the nanobubbles to enhance contrast imaging was confirmed both by in vitro and in vivo methods. The nanobubbles fabricated by the optimized method can be used as ultrasound contrast agents for molecular-targeted tumor imaging in the future. |
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