Fig. 1. Schematics of SPIONs assembled magnetic nanobubbles and their application for neural stem cell labeling with different methods. SPIONs: Superparagmagnetic iron oxide nanoparticles.

Fig. 2. Assembled parameters and characterization of assembled magnetic nanobubbles. (a) Effect of compression speed (a1-1) for size distribution; (a1-2) for count rate of bubbles and peak ratio of free SPIONs), compression cycles (a2-1) for size distribution; (a2-2) for count rate of bubbles and peak ratio of free SPIONs) and the concentration of SPIONs (a3-1) for size distribution; (a3-2) for peak ratio of free SPIONs) on assembled nanobubbles; (b) size distribution (b1) and zeta potential (b2) of nanobubbles, nanoparticles and assembled nanobubbles by NanoSizer. All bars represent mean ± SD, and three independent experiments (n = 3) were performed; (c) The stability of assembled magnetic nanobubbles at room temperature (25 °C).

Fig. 3. Morphologies of assembled magnetic nanobubbles. (a) TEM images of SPIONs; (b) TEM images of assembled nanobubbles, and the top left corner shows the magnification of one sample; (c) SEM images of assembled nanobubbles, and the top left corner shows the magnification of one sample; (d) analysis of atomic percentage and mass percentage in assembled nanobubbles by EDS module.

Fig. 4. Mechanism of assembled process for assembled nanobubbles. (a) SPIONs in water solution before compression; (b) a gas layer deposited on the surface of the nanoparticles during repeat compressions; (c) attractions between nanoparticles and nanobubbles; (d) the assembled nanobubbles structures.

Fig. 5. MR and ultrasound imaging of assembled nanobubbles. (a) Fe concentration-dependent T2-weighted magnetic resonance images in a 7.0 T MR scanner, and the average mean gray value of T2 signal (b); (c) ultrasound imaging of assembled nanobubbles, and mean contrast enhanced grayscale of ultrasound imaging (d). All bars represent mean ± SD, and three independent experiments (n = 3) were performed; and the statistical significance is indicated by ***p < 0.001 and ****p < 0.0001.

Fig. 6. Neural stem cell (NSCs) labeling in vitro. (a) Cell viability under different ultrasound acoustic pressure (2.50, 4.40, 6.00, 7.65 kPa); stability of assembled nanobubbles under ultrasound exposure (b), and in cell incubator at different time points (0, 6, 14, 24, 48 h) (c); (d) Prussian blue staining of no treatment of control cells and cells treated by nanoparticles loaded nanobubbles under the 4.40 kpa ultrasound exposure for 40 s; (e) iron concentration in one cell treated with different concentrations of iron in assembled nanobubbles or with different labeling method, and measured by ICP-OES; (f) TEM images of NSCs with and without nanoparticles loaded nanobubbles treatment. A higher-magnification image of the indicated portion is shown in the right panel. All bars represent mean ± SD, and three independent experiments (n = 3) were performed; and the statistical significance is indicated by **p < 0.01 and *p < 0.05. NSCs: Neural stem cells; SPIONs: Superparamagnetic iron oxide nanoparticles.