Nanobubbles for enhanced ultrasound imaging of tumors

Abstract

The fabrication and initial applications of nanobubbles (NBs) have shown promising results in recent years. A small particle size is a basic requirement for ultrasound contrast-enhanced agents that penetrate tumor blood vessel pores to allow for targeted imaging and therapy. However, the nanoscale size of the particles used has the disadvantage of weakening the imaging ability of clinical diagnostic ultrasound. In this work, we fabricated a lipid NBs contrast-enhanced ultrasound agent and evaluated its passive targeting ability in vivo. The results showed that the NBs were small (436.8 ± 5.7 nm), and in vitro ultrasound imaging suggested that the ultrasonic imaging ability is comparable to that of microbubbles (MBs). In vivo experiments confirmed the ability of NBs to passively target tumor tissues. The NBs remained in the tumor area for a longer period because they exhibited enhanced permeability and retention. Direct evidence was obtained by direct observation of red fluorescence-dyed NBs in tumor tissue using confocal laser scanning microscopy. We have demonstrated the ability to fabricate NBs that can be used for the in vivo contrast-enhanced imaging of tumor tissue and that have potential for drug/gene delivery.

Keywords: contrast agent; phospholipids; tumor-targeted; ultrasound.

Figure 1

Formation and structural transitions of nanobubbles for ultrasonic imaging and tumor targeting. Abbreviations: DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphate; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; PEG-DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[biotinyl(polyethylene glycol)2000].

Figure 2

Custom-made 2% (w/v) agarose mold for use with in vitro ultrasound imaging and the in vitro experimental setup.

Figure 3

Particle size and morphology of the nanobubbles. The diameter distribution was measured using dynamic light scattering in the nanobubbles (A) and microbubbles (B). The surface morphology of the nanobubbles was visualized using scanning electron microscopy (C).

Figure 4

In vitro cytotoxicity of various concentrations of nanobubbles in RM-1 cells determined using the MTT assay. The hemolysis rate of erythrocyte suspensions incubated in the presence of various amounts of nanobubbles.

Figure 5

Representative in vitro ultrasonic images (A) of nanobubbles (NBs) and microbubbles (MBs) at high-frequency diagnostic ultrasound in contrast pulse sequencing mode. (B) Quantitative gray-scale ultrasonic intensity (ratio change over gas-free water). The initial gray-scale value was obtained from gas-free water in the sample wells. NBs presented similar gray-scale intensity to MBs at 7 MHz (P = 0.134). (C) Pre- and postdestruction of NBs low-frequency ultrasound exposure.

Figure 6

Contrast pulse sequencing-mode images of various organs of normal rats. Images after nanobubble injection (right) showed obvious contrast enhancement in the heart, kidney and liver of Sprague–Dawley rats compared with preinjection images (left).

Figure 7

In vivo passive tumor targeting. Representative subcutaneous tumor images before (blue dotted line) and after the injection of nanobubbles (NBs) (A) compared with microbubbles (MBs) (B) at various time points (0, 0.5, 1, 5, 10, and 15 minutes). The corresponding time–intensity curve of tumor enhancement after injection of the contrast agent (C).

Figure 8

Confocal laser-scanning microscopy images of frozen sections after nuclear labeling. A considerable number of DiI-labeled nanobubbles are observed in the intercellular space (A), whereas DiI-labeled microbubbles are hardly visible in tumors (B). Both DiI-labeled nanobubbles and microbubbles were difficult to detect in skeletal muscle (C and D).