Formulation and characterization of echogenic lipid-Pluronic nanobubbles

Abstract

The advent of microbubble contrast agents has enhanced the capabilities of ultrasound as a medical imaging modality and stimulated innovative strategies for ultrasound-mediated drug and gene delivery. While the utilization of microbubbles as carrier vehicles has shown encouraging results in cancer therapy, their applicability has been limited by a large size which typically confines them to the vasculature. To enhance their multifunctional contrast and delivery capacity, it is critical to reduce bubble size to the nanometer range without reducing echogenicity. In this work, we present a novel strategy for formulation of nanosized, echogenic lipid bubbles by incorporating the surfactant Pluronic, a triblock copolymer of ethylene oxide copropylene oxide coethylene oxide into the formulation. Five Pluronics (L31, L61, L81, L64 and P85) with a range of molecular weights (M(w): 1100 to 4600 Da) were incorporated into the lipid shell either before or after lipid film hydration and before addition of perfluorocarbon gas. Results demonstrate that Pluronic-lipid interactions lead to a significantly reduced bubble size. Among the tested formulations, bubbles made with Pluronic L61 were the smallest with a mean hydrodynamic diameter of 207.9 +/- 74.7 nm compared to the 880.9 +/- 127.6 nm control bubbles. Pluronic L81 also significantly reduced bubble size to 406.8 +/- 21.0 nm. We conclude that Pluronic is effective in lipid bubble size control, and Pluronic M(w), hydrophilic-lipophilic balance (HLB), and Pluronic/lipid ratio are critical determinants of the bubble size. Most importantly, our results have shown that although the bubbles are nanosized, their stability and in vitro and in vivo echogenicity are not compromised. The resulting nanobubbles may be better suited for contrast enhanced tumor imaging and subsequent therapeutic delivery.

Figure 1

Bubble size in the presence of 0.006, 0.06, 0.6, and 6 mg/mL of Pluronic that was incorporated into the bubble formulation before (A: prefilm) or after (B: postfilm) lipid film hydration (mean ± SEM; n = 3). The symbol † indicates statistically significant difference compared to control (P: 0.001–0.01).

Figure 2

Bubble size distribution change as a function of time. Data presented for control bubbles and bubbles with 0.6 mg/mL of Pluronic (mean ± SEM; n = 3). (A) Prefilm; (B) postfilm.

Figure 3

Bubble concentration change as a function of time (A, prefilm; B, postfilm; P: 0.0001–0.023). Data presented as mean ± SEM (n = 3). The symbol ¥ indicates the only condition that showed no significant difference at t = 60 min vs t = 0 (P = 0.1).

Figure 4

Representative control, Pluronic bubble grayscale ultrasound images in vitro in custom-made agarose gel mold and experimental setup (A); dashed line indicates sample well; (B) US image of H₂O; (C) bubbles with 0.6 mg/mL of L31, L61, L81, L64 and P85 at same dilutions; (D) control, bubbles with 0.6 mg/mL of L31, L61, L81, L64 and P85 at equivalent bubble concentrations.

Figure 5

Quantitative analysis of grayscale ultrasound signal intensity of bubbles in the presence of 0.006, 0.06, 0.6, and 6 mg/mL of Pluronic that were incorporated in the formulation before or after lipid film hydration (mean ± SEM; n = 3). The symbol * indicates statistically significantly higher compared to control (P: 0.0006–0.008); and ‡ indicates statistically significant differences compared to postfilm bubble signals under the same conditions (P = 0.003). (A) L31; (B) L61; (C) L81; (D) L64; (E) P85.

Figure 6

In vitro bubble stability. (A) Representative ultrasound images of control microbubbles and (B) L61 nanobubbles over 30 min; (C) quantatitative grayscale ultrasound signal intensity (% of initial value). The initial values of the bubble grayscale signal intensities were 79.1 ± 3.0 for control and 74.8 ± 16.3 for L61 bubbles (mean ± SEM; n = 3). The symbol * indicates statistically significant difference compared to initial value (P = 0.0006).

Figure 7

Bubble performance in vivo in rat tumor (perfusion imaging). (A) Representative subcutaneous tumor; (B) an example of the mosaic image used to determine quality of fit for the registration; (C) ultrasound images at t = 0, 10 s, 5 and 20 min after injection of control or L61 bubbles; dotted lines outline the tumors; (D) quantitative summary of tumor enhancement after contrast administration presented as fold of increase in signal intensity relative to baseline images.

Figure 8

Tumor microflow imaging. (A) Representative subcutaneous tumor and experimental setup; (B) microflow images of tumor after L61 nanobubbles; (C) the same tumor after control microbubble administration. Dashed lines indicate the tumor location. Baseline image is the first video frame immediately after flash.

Figure 9

Bubble size dependence on Pluronic concentration. (A) L61; (B) L81.