Functional nanocrystal as effective contrast agents for dual-mode imaging: Live-cell sonoluminescence and contrast-enhanced echography

Abstract

In the context of molecular imaging, the present work explores an innovative platform made of lipid-coated nanocrystals as contrast-enhanced agent for both ultrasound imaging and sonoluminescence. At first, the dynamics of gas bubbles generation and cavitation under insonation with either pristine or lipid-coated nanocrystals (ZnO-Lip) are described, and the differences between the two colloidal systems are highlighted. These ZnO-Lip show an unprecedented ability to assist cavitation, which is reflected in enhanced sonoluminescent light emission with respect to the pristine nanocrystals or the pure water. Highly defined and sharp sonoluminescent images of cultured cells are indeed obtained, for the first time, when ZnO-Lip are used. Furthermore, ZnO-Lip were adopted as a nanosized agent for contrast-enhanced ultrasound imaging, i.e. echography, first in solutions, and then on ex-vivo tissues. A prolonged over time and bright imaging effect is observed when adopting the developed nanoparticles. Furthermore, their nanometric size and potential targeting with biomolecules would allow ease extravasation and tissue or even cell penetration, achieving enhanced-contrast imaging. Finally, the stimuli-responsive therapeutic applications of ZnO-Lip against tumors is overviewed, aiming to achieve a fully theranostic approach.

Keywords: Bubble cavitation; Echographic nano-contrast agent; Inertial cavitation; Sonoluminescence; Zinc oxide.

Graphical abstract

Fig. 1

Schematic representation of the acquisition and analysis of sonoluminescent images.

Fig. 2

(A) Schematic representation of ZnO and ZnO-Lip structures and composition. (B) TEM images of ZnO NCs and (C) ZnO-Lip. (D) XRD analysis of ZnO NCs and (E) DLS Number measurement of ZnO and ZnO-Lip in water at the concentration of 100 μg/mL.

Fig. 3

(A) Schematic representation and ultra-high-speed images of ultrasound driven bubbles, initially trapped at the surface of ZnO particles. (B) Acoustic attenuation spectra measured for a suspension of ZnO NCs at a peak-negative acoustic pressure amplitude of 95 kPa measured successively for 6 times. (C) Bubble radius in time and (D) bubble amplitude in frequency obtained with MATLAB analysis of representative recorded video.

Fig. 4

(A) Schematic representation and recorded images of bubble dynamics in presence of ZnO-Lip. (B) Bubble attenuation signals in presence of ZnO-Lip at 95 kPa measured in subsequent pressure cycles. DLS Intensity measurement of ZnO and ZnO-Lip (C) pre and (D) post 120 s of ultrasound exposure at 2 W/cm², 1 MHz, 100 % DC.

Fig. 5

(A) Sonoluminescence signal obtained in pure water and water in presence of ZnO with a concentration of (B) 100 μg/mL and (C) 200 μg/mL, and in presence of ZnO-Lip with a concentration of (D) 100 μg/mL and (E) 200 μg/m. The sonoluminescence signal of pure water irradiated with US was subtracted as background from each image. Intensity pixel distribution of all the sonoluminescence images obtained with (F) ZnO and ZnO-Lip, of the images obtained with (G) different concentration of ZnO, of the images obtained with (H) different concentration of ZnO-Lip, of the images obtained with (I) 100 μg/mL of ZnO and ZnO-Lip and of the images obtained with (J) 100 μg/mL of ZnO and ZnO-Lip. Each tested solution was irradiated for 120 s with 2 W/cm², 1 MHz and 100 %DC.

Fig. 6

(A) Sonoluminescence signal obtained in complete RPMI cell culture medium, and complete RPMI cell culture medium in presence of ZnO with a concentration of (B) 100 μg/mL and (C) 200 μg/mL, and in presence of ZnO-Lip with a concentration of (D) 100 μg/mL and (E) 200 μg/m. The sonoluminescence signal of pure water irradiated with US was subtracted as background from each image. Intensity pixel distribution of all the sonoluminescence images obtained with (F) ZnO and ZnO-Lip, of the images obtained with (G) different concentration of ZnO, of the images obtained with (H) different concentration of ZnO-Lip, of the images obtained with (I) 100 μg/mL of ZnO and ZnO-Lip and of the images obtained with (J) 100 μg/mL of ZnO and ZnO-Lip. Each tested solution was irradiated for 120 s with 2 W/cm², 1 MHz and 100 %DC.

Fig. 7

HT-29 cells, in complete RPMI cell culture medium, captured by CCD camera after 120 s of acquisition. (A) and (C) show the images of cells acquired with ambient light, (B) is obtained in the dark thank to the sonoluminescent (SL) signal, and (D) is obtained in the dark thank to the light emitted by the sonoluminescent phenomenon augmented by the presence of 100 μg/mL of ZnO-Lip. Samples (B) and (D) were irradiated for 120 s with 2 W/cm², 1 MHz and 100 %DC.

Fig. 8

(A) Echographic contrast signal of water, water with 100 μg/mL of ZnO and ZnO-Lip and (B) complete growth medium, i.e. RPMI, RPMI with 100 μg/mL of ZnO and ZnO-Lip when irradiated with ultrasound equal to 2 W/cm², 1 MHz and 100 % DC. White circles indicated the areas with the highest contrast signal.

Fig. 9

Echographic contrast signal of a mock tissue injecting water (at time 0), ZnO suspension (at time 0 and at minute 5) and ZnO-Lip suspension (at minutes 5 and 10). The analysis over time at (A) time 0, (B) after 5 min, and (C) after 10 min allowed to evaluate the signal persistence.

Fig. 10

Echographic contrast signal of an ex-vivo tissue pre and post injection of water, and ZnO and ZnO-Lip suspensions.