Activatable Hybrid Polyphosphazene-AuNP Nanoprobe for ROS Detection by Bimodal PA/CT Imaging

Abstract

Overproduction of reactive oxygen species (ROS) is often related to inflammation or cancer and can cause tissue damage. Probes that have been previously reported to image ROS typically rely on imaging techniques that have low depth penetration in tissue, thus limiting their use to superficial disease sites. We report herein a novel formulation of hybrid nanogels loaded with gold nanoparticles (AuNP) to produce contrast for computed tomography (CT) and photoacoustics (PA), both being deep-tissue imaging techniques. The polyphosphazene polymer has been designed to selectively degrade upon ROS exposure, which triggers a switch-off of the PA signal by AuNP disassembly. This ROS-triggered degradation of the nanoprobes leads to a significant decrease in the PA contrast, thus allowing ratiometric ROS imaging by comparing the PA to CT signal. Furthermore, ROS imaging using these nanoprobes was applied to an in vitro model of inflammation, that is, LPS-stimulated macrophages, where ROS-triggered disassembly of the nanoprobe was confirmed via reduction of the PA signal. In summary, these hybrid nanoprobes are a novel responsive imaging agent that have the potential to image ROS overproduction by comparing PA to CT contrast.

Keywords: ROS; computed tomography; gold nanoparticles; photoacoustic; polyphosphazene.

Figure 1.

Assembly of AuNP in nanogels that selectively degrade under ROS exposure and release free AuNP for imaging of ROS with PA.

Figure 2.

(A)–(C) representative TEM of AuNP (left), PPB NP (center), and PCPP NP (right). (D) UV–visible spectra of the PPB NP (red) compared to the free AuNP (orange) and control PCPP NP (blue); (E) FT-IR spectrum of ROS-sensitive NP (red) compared to the free PPB polymer (grey) and PCPP NP (blue).

Figure 3.

Effects of 10 mM H₂O₂ on the degradation of PPB NP and the control PCPP NP after 48 h. (A) Cumulative gold released determined by ICP-OES. (B) UV–visible spectra. Representative TEM of (C) PPB NP in 10 mM H₂O₂, (D) PPB NP without H₂O₂, and (E) PCPP NP in 10 mM H₂O₂. The symbol *** indicates a statistically significant difference at p < 0.001.

Figure 4.

Effect of H₂O₂ incubation for 48 h with PPB and PCPP NPs on their (A) PA contrast and (B) CT contrast. (C) Percentage of contrast enhancement in CT (blue) and PA (red) of the PPB or PCPP NP to control. Experiments done at a gold concentration of 1 mg·mL⁻¹. The symbols ** and ‘ns’ indicate either a statistically significant difference at p < 0.01 or nonstatistically significant differences, respectively. Error bars represent standard deviations after conversion to percentages.

Figure 5.

Detection of ROS in a stimulated RAW 264.7 monocyte model of inflammation. Representative TEM images of normal RAW 264.7 cells incubated with (A) PPB NP or (B) PCPP NP, and LPS-stimulated RAW 264.7 monocyte cells after 4 h incubation with (C) the ROS-sensitive NP or (D) the control PCPP NP. The scale bars in the insets corresponds to 200 nm.

Figure 6.

Influence of LPS stimulation on PA and CT contrast in macrophages incubated with PPB NP or the control PCPP NP. (A) PA images, (B) CT images, and (C) percentage of contrast enhancement in CT (blue) and PA (red) of the normal or stimulated cells incubated with PPB or PCPP NP. The symbols ** and ‘ns’ indicate either a statistically significant difference at p < 0.01 or nonstatistically significant differences, respectively. Error bars represent standard deviations after conversion to percentages.

Scheme 1.

Degradation Pathways of PCPP (Upper) and the ROS-Sensitive PPB (Lower) Yielding Neutral pH and Nontoxic Byproducts