An azo substituted quinoline-malononitrile enzyme-activable aggregation-induced emission nanoprobe for hypoxia imaging

Abstract

The development of efficient aggregation-induced emission (AIE) active probes is crucial for disease diagnosis, particularly for tumors and cardiovascular diseases. Current AIE-active probes primarily focus on improving their water solubility to resist aggregation, thereby achieving an initial fluorescence-off state. However, the complex biological environment can cause undesirable aggregation, resulting in false signals. To address this issue, we have ingeniously introduced an azo group into the AIE luminogen (AIEgen), developing a reductase-activated AIE probe, Azo-quinoline-malononitrile (QM)-PN, for imaging hypoxic environments. In this probe, the azo group promotes intramolecular motion through rapid E/Z isomerization, causing the excited state energy to dissipate via non-radiative decay, thus turning off the initial fluorescence. In the presence of reductase, Azo-QM-PN is reduced and cleaved to produce the hydrophobic AIEgen NH₂-QM-PN, which subsequently aggregates and generates an in situ AIE signal, thereby imaging the hypoxic environment with reductase. Encapsulation of Azo-QM-PN with DSPE-PEG₂₀₀₀ results in the formation of the nanoprobe Azo-QM-PN NPs, which can effectively penetrate cell membranes, specifically illuminate tumor cells, monitor fluctuations in azo reductase levels, and deeply penetrate and image multicellular tumor spheroids, demonstrating potential for hypoxic tumor imaging. Additionally, the nanoprobe Azo-QM-PN NPs can selectively image hypoxic atherosclerotic plaque tissues, showing potential for detecting atherosclerosis. Therefore, in this study, we successfully developed an enzyme-activated AIE probe for imaging hypoxic environments, laying the foundation for further clinical applications.

Keywords: AIE‐active; fluorescent probe; hypoxia imaging.

FIGURE 1

Design strategy for azo‐derived AIE‐active probes for hypoxia imaging. (a) Molecular structure of Azo‐QM‐PN and its response mechanism with reductase. (b) Encapsulation of Azo‐QM‐PN with DSPE‐PEG₂₀₀₀ to prepare AIE nanoprobe Azo‐QM‐PN NPs, enhancing cellular penetration efficiency. AIE, aggregation‐induced emission; QM, quinoline‐malononitrile.

FIGURE 2

Spectral properties of NH₂‐QM‐PN and Azo‐QM‐PN. (a, d) Absorbance of Azo‐QM‐PN and NH₂‐QM‐PN in water, respectively. (b, e) Emission spectra of Azo‐QM‐PN and NH₂‐QM‐PN with different water fractions (f _w ) in the mixture of the THF‐water system. (c, f) I/I ₀ plots of Azo‐QM‐PN and NH₂‐QM‐PN, where I is the fluorescence intensity at 660 nm and I ₀ is the fluorescence intensity of Azo‐QM‐PN and NH₂‐QM‐PN in 0% water. QM, quinoline‐malononitrile.

FIGURE 3

Fluorescence response of Azo‐QM‐PN to Na₂S₂O₄. (a) Time‐dependent response spectra of Azo‐QM‐PN (10 μM) to sodium dithionite (50 μM). (b) I/I ₀ plot of Azo‐QM‐PN in THF/water mixtures (THF: water = 1: 4, v/v), λ _ex = 460 nm. I ₀ presents the initial fluorescence intensity of Azo‐QM‐PN in the mixed solution. (c) High‐resolution mass spectrometry was used to validate the degradation mechanism of Azo‐QM‐PN. QM, quinoline‐malononitrile.

FIGURE 4

Imaging hypoxic tumor cells. (a) Preparation of Azo‐QM‐PN NPs. (b, c) Dynamic light scattering (DLS) of Azo‐QM‐PN and Azo‐QM‐PN NPs. (d) Cell viability of Azo‐QM‐PN and Azo‐QM‐PN NPs. (e, f) Imaging normoxia and hypoxia cells after incubating with Azo‐QM‐PN NPs for different times. (g) Time‐dependent fluorescence intensity of normoxia and hypoxia cells. (h, i) Hypoxic HeLa cells treated with or without diphenyl iodide chloride (DPI, an inhibitor of azoreductase) and their fluorescence intensity statistics. (j) Fluorescence images of HeLa multicellular tumor spheroids treated with Azo‐QM‐PN NPs at different depths. QM, quinoline‐malononitrile.

FIGURE 5

Fluorescence imaging of frozen sections of the tricuspid valve and aortic arch in atherosclerotic mice. (a) Schematic diagram of imaging. (b) and (d) Fluorescence imaging of frozen sections of the tricuspid valve and aortic arch. (c) and (e) Fluorescence intensity changes.