Reversibly Photoswitching Upconversion Nanoparticles for Super-Sensitive Photoacoustic Molecular Imaging

Abstract

Photoacoustic (PA) imaging uses light excitation to generate the acoustic signal for detection and improves tissue penetration depth and spatial resolution in the clinically relevant depth of living subjects. However, strong background signals from blood and pigments have significantly compromised the sensitivity of PA imaging with exogenous contrast agents. Here we report a nanoparticle-based probe design that uses light to reversibly modulate the PA emission to enable photoacoustic photoswitching imaging (PAPSI) in living mice. Such a nanoprobe is built with upconverting nanocrystals and photoswitchable small molecules and can be switched on by NIR light through upconversion to UV energy. Reversibly photoswitching of the nanoprobe reliably removed strong tissue background, increased the contrast-to-noise ratio, and thus improved imaging sensitivity. We have shown that PAPSI can image 0.05 nM of the nanoprobe in hemoglobin solutions and 10⁴ labeled cancer cells after implantation in living mice using a commercial PA imager.

Keywords: Imaging Agents; Near-infrared; Photoacoustic Imaging; Reversible Photoswitching; Upconversion Nanoparticles.

Figure 1.

Characterization of the photoswitchable small molecules (3ThacacH) and UCNPs. a) Absorption spectra of 3ThacacH in CHCl₃ (0.1 mM) when it was switched ‘ON’ from the initial open form (black) to the closed form (red) by 365 nm light (4 W UV lamp, 10 s) and then switched OFF to the open form (blue) by 640 nm laser (10 mW/cm², 10 s). Insert: The color of the sample solution changed from colorless to green then back to colorless when switched ON and OFF. b) Evolution of the absorption intensity of 3ThacacH at 640 nm when it was repeatedly switched ON/OFF by 365 nm and 640 nm light. c) TEM images of the UCNP. Left: The NaYF₄ UCNP core doped with Yb³⁺ and Tm³⁺; Right: The UCNP core coated with an inert NaYF₄ shell. d-e) Overlay of the emission spectrum of the UCNP and the absorption spectrum of 3ThacacH in d) open or e) closed form. f) 3ThacacH (0.1 mM in CHCl₃) was switched ON when mixed with the UCNP (0.5 mg mL⁻¹) and excited with CW 980 nm laser (average power density at 3 W/cm²).

Figure 2.

Synthesis and characterization of the photoswitchable nanoprobe. a) Schematic of the photoswitchable nanoprobe and chemical structure of the amphiphilic polymer. b) Hydrodynamic diameter of the photoswitchable nanoprobe. c) Kinetic of the nanoprobe (2.5 nM in water) photoswitching by 980 nm laser (average power density 3 W/cm²) and 640 nm laser (10 mW/cm²). d) Absorbance of the nanoprobe (2.5 nM in water) at 640 nm upon irradiation by 980 nm laser for 1 min at various average power densities (3 to 0.024 W/cm²). e) A PAPSI cycle: A 980 nm laser switches ON the probe (1 min), and a 680 nm pulsed laser (7 ns pulses, 3 mJ, 20 Hz) collects PA image and also gradually switches OFF the probe (1.9 min). f) Representative OFF/ON PAPSI images of the nanoprobe (5 nM, 100 μL) in a transparent plastic tube. g) Normalized PA signal amplitude of PAPSI images in f). h-i) PAPSI of the nanoprobe mixed with Hb solution in a transparent plastic tube at various nanoprobe concentrations (h): 2.5 nM and (i): 1.25 to 0.05 nM. Scale bars: 2 mm.

Figure 3.

Cell labeling with the nanoprobe and PAPSI in the cells. a) Fluorescence images of the HeLa cells incubated with the nanoprobe (0.125 nM) for 2, 6, or 10 h at 37 °C. Scale bar: 20 μm. b, c) Number of nanoprobes taken up by the cells determined by ICP-MS at b) different incubation time (2–10 h) with the same probe concentration (0.125 nM) or c) different probe concentrations (0.0625–2 nM) with the same incubation time (6 h). Results are presented as Mean ± SD (three wells per group in 6-well cell culture plates). d-g) PAPSI images of labeled HeLa cells (2 × 10⁴ NPs per cell) in 10 μL PBS (pH 7.4) in a transparent plastic tube at indicated cell number. d): 10⁶; e): 10⁵, f): 2 × 10⁴; g): 5 × 10³ with indicated PAPSI cycle number. Scale bars: 2 mm.

Figure 4.

Tracking of HeLa cells labeled with the PAPSI nanoprobe in the living mice. a) 10⁶, b) 10⁵, c) 10⁴ labeled cells were subcutaneously injected into the back of the NU/NU nude mice (three mice per group) and imaged by PAPSI with the cycle number of 5, 10, and 22, respectively. For each group, the stacked image of the differential images in all PAPSI cycles (“Summed”) is demonstrated and overlaid with the endogenous PA signals (the OFF image of the first PAPSI cycle). The white dashed circles indicate the predetermined position of the injected cells. Scale bar: 2 mm.

Scheme 1.

Principle of PAPSI and design of the photoswitchable nanoprobe. a) The working principle of PAPSI. PA imaging is first taken at the PA inactive state of the nanoprobe to collect the background signal. Then the switching laser (λ₁) is applied to turn ON the probe to PA active state, followed by another PA imaging. The differential image produced by subtraction should only represent the probe signal. Then the probe is switched back to PA inactive state by another laser (λ₂) to start the next cycle of PA imaging. Many differential images can be stacked to increase the probe signal and improve PA imaging sensitivity. b) Design of the NIR photoswitchable nanoprobe. A photoswitchable small molecule whose NIR absorption can be switched ON by UV light and switched OFF by NIR light (λ₂) is combined with UCNP to form the nanoprobe. The UCNP can convert NIR light (λ₁) to UV light and switch ON the photoswitchable small molecule. c) Chemical structure of the photoswitchable small molecule (3ThacacH) that can be switched between the open form to the closed form by UV and red light.