Rare-Earth-Doped Nanoparticles for Short-Wave Infrared Fluorescence Bioimaging and Molecular Targeting of αVβ3-Expressing Tumors

Abstract

The use of short-wave infrared (SWIR) light for fluorescence bioimaging offers the advantage of reduced photon scattering and improved tissue penetration compared to traditional shorter wavelength imaging approaches. While several nanomaterials have been shown capable of generating SWIR emissions, rare-earth-doped nanoparticles (REs) have emerged as an exceptionally bright and biocompatible class of SWIR emitters. Here, we demonstrate SWIR imaging of REs for several applications, including lymphatic mapping, real-time monitoring of probe biodistribution, and molecular targeting of the α_vβ₃ integrin in a tumor model. We further quantified the resolution and depth penetration limits of SWIR light emitted by REs in a customized imaging unit engineered for SWIR imaging of live small animals. Our results indicate that SWIR light has broad utility for preclinical biomedical imaging and demonstrates the potential for molecular imaging using targeted REs.

Keywords: NIR-II; SWIR; advances in optical probes; cancer detection imaging; integrin; nanoparticle; near-infrared imaging; short-wave infrared.

Figure 1.

A, Rare-earth-doped nanoparticles display spherical morphology and homogenous size distribution as shown by TEM. B, Dynamic light scattering confirms uniform RE particle size distribution. C, Upon NIR excitation at 975 nm, REs displayed bright, narrow emission at 1536 nm. D, energy dispersive X-ray spectroscopy (EDS) profile of REs confirm the elemental composite of the NaYF4 nanoparticles and confirm the presence of Er, the rare-earth dopant and SWIR emitter. E, polyethylene glycolylated REs display narrow size distribution in aqueous solution and are noticeably larger in diameter than assynthesized Res. F, Transmission electron microscopy imaging of PEGylated REs show a crystalline nanoparticle encased in an amorphous shell with the boundary indicated by the red line. DLS indicates dynamic light scattering; NIR, near infrared; PEG, polyethylene glycol; RE, rare-earth doped nanoparticles; SWIR, short-wave infrared; TEM, transmission electron microscopy; YF, yttrium fluoride.

Figure 2.

A, Schematic of the small animal SWIR imaging system. B, PET chamber filled with REs and excited at 975 nm. Rod diameters counterclockwise from bottom are 4.8 mm, 4.0 mm, 3.2 mm, 2.4 mm, 1.6 mm, and 1.2 mm. C, Short-wave infrared signal from REs progressively diminished through increasing depth of phantom tissue, yet signal was still detected through 1.0 cm of tissue. D, Effects of increasing phantom depth on the SWIR resolution reveals point fluorescence could be distinguished with millimeter precision using the 4.8-mm-diameter rods as SWIR emission points. RE indicates rare-earth-doped nanoparticles; SWIR, short-wave infrared.

Figure 3.

A, Representative image showing tracking of the lymphatic vascular in the hind leg of a mouse using REs injected into the footpad. B, Raw SWIR image used to assess the resolution of SWIR signal tracked through the lymphatic vasculature. C, Reveals submillimeter, micron resolution. D, Representative SWIR image of PEGylated REs intravenously injected into a mouse exhibiting a 4T1 tumor reveals intense SWIR emissions emitted from the liver, spleen, and lymph nodes as well as certain vasculature and passive tumor accumulation. RE indicates rare-earth doped nanoparticles; SWIR, short-wave infrared; PEG, polyethylene glycol.

Figure 4.

A, Targeting the α_Vβ₃ receptor using RGD and RAD functionalized REs. Targeting of functionalized REs was first assessed and quantified in vitro using MDA-MB-231 and MCF-7 cell lines exhibiting varying expression of αVβ3. B, Short-wave infrared signal was captured using the small animal imaging system and tracked over 120 hours. C, Bioluminescence and X-ray imaging were used to identify location of the luciferase expressing U87 tumor xenografts in relation to the mouse’s anatomy. While SWIR signals were broadly distributed throughout the animals’ bodies at earlier time points before being cleared at 120 hours, tumors in mice injected with RGD-REs showed enhancement of SWIR at 48 hours. Compared to background tissue, RGD-REs showed over a 15-fold signal enhancement compared to tumors in mice injected with RAD-REs (mean values ± SEM; n = 3; *P < .01) determined by Student t test; A). RE indicates rare-earth doped nanoparticles; SEM, standard error of mean; SWIR, short-wave infrared.