Abstract
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Objectives Fluorescence imaging in the second near-infrared window (NIR-II, 1000 - 1700 nm) instead of the traditional NIR-I window (650 - 950 nm) has been attracting considerable attention for medical imaging because of its superior temporal resolutions, reduced scattering, deep tissue penetration, and negligible tissue autofluorescence. As an emerging hybrid molecular imaging modality, Photoacoustic (PA) imaging integrates optical excitation with ultrasonic detection and takes advantage of the respective strengths of optical and acoustic imaging to provide tomographic images of abnormalities with excellent spatial resolution. Our development of multifunctional dual-modal imaging probes aims to integrate the benefits both NIR-II fluorescence and photoacoustic imaging, with the ultimate goal of improving overall diagnosis of cancer such as thyroid carcinoma.
Methods Herein we designed a donor-acceptor chromophore based nanoparticle (DAP) which has an absorption in NIR-I window and a fluorescence peak in NIR-II region. The resultant dual-modal nanoprobes composed of D-π-A-π-D type chromophores were PEGylated through nanoprecipitation. The surface of dual nanoprobes was then conjugated with EGFR Affibody (Ac-Cys-ZEGFR:1907) for selectively targeting EGFR-positive thyroid cancers. Affibody-DAPs were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Specific uptake and cellular internalization of EGFR-DAPs were evaluated by PA and NIR-II fluorescence imaging. PA and fluorescent signals were measured by an Endra system and a customized NIR-II fluorescence imaging system (Princeton Instruments). To characterize their in vivo behavior and tumor targeting properties, the dual PA and NIR-II fluorescence imaging and ex-vivo biodistribution were performed in FTC-133 thyroid tumor-bearing nude mice (two groups, each group n = 4).
Results The resultant Affibody-DAPs are highly monodisperse particles with an average size of 24 nm, strong absorption in the NIR-I window (at c.a. 680 nm) and a relatively high fluorescence in the NIR-II region (peak maximum at 1000 nm). They also showed significantly enhanced photoacoustic and NIR-II fluorescence contrast effects in both phantom and small animal imaging experiments. Compared to single water-soluble chromophore, the chromophore-incorporated nanoprobes possessed much higher quantum yield (~40x) and brightness at the same excitation wavelength. NIR-II fluorescence imaging is capable of a fast wide-field acquisition rate (10 to 100 frames per second), and PA imaging has high diffraction-limited spatial resolution (20~50 microns). The PA and NIR-II fluorescence signals produced by the Affibody-DAPs were highly linear and correlated with each other. The studies of in vivo pharmacokinetics and biodistribution of nanoprobes showed that Affibody-DAPs had high liver and spleen uptakes. Hepatic clearance was observed and associated with the elimination of the nanoprobes from mice. The PA imaging provided functional and molecular information of tumors with high spatial resolution, which was well correlated with the results obtained by NIR-II fluorescent images.
Conclusions The multifunctional donor-acceptor chromophore based nanoparticles display the unique ability to combine PA and NIR-II fluorescence imaging for tumor detection. Affibody-conjugated probes have the capability to bind EGFR-positive tumors in FTC-133 subcutaneous mouse model with relatively high photoacoustic and fluorescence signals. With the combination of high spatial resolution and excellent temporal resolution, targeted photoacoustic/NIR-II fluorescence imaging with dual modal contrast agents allow us to specifically image and detect thyroid carcinomas at the early stage in an accurate and quantitative manner.