Innovative NIR fluorescent probes for an improved tumor detection in vivo

Abstract

There is a great demand for the development of highly fluorescent and specific probes that enable a sensitive and early detection of malignancies and a separation from healthy tissue in a non-invasive way by in vivo imaging. The main drawback of fluorescence imaging in vivo is the autofluorescence, which hampers the detection of probe-derived signals. In this study, two novel fluorescence probes were investigated concerning their suitability for visualization of tumors in mice: a pH-sensitive dye, CypHer5E conjugated to the tumor specific antibody Herceptin (pH-Her) as well as polystyrene nanoparticles (NPs) of different sizes (15, 25, and 100 nm) and surface modifications (NH2, polyethylene glycol (PEG), and Herceptin) loaded with the broadband fluorophore Itrybe. For this purpose, time-domain near-infrared (NIR) fluorescence imaging was applied in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast tumor xenografts. The pH-sensitivity and functionality of pH-Her was confirmed by spectroscopic measurements and in cell-based assays. In HER2-positive tumor-bearing mice, pH-Her, which increases fluorescence in the acidic environment of the tumor only, combined with subtraction of autofluorescence led to an increased contrast to noise ratio (CNR) and thereby an improved sensitivity of tumor detection, when compared to the always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). In contrast, LT-gated imaging with pH-Her and Alexa-Her did not improve tumor-detection sensitivity in vivo. The broad excitation and emission spectra of Itrybe enclosed in NPs enable excitation and sensitive detection of this dye at different wavelengths in the NIR region, in vivo. Furthermore, Herceptin-conjugated 100 nm NPs bind specifically to HER2 as demonstrated in immunoassays as well as on KPL-4 tumor cells and -sections in vitro. However, a certain amount of unspecific binding/uptake was observed. In vivo accumulation of PEGylated NPs of all sizes was detected in MDA-MB-231- but not in KPL-4 tumors as shown by ex vivo scans of excised tumors. Biodistribution analyses in healthy mice illustrated a high and rapid uptake of NPs of all sizes in the liver, accompanied by a fast elimination of NPs from the blood, thereby reducing efficient tumor accumulation. In conclusion, the pH-sensitive dye, CypHer5E in combination with tumor-specific ligands is highly suitable for sensitive in vivo monitoring of tumors and may also be a promising tool for the detection of weak signals deriving e.g. from small metastatic lesions. Highly fluorescent Itrybe-loaded NPs have great potential for imaging applications as they provide 3D platforms for various modifications and thus help to address different biological questions in vivo. However, the bioavailability of Itrybe-loaded NPs has to be improved by modification of their surface or composition to enable efficient tumor targeting in vivo.