Shortwave infrared emitting multicolored nanoprobes for biomarker-specific cancer imaging in vivo

Abstract

Background The ability to detect tumor-specific biomarkers in real-time using optical imaging plays a critical role in preclinical studies aimed at evaluating drug safety and treatment response. In this study, we engineered an imaging platform capable of targeting different tumor biomarkers using a multi-colored library of nanoprobes. These probes contain rare-earth elements that emit light in the short-wave infrared (SWIR) wavelength region (900–1700 nm), which exhibits reduced absorption and scattering compared to visible and NIR, and are rendered biocompatible by encapsulation in human serum albumin. The spectrally distinct emissions of the holmium (Ho), erbium (Er), and thulium (Tm) cations that constitute the cores of these nanoprobes make them attractive candidates for optical molecular imaging of multiple disease biomarkers. Methods SWIR-emitting rare-earth-doped albumin nanocomposites (ReANCs) were synthesized using controlled coacervation, with visible light-emitting fluorophores additionally incorporated during the crosslinking phase for validation purposes. Specifically, HoANCs, ErANCs, and TmANCs were co-labeled with rhodamine-B, FITC, and Alexa Fluor 647 dyes respectively. These Rh-HoANCs, FITC-ErANCs, and 647-TmANCs were further conjugated with the targeting ligands daidzein, AMD3100, and folic acid respectively. Binding specificities of each nanoprobe to distinct cellular subsets were established by in vitro uptake studies. Quantitative whole-body SWIR imaging of subcutaneous tumor bearing mice was used to validate the in vivo targeting ability of these nanoprobes. Results Each of the three ligand-functionalized nanoprobes showed significantly higher uptake in the targeted cell line compared to untargeted probes. Increased accumulation of tumor-specific nanoprobes was also measured relative to untargeted probes in subcutaneous tumor models of breast (4175 and MCF-7) and ovarian cancer (SKOV3). Preferential accumulation of tumor-specific nanoprobes was also observed in tumors overexpressing targeted biomarkers in mice bearing molecularly-distinct bilateral subcutaneous tumors, as evidenced by significantly higher signal intensities on SWIR imaging. Conclusions The results from this study show that tumors can be detected in vivo using a set of targeted multispectral SWIR-emitting nanoprobes. Significantly, these nanoprobes enabled imaging of biomarkers in mice bearing bilateral tumors with distinct molecular phenotypes. The findings from this study provide a foundation for optical molecular imaging of heterogeneous tumors and for studying the response of these complex lesions to targeted therapy.

Shortwave‐Infrared‐Emitting Nanoprobes for CD8 Targeting and In Vivo Imaging of Cytotoxic T Cells in Breast Cancer

“…SWIR‐emitting nanocomposites have multispectral capabilities based on the RE dopant. [ 22,27 ] Future studies can address the issue of delineating signal from nonspecific binding through multispectral imaging using a mix of differently doped CD8‐targeted and isotype control‐conjugated ReANCs to unravel the actual signal from that of nonspecific uptake through spectral unmixing. [ 50 ]…”

“…This is consistent with similar studies that have reported nonlinear relationships between ligand density and target cell binding. [24,27,36] Among the tested CD8-ANCs, those conjugated with a loading density of 28.6 μg IgG mg À1 HSA had the most significant binding effect on CTLs, followed by a loading density of 42.9 μg IgG mg À1 HSA. We selected nanocomposites with the loading density of 28.6 μg IgG mg À1 HSA for all other experiments based on these results.…”

“…SWIR-emitting nanocomposites have multispectral capabilities based on the RE dopant. [22,27] Future studies can address the issue of delineating signal from nonspecific binding through multispectral imaging using a mix of differently doped CD8-targeted and isotype control-conjugated ReANCs to unravel the actual signal from that of nonspecific uptake through spectral unmixing. [50] CD8-targeted ReANCs can be used for monitoring of genetically engineered immune cells in an adoptive cellular therapy setting and track their trafficking to immunogenic tumors.…”

“…[22] Compared to the light emitted by visible or NIR-I (λ = 700-1000 nm) probes, SWIR light is less scattered by tissue, and the captured signal is not confounded by tissue autofluorescence, thus allowing for greater tissue penetration depth (up to 1 cm deep in a mouse) with high signal-to-noise ratios. [23][24][25] Drug-binding pockets and functional groups amenable to chemical conjugation on the albumin shell allow for functionalization, or targeting to disease-specific cellular markers, [26,27] facilitating precision targeting and in vivo imaging of small cellular clusters. [23,24] Tumor-targeted ReANCs have been shown to detect tumors earlier than imaging modalities such as MRI and can monitor therapy response in real time in small animal models of human cancer.…”

Review of in vivo optical molecular imaging and sensing from x-ray excitation

“…50,53,54 There is a high volume of research in this area, and multicolor emitters are available. 55,56 Detectors for SWIR wavelengths are often different than those for the red or NIR wavelengths, 57 and so it is feasible to use separated detectors for parallelization or spectral decomposition through a spectrometer. 58,59 Time-gated detection can be coupled with wavelength-based separation of the signals as well, to further maximize the ability to detect signals at the same time, and scintillation-based signals can be orders of magnitude faster than organic fluorescence, which in turn is orders of magnitude faster than phosphorescent signals.…”