The near-infrared-IIb (NIR-IIb) (1,500–1,700 nm) window is ideal for deep-tissue optical imaging in mammals, but lacks bright and biocompatible probes. Here, we developed biocompatible cubic-phase (α-phase) erbium-based rare-earth nanoparticles (ErNPs) exhibiting bright downconversion luminescence at ~1,600 nm for dynamic imaging of cancer immunotherapy in mice. We used ErNPs functionalized with cross-linked hydrophilic polymer layers attached to anti-PD-L1 (programmed cell death-1 ligand-1) antibody for molecular imaging of PD-L1 in a mouse model of colon cancer and achieved tumor-to-normal tissue signal ratios of ~40. The long luminescence lifetime of ErNPs (~4.6 ms) enabled simultaneous imaging of ErNPs and lead sulfide quantum dots emitting in the same ~1,600 nm window. In vivo NIR-IIb molecular imaging of PD-L1 and CD8 revealed cytotoxic T lymphocytes in the tumor microenvironment in response to immunotherapy, and altered CD8 signals in tumor and spleen due to immune activation. The cross-linked functionalization layer facilitated 90% ErNP excretion within 2 weeks without detectable toxicity in mice.
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Hong, G. et al. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nat. Photonics 8, 723–730 (2014).
Antaris, A. L. et al. A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging. Nat. Commun. 8, 15269 (2017).
Yang, Q. et al. Donor engineering for NIR-II molecular fluorophores with enhanced fluorescent performance. J. Am. Chem. Soc. 140, 1715–1724 (2018).
Liu, Z. et al. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotech. 2, 47–52 (2006).
Wan, H. et al. A bright organic NIR-II nanofluorophore for three-dimensional imaging into biological tissues. Nat. Commun. 9, 1171 (2018).
Bruns, O. T. et al. Next-generation in vivo optical imaging with short-wave infrared quantum dots. Nat. Biomed. Eng. 1, 0056 (2017).
Naczynski, D. J. et al. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 4, 2199 (2013).
Diao, S. et al. Fluorescence imaging in vivo at wavelengths beyond 1500 nm. Angew. Chem. 127, 14971–14975 (2015).
Zhang, M. et al. Bright quantum dots emitting at ~1,600 nm in the NIR-IIb window for deep tissue fluorescence imaging. Proc. Natl Acad. Sci. USA 115, 6590–6595 (2018).
Diao, S. et al. Biological imaging without autofluorescence in the second near-infrared region. Nano Res. 8, 3027–3034 (2015).
Franke, D. et al. Continuous injection synthesis of indium arsenide quantum dots emissive in the short-wavelength infrared. Nat. Commun. 7, 12749 (2016).
Shao, W. et al. Tunable narrow band emissions from dye-sensitized core/shell/shell nanocrystals in the second near-infrared biological window. J. Am. Chem. Soc. 138, 16192–16195 (2016).
Zou, W., Wolchok, J. D. & Chen, L. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: mechanisms, response biomarkers, and combinations. Sci. Transl. Med. 8, 328rv4 (2016).
Okazaki, T. et al. A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat. Immunol. 14, 1212–1218 (2013).
Sheng, W. et al. LSD1 ablation stimulates anti-tumor immunity and enables checkpoint blockade. Cell 174, 549–563.e519 (2018).
Butte, M. J. et al. Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 27, 111–122 (2007).
Hu, Q. et al. Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy. Nat. Biomed. Eng. 2, 831–840 (2018).
Powles, T. et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515, 558–562 (2014).
Sagiv-Barfi, I. et al. Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK. Proc. Natl Acad. Sci. USA 112, E966–E972 (2015).
Lau, J. et al. Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice. Nat. Commun. 8, 14572 (2017).
Bensch, F. et al. 89Zr-atezolizumab imaging as a non-invasive approach to assess clinical response to PD-L1 blockade in cancer. Nat. Med. 24, 1852–1858 (2018).
Herbst, R. S. et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515, 563–567 (2014).
Mall, S. et al. Immuno-PET imaging of engineered human T cells in tumors. Cancer Res. 76, 4113–4123 (2016).
Nedrow, J. R. et al. Imaging of programmed cell death ligand 1: impact of protein concentration on distribution of anti-PD-L1 SPECT agents in an immunocompetent murine model of melanoma. J. Nucl. Med. 58, 1560–1566 (2017).
Wan, H. et al. Developing a bright NIR-II fluorophore with fast renal excretion and its application in molecular imaging of immune checkpoint PD-L1. Adv. Funct. Mater. 28, 1804956 (2018).
Balar, A. V. et al. Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial. Lancet 389, 67–76 (2017).
Daud, A. I. et al. Programmed death-ligand 1 expression and response to the anti-programmed death 1 antibody pembrolizumab in melanoma. J. Clin. Oncol. 34, 4102–4109 (2016).
Shen, X. & Zhao, B. Efficacy of PD-1 or PD-L1 inhibitors and PD-L1 expression status in cancer: meta-analysis. BMJ 362, k3529 (2018).
Hong, G. et al. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 18, 1841–1846 (2012).
Zhu, S. et al. Molecular imaging of biological systems with a clickable dye in the broad 800- to 1,700-nm near-infrared window. Proc. Natl Acad. Sci. USA 114, 962–967 (2017).
Fan, Y. et al. Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging. Nat. Nanotech. 13, 941–946 (2018).
Wang, W. et al. Molecular cancer imaging in the second near-infrared window using a renal-excreted NIR-II fluorophore-peptide probe. Adv. Mater. 30, 1800106 (2018).
Antaris, A. L. et al. A small-molecule dye for NIR-II imaging. Nat. Mater. 15, 235–242 (2015).
Zhong, Y. et al. Boosting the down-shifting luminescence of rare-earth nanocrystals for biological imaging beyond 1500 nm. Nat. Commun. 8, 737 (2017).
Wang, R., Li, X., Zhou, L. & Zhang, F. Epitaxial seeded growth of rare-earth nanocrystals with efficient 800 nm near-infrared to 1525 nm short-wavelength infrared downconversion photoluminescence for in vivo bioimaging. Angew. Chem. Int. Ed. 53, 12086–12090 (2014).
Wang, F. et al. Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping. Nature 463, 1061–1065 (2010).
Klier, D. T. & Kumke, M. U. Analysing the effect of the crystal structure on upconversion luminescence in Yb3+,Er3+-co-doped NaYF4 nanomaterials. J. Mater. Chem. C 3, 11228–11238 (2015).
Sommerdijk, J. L. Influence of host lattice on the infrared-excited visible luminescence in Yb3+, Er3+-doped fluorides. J. Lumin. 6, 61–67 (1973).
Chen, G. et al. Upconversion emission enhancement in Yb3+/Er3+-codoped Y2O3 nanocrystals by tridoping with Li+ ions. J. Phys. Chem. C 112, 12030–12036 (2008).
Li, D. et al. Efficient luminescence enhancement of Gd2O3:Ln3+ (Ln = Yb/Er, Eu) NCs by codoping Zn2+ and Li+ inert ions. Opt. Mater. Express 7, 329–340 (2017).
Yu, A. K. et al. Luminescence of rare-earth ions and intrinsic defects in Gd2O3 matrix. J. Phys. Conf. Series 741, 012089 (2016).
Lu, Y. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photonics 8, 32–36 (2013).
Dang, X. et al. Layer-by-layer assembled fluorescent probes in the second near-infrared window for systemic delivery and detection of ovarian cancer. Proc. Natl Acad. Sci. USA 113, 5179–5184 (2016).
Chatterjee, S. et al. A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors. Oncotarget 7, 10215–10227 (2016).
Chatterjee, S., Lesniak, W. G. & Nimmagadda, S. Noninvasive imaging of immune checkpoint ligand PD-L1 in tumors and metastases for guiding immunotherapy. Mol. Imaging 16, 1 (2017).
Seidel, J. A., Otsuka, A. & Kabashima, K. Anti-PD-1 and anti-CTLA-4 therapies in cancer: mechanisms of action, efficacy, and limitations. Front. Oncol. 8, 86 (2018).
Brahmer, J. R. et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465 (2012).
Jain, R. K. & Stylianopoulos, T. Delivering nanomedicine to solid tumors. Nat. Rev. Clin. Oncol. 7, 653–664 (2010).
Liu, C., Hou, Y. & Gao, M. Are rare-earth nanoparticles suitable for in vivo applications? Adv. Mater. 26, 6922–6932 (2014).
Wang, X. et al. Single ultrasmall Mn2+-doped NaNdF4 nanocrystals as multimodal nanoprobes for magnetic resonance and second near-infrared fluorescence imaging. Nano Res. 11, 1069–1081 (2018).
Mai, H.-X. et al. High-quality sodium rare-earth fluoride nanocrystals: controlled synthesis and optical properties. J. Am. Chem. Soc. 128, 6426–6436 (2006).
Ma, Z. et al. Near-infrared IIb fluorescence imaging of vascular regeneration with dynamic tissue perfusion measurement and high spatial resolution. Adv. Funct. Mater. 28, 1803417 (2018).
Wang, F. et al. Light-sheet microscopy in the near-infrared II window. Nat. Methods 16, 545–552 (2019).
Boyer, J.-C. & van Veggel, F. C. J. M. Absolute quantum yield measurements of colloidal NaYF4: Er3+, Yb3+ upconverting nanoparticles. Nanoscale 2, 1417–1419 (2010).
This study was supported by the National Institutes of Health (grant no. DP1-NS-105737).
The authors declare no competing interests.
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Supplementary Figs. 1–26 and Table 1.
Real-time NIR-IIb in vivo dynamic imaging of ErNP-labeled cerebral blood flow in a BALB/c mouse brain on 970 nm LED lamp excitation (15 mW cm−2). Total video time: 33.2 s, frame rate: 30 fps. Similar results for n > 3 independent experiments.
Real-time NIR-IIb in vivo dynamic imaging distinguished the vessels in mouse scalp and under the skull. Similar results for n > 3 independent experiments.
Ultra-fast NIR-IIb in vivo dynamic imaging of ErNP-labeled hindlimb blood flow in a BALB/c mouse on 980 nm diode laser excitation (100 mW cm−2). Total video time: 13.2 s, frame rate: 90 fps. Similar results for n > 3 independent experiments.
In vivo two-plex rotation imaging of a CT-26 tumor mouse intravenously injected with ErNPs-aPDL1 and PbS-aCD8 at 0 h, 24 h and 48 h post injection. Similar results for n = 3 independent experiments
In vivo rotation imaging of a CT-26 tumor mouse intravenously injected with PbS-aCD8 at 24 h post injection. Similar results for n = 3 independent experiments.
In vivo two-plex rotation imaging of a 4T1 tumor mouse intravenously injected with ErNPs-aPDL1 and PbS-aCD8 at 24 h post injection. Similar results for n = 3 independent experiments.
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Zhong, Y., Ma, Z., Wang, F. et al. In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles. Nat Biotechnol 37, 1322–1331 (2019). https://doi.org/10.1038/s41587-019-0262-4
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