In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles

Abstract

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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Fig. 1: Ultra-bright ~1,550 nm NIR-IIb luminescence of Zn-doped α-ErNPs.
Fig. 2: Biocompatible, rapid-excretable α-ErNPs for real-time NIR-IIb imaging under low-power LED excitation.
Fig. 3: ErNPs-aPDL1 mAb complex for in vivo PD-L1 molecular imaging and immunotherapy.
Fig. 4: In vivo two-plex NIR-IIb molecular imaging of immune responses using ErNPs-aPDL1 and PbS-aCD8 at the same ~1,600 nm emission range.
Fig. 5: In vivo two-plex NIR-IIb molecular imaging of PDL1 and CD8+ CTLs for assessing immune activation and responses.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. 1.

    Hong, G. et al. Through-skull fluorescence imaging of the brain in a new near-infrared window. Nat. Photonics 8, 723–730 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Antaris, A. L. et al. A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging. Nat. Commun. 8, 15269 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Yang, Q. et al. Donor engineering for NIR-II molecular fluorophores with enhanced fluorescent performance. J. Am. Chem. Soc. 140, 1715–1724 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Liu, Z. et al. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotech. 2, 47–52 (2006).

    Google Scholar 

  5. 5.

    Wan, H. et al. A bright organic NIR-II nanofluorophore for three-dimensional imaging into biological tissues. Nat. Commun. 9, 1171 (2018).

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Bruns, O. T. et al. Next-generation in vivo optical imaging with short-wave infrared quantum dots. Nat. Biomed. Eng. 1, 0056 (2017).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Naczynski, D. J. et al. Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nat. Commun. 4, 2199 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Diao, S. et al. Fluorescence imaging in vivo at wavelengths beyond 1500 nm. Angew. Chem. 127, 14971–14975 (2015).

    Google Scholar 

  9. 9.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Diao, S. et al. Biological imaging without autofluorescence in the second near-infrared region. Nano Res. 8, 3027–3034 (2015).

    CAS  Google Scholar 

  11. 11.

    Franke, D. et al. Continuous injection synthesis of indium arsenide quantum dots emissive in the short-wavelength infrared. Nat. Commun. 7, 12749 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    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).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    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).

    CAS  PubMed  Google Scholar 

  15. 15.

    Sheng, W. et al. LSD1 ablation stimulates anti-tumor immunity and enables checkpoint blockade. Cell 174, 549–563.e519 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Powles, T. et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515, 558–562 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Herbst, R. S. et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 515, 563–567 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Mall, S. et al. Immuno-PET imaging of engineered human T cells in tumors. Cancer Res. 76, 4113–4123 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    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).

    Google Scholar 

  26. 26.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    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).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Hong, G. et al. Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nat. Med. 18, 1841–1846 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Fan, Y. et al. Lifetime-engineered NIR-II nanoparticles unlock multiplexed in vivo imaging. Nat. Nanotech. 13, 941–946 (2018).

    CAS  Google Scholar 

  32. 32.

    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).

    Google Scholar 

  33. 33.

    Antaris, A. L. et al. A small-molecule dye for NIR-II imaging. Nat. Mater. 15, 235–242 (2015).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Zhong, Y. et al. Boosting the down-shifting luminescence of rare-earth nanocrystals for biological imaging beyond 1500 nm. Nat. Commun. 8, 737 (2017).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    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).

    CAS  Google Scholar 

  36. 36.

    Wang, F. et al. Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping. Nature 463, 1061–1065 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    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).

    CAS  Google Scholar 

  38. 38.

    Sommerdijk, J. L. Influence of host lattice on the infrared-excited visible luminescence in Yb3+, Er3+-doped fluorides. J. Lumin. 6, 61–67 (1973).

    CAS  Google Scholar 

  39. 39.

    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).

    CAS  Google Scholar 

  40. 40.

    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).

    CAS  Google Scholar 

  41. 41.

    Yu, A. K. et al. Luminescence of rare-earth ions and intrinsic defects in Gd2O3 matrix. J. Phys. Conf. Series 741, 012089 (2016).

    Google Scholar 

  42. 42.

    Lu, Y. et al. Tunable lifetime multiplexing using luminescent nanocrystals. Nat. Photonics 8, 32–36 (2013).

    Google Scholar 

  43. 43.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Chatterjee, S. et al. A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors. Oncotarget 7, 10215–10227 (2016).

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    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).

    CAS  Google Scholar 

  46. 46.

    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).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Jain, R. K. & Stylianopoulos, T. Delivering nanomedicine to solid tumors. Nat. Rev. Clin. Oncol. 7, 653–664 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Liu, C., Hou, Y. & Gao, M. Are rare-earth nanoparticles suitable for in vivo applications? Adv. Mater. 26, 6922–6932 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    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).

    CAS  Google Scholar 

  51. 51.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    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).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Wang, F. et al. Light-sheet microscopy in the near-infrared II window. Nat. Methods 16, 545–552 (2019).

    PubMed  PubMed Central  Google Scholar 

  54. 54.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was supported by the National Institutes of Health (grant no. DP1-NS-105737).

Author information

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Authors

Contributions

H. Dai and Y.Z. conceived and designed the experiments. Y.Z., Z.M., F.W., X.W., Y.Y., Y.L., X.Z. and J.L. performed the experiments. Y.Z., Z.M., F.W., X.W., Y.Y., Y.L., X.Z., J.L., H. Du, M.Z., Q.C., S.Z., Q.S., H.W., Y.T., Q.L., W.W., K.C.G. and H. Dai analyzed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Hongjie Dai.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–26 and Table 1.

Reporting Summary

Supplementary Video 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.

Supplementary Video 2

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.

Supplementary Video 3

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.

Supplementary Video 4

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

Supplementary Video 5

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.

Supplementary Video 6

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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