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Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo


Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and already this has been used widely to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling the self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employ an optimized first-order bioorthogonal cyclization reaction to control the self-assembly of a fluorescent small molecule, and demonstrate its in vivo applicability by imaging caspase-3/7 activity in human tumour xenograft mouse models of chemotherapy. The fluorescent nanoparticles assembled in situ were imaged successfully in both apoptotic cells and tumour tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo.

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Figure 1: Illustration of the mechanism of in vivo imaging by C-SNAF of caspase-3/7 activity in human tumour xenograft mouse models.
Figure 2: In vitro characterization of the C-SNAF probe.
Figure 3: Imaging of caspase-3/7 activity in STS-treated cancer cells with C-SNAF.
Figure 4: 3D-SIM imaging of self-assembled fluorescent nanoaggregates in cells.
Figure 5: Non-invasive imaging of apoptosis in tumour-bearing mice treated with DOX.
Figure 6: Correlation of enhanced C-SNAF macrocyclization and tissue retention with caspase-3 activation and tumour response to therapy.


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This work was supported by the Stanford University National Cancer Institute (NCI) Centers of Cancer Nanotechnology Excellence (1U54CA151459-01), the NCI ICMIC@Stanford (1P50CA114747-06) and an Institutional Development Award from the Department of Defense Breast Cancer Research Program (W81XWH-09-1-0057). A.J.S. is supported by a postdoctoral fellowship from the Susan Komen Breast Cancer Foundation. We thank A. Olson at the Neuroscience Microscopy Service in Stanford University for assistance with 3D-SIM imaging.

Author information




D.Y. performed all the compound syntheses and characterizations, collected enzymatic reactions and carried out the cell imaging. D.Y. and A.J.S. performed the 3D-SIM studies. A.J.S. set up the animal model. A.J.S. and D.Y. performed the in vivo studies and A.J.S. analysed the data. D.Y. and S.S.T. set up the apoptotic cell model. D.Y. and L.C. performed the flow-cytometry studies and analysed the data. G.T. performed the TEM experiment. L.T. carried out the immunohistochemistry staining of the tumour tissue. All authors discussed the results and commented on the manuscript. D.Y., A.J.S., D.W.F. and J.R. co-wrote the paper.

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Correspondence to Jianghong Rao.

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The authors declare competing financial interests: Stanford University has filed a provisional patent application (serial number 61/869,223) to protect part of the technology described in the study.

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Ye, D., Shuhendler, A., Cui, L. et al. Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo. Nature Chem 6, 519–526 (2014).

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