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Near-IR photoactivation using mesoporous silica–coated NaYF4:Yb,Er/Tm upconversion nanoparticles

Nature Protocols volume 11, pages 688–713 (2016)Cite this article

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Abstract

Photoactivation is a process in which light is used to 'activate' photolabile therapeutics. As a therapeutic strategy, its advantages are that it is noninvasive and that a high degree of spatial and temporal control is possible. However, conventional photoactivation techniques are hampered by the limited penetration depth of the UV and visible lights to which the photosensitive compounds are responsive. Here we describe a protocol for the use of upconversion nanoparticles (UCNs) as light transducers to convert deeply penetrating near-infrared (NIR) light to UV-visible wavelengths matching that of the absorption spectrum of photosensitive therapeutics. This allows the use of deep-penetrating and biologically friendly NIR light instead of low-penetrating and/or toxic visible or UV lights for photoactivation. In this protocol, we focus on two photoactivation applications: photodynamic therapy (PDT) and photoactivated control of gene expression. We describe how to prepare and characterize the UCNs, as well as how to check their function in biochemical assays and in cells. For both applications, the UCNs are coated with mesoporous silica for easy loading of the therapeutics. For PDT, the UCNs are coated with polyethylene glycol (PEG) for stabilization and folic acid for tumor targeting and then loaded with photosensitizers that would be expected to kill cells by singlet oxygen production; the nanoparticles are injected intravenously. For photoactivated control of gene expression, knockdown of essential tumor genes is achieved using UCNs loaded with caged nucleic acids, which are injected intratumorally. The whole process from nanoparticle synthesis to animal studies takes ∼36 d.

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Figure 1: Schematic illustration of delivering UCNs loaded with photosensitive nucleic acids and photoactivating them using upconverted UV light from NIR-to-UV UCNs.
Figure 2: Flowchart illustrating the experimental design of the synthesis of mesoporous silica–coated NaYF4:Yb,Er/Tm UCNs for photoactivation applications.
Figure 3: Schematic illustration of the laser setup used for irradiating small animals and cells.
Figure 4: Photographs of the reaction mixture at various stages of the UCN synthesis.
Figure 5: Characterizations of nanoparticles.
Figure 6: Appearance of mesoporous silica–coated UCNs loaded with photosensitizers.
Figure 7: ABDA fluorescence decay representing 1O2 production from 980-nm laser-irradiated UCNs.
Figure 8: ROS production in cultured mammalian cells.
Figure 9: Viability of cells 48 h after treatment.
Figure 10: Assessing in vivo targeting of UCNs to tumor tissues.
Figure 11: Assessing tumor cell death by apoptosis.
Figure 12: Assessing PDT efficacy based on the inhibition of tumor growth in mice treated with folic acid-and-PEG-modified UCNs and exposure to the 980-nm laser.
Figure 13: Effect of GFP knockdown in cells.
Figure 14: Effect of STAT3 knockdown in a murine model of melanoma.

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Acknowledgements

We thank S.S. Lucky for the helpful discussion. We also thank S. Jayakumar for technical support in the schematics. Y.Z. received funding support from the Agency for Science, Technology and Research (A*STAR) Biomedical Research Council (grant nos. R-397-000-062-305 and R-397-000-119-305), the Biomedical Engineering Programme (grant no. R-397-000-128-305), the National Medical Research Council (NMRC, grant nos. CBRG13nov052 and R-397-000-199-511), the National Natural Science Foundation of China (grant no. 31328009) and the National University of Singapore. A.B. is a recipient of the NGS scholarship from NUS graduate school (NGS) for Integrative Sciences and Engineering, National University of Singapore.

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  1. Muthu Kumara Gnanasammandhan and Niagara Muhammad Idris: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore

    Muthu Kumara Gnanasammandhan, Niagara Muhammad Idris, Akshaya Bansal, Kai Huang & Yong Zhang

  2. NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore

    Akshaya Bansal & Yong Zhang

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  1. Muthu Kumara Gnanasammandhan
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Contributions

All authors conceived and designed the experiments, analyzed the data and wrote the paper. K.H. prepared the UCN constructs and their characterizations. N.M.I. performed the photosensitizer loading into UCN constructs and validated them in in vitro and in vivo PDT studies. M.K.G. and A.B. performed the nucleic acid loading into UCN constructs and validated them in in vitro and in vivo studies on photoactivated control of gene expression. Y.Z. supervised the project. All authors commented on the manuscript at all stages.

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Correspondence to Yong Zhang.

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Gnanasammandhan, M., Idris, N., Bansal, A. et al. Near-IR photoactivation using mesoporous silica–coated NaYF4:Yb,Er/Tm upconversion nanoparticles. Nat Protoc 11, 688–713 (2016). https://doi.org/10.1038/nprot.2016.035

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