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Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery

Abstract

Ferritins are spherical iron storage proteins within cells, composed of 24 subunits of two types, heavy-chain ferritin (HFn) and light-chain ferritin. Ferritins auto-assemble naturally into hollow nanocages with an outer diameter of 12 nm and an interior cavity 8 nm in diameter. Since the intrinsic tumor-targeting property of human HFn was first reported in 2012, HFn has been extensively explored for tumor-targeted delivery of anticancer drugs and diagnostic molecules, including radioisotopes and fluorophores, as well as inorganic nanoparticles (NPs) and chemotherapeutic drugs. This protocol provides four detailed procedures describing how to load four types of cargoes within HFn nanocages that are capable of accurately controlling cargo loading: synthesis of inorganic metal nanoparticles within the cavity of a wild-type human HFn nanocage (Procedure 1, requires ~5 h); loading of doxorubicin into the cavity of a wild-type human HFn nanocage (Procedure 2, requires ~3 d); loading Gd3+ into the cavity of a genetically engineered human HFn nanocage (Procedure 3, requires ~20 h); and loading 64Cu2+ radioisotope into the cavity of a genetically engineered human HFn nanocage (Procedure 4, requires ~3 h). Subsequent use of these HFn-based formulations is advantageous as they have intrinsic tumor-targeting capability and lack immunogenicity. Human HFn generated as described in this protocol can therefore be used to deliver therapeutic drugs and diagnostic signals as multifunctional nanomedicines.

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Fig. 1: Characterization of HFn encapsulation of metal oxide NPs.
Fig. 2: Characterization of HFn nanocage encapsulation of doxorubicin (Dox).
Fig. 3: Characterization of HFn–His nanocage encapsulation of Gd3+ or 64Cu2+.
Fig. 4: Characterization of HFn and HFn–His nanocages.

Data availability

Source data are provided with this paper.

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Acknowledgements

This work was supported by the following grants: National Key R&D Program of China (2017YFA0205501), National Natural Science Foundation of China (81722024 and 81571728), Youth Innovation Promotion (2014078 and Y201819) and Shenzhen Key Technologies R&D general program (Shenzhen Science & Technology Innovation, 2020, NO. 194).

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All authors contributed to developing this protocol and writing this paper. M.L. supervised the project.

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Correspondence to Minmin Liang.

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Peer review information Nature Protocols thanks Pierpaolo Ceci, Insan Kim, Ichiro Yamashita and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Fan, K. et al. Nat. Nanotechnol. 7, 459–464 (2012): https://doi.org/10.1038/nnano.2012.90

Liang, M. et al. Proc. Natl Acad. Sci. 111, 14900–14905 (2014): https://doi.org/10.1073/pnas.1407808111

Zhao, Y. et al. ACS Nano, 10, 4184–4191(2016): https://doi.org/10.1021/acsnano.5b07408

Liang, M. et al. ACS Nano 12, 9300–9308 (2018): https://doi.org/10.1021/acsnano.8b04158

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Zhang, J., Cheng, D., He, J. et al. Cargo loading within ferritin nanocages in preparation for tumor-targeted delivery. Nat Protoc 16, 4878–4896 (2021). https://doi.org/10.1038/s41596-021-00602-5

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