Metal ion assisted interface re-engineering of a ferritin nanocage for enhanced biofunctions and cancer therapy

Abstract

The bottom-up self-assembly of protein subunits into supramolecular nanoarchitectures is ubiquitously exploited to recapitulate and expand the features of natural proteins to advance nanoscience in medicine. Various chemical and biological re-engineering approaches are available to render diverse functions in the given proteins. They are, unfortunately, capable of compromising protein integrity and stability after extensive modifications. In this study, we introduce a new protein re-engineering method, metal ion assisted interface re-engineering (MAIR), to serve as a robust and universal strategy to extend the functions of self-assembly proteins by boosting structural features to advance their diverse biomedical applications. In particular, the MAIR strategy was applied to a widely used natural protein, ferritin, as a model protein to coordinate with copper ions in its mutagenic artificial metal binding domain. Structure directed rational protein mutagenesis was carried out at the C2 interface amino acid residues of the ferritin subunit for metal ion coordination site optimization. Copper binding at the artificial binding pocket was highly specific over the other divalent ions present in physiological fluids, and the structurally embedded copper ion in turn strengthened the overall protein integrity and stability. In the presence of isotopic copper-64, the interface re-engineered ferritin worked as a chelator-free molecular nanoprobe with an extraordinarily high specific activity to allow PET imaging of tumors in live animals. We also found that the re-engineered ferritin coordinating with copper ions demonstrates high drug loading capacity of a widely used anti-cancer agent, doxorubicin (DOX), to achieve significant drug retention at the tumor site and enhance tumor regression for improved anti-cancer effects. The MAIR approach, thus, exploited the copper ion to facilitate efficient one-step labeling of mutant ferritin derivatives for simultaneous molecular imaging and drug delivery. The reported interface re-engineering strategy provides an unparalleled opportunity to expand protein biofunctions to serve as a new theranostic agent in cancer research.

Figure 1

(A) Computational simulation of three interface re-engineered ferritin variants with copper chelation. (B) Magnified protein ribbon structures of two potent permutated ferritin variants coordinated with copper. The amino acid residues associated with copper coordination are labelled. (C) Computational calculation of interface atom number and interface area of different ferritin variants.

Figure 2

(A) TEM images of ferritin variants with uranyl acetate negative staining. Scale bar=100 nm. (B) Dynamic light scattering study of the nanocage size of mutant B ferritin under heating (60 °C) and different pH values in buffers. (C) Circular dichroism spectra of native ferritin and mutant B ferritin in PBS (0.5 mg/mL). (D) Radioactive ⁶⁴Cu incorporation ratio of ferritin variants under different concentrations. (E) ⁶⁴Cu incorporation stability study of ferritin variants in mouse serum over 24 h. (F) ⁶⁴Cu incorporation stability study of ferritin variants in the presence of strong metal chelator, EDTA, in mouse serum over 24 h. (G–I) Divalent metal ion competition with ⁶⁴Cu in ferritin variants.

Figure 3

(A) Cancer cell (U87MG) uptake of ferritin variants labeled with Cy5.5 fluorophore. Scale bar = 20 μm (B) Magnified confocal laser scanning imaging (CLSM) (Z-section) of intracellular uptake of mutant B ferritin after 2 h incubation. Scale bar = 10 μm. Blue: nucleus counterstained with DAPI; Red: Cy5.5 labeled mutant B ferritin; Green: F-actin stained with Alexa488 conjugated phalloidin. (C) Flow cytometry analysis of cellular uptake of mutant B ferritin at different concentrations. (D) MTT assay of mutant B ferritin with cancer cells (n = 6).

Figure 4

(A) Pharmacokinetics of ⁶⁴Cu coordinated mutant B ferritin and native ferritin in healthy balb/c mice over 72 h (n = 3/group). (B) Biodistribution of ⁶⁴Cu coordinated mutant B ferritin and native ferritin in different major organs in healthy balb/c mice at 24 h postinjection (n = 3/group). (C) PET images of ⁶⁴Cu coordinated mutant B ferritin (left panel) and free ⁶⁴Cu²⁺ (right panel) in U87MG tumor mice. (D) Biodistribution of ⁶⁴Cu coordinated mutant B ferritin and free ⁶⁴Cu²⁺ in U87MG tumor mice at 24 h postinjection (n = 3/group). **P<0.01. (E) Quantitative analysis of tumor uptake of ⁶⁴Cu coordinated mutant B ferritin and free ⁶⁴Cu²⁺ at different time points (n = 3/group). *P<0.05; **P<0.01; ***P<0.001. (F) Quantitative analysis of signals from the heart of ⁶⁴Cu coordinated mutant B ferritin and free ⁶⁴Cu²⁺ in U87MG tumor mice at different time points (n = 3/group). *P<0.05; **P<0.01; ***P<0.001.

Figure 5

(A) PET imaging of ⁶⁴Cu coordinated mutant B ferritin (upper panel) and native ferritin (lower panel) of U87MG tumor mice at different time points. An equivalent of 150 μCi radioactivity was injected into each mouse. (B) Quantitative analysis of tumor accumulation of ⁶⁴Cu coordinated mutant B and native ferritin nanoprobes at different time points (n = 3/group). (C) Quantitative analysis of liver retention of ⁶⁴Cu coordinated mutant B and native ferritin nanoprobes at different time points (n = 3/group). (D) Biodistribution of ⁶⁴Cu coordinated mutant B and native ferritin nanoprobes at 24 h postinjection (n = 3/group). **P<0.05.

Figure 6

(A) Schematic illustration of Mutant B@DOX preparation. Cu²⁺ was coordinated with DOX first, then the DOX-Cu²⁺ complexes were encapsulated into the Mutant B ferritin. (B) Tumor growth curves under various treatments. (C) Survival rates of tumor xenograft mice from different groups. (D) The body weight of mice from different groups. (E) H&E stained tumor histological sections from different groups on the 7^th day. Scale bar = 200 μm. **P<0.05, ***P<0.01.