A new and efficient procedure to load bioactive molecules within the human heavy-chain ferritin nanocage

Abstract

For their easy and high-yield recombinant production, their high stability in a wide range of physico-chemical conditions and their characteristic hollow structure, ferritins (Fts) are considered useful scaffolds to encapsulate bioactive molecules. Notably, for the absence of immunogenicity and the selective interaction with tumor cells, the nanocages constituted by the heavy chain of the human variant of ferritin (hHFt) are optimal candidates for the delivery of anti-cancer drugs. hHFt nanocages can be disassembled and reassembled in vitro to allow the loading of cargo molecules, however the currently available protocols present some relevant drawbacks. Indeed, protein disassembly is achieved by exposure to extreme pH (either acidic or alkaline), followed by incubation at neutral pH to allow reassembly, but the final protein recovery and homogeneity are not satisfactory. Moreover, the exposure to extreme pH may affect the structure of the molecule to be loaded. In this paper, we report an alternative, efficient and reproducible procedure to reversibly disassemble hHFt under mild pH conditions. We demonstrate that a small amount of sodium dodecyl sulfate (SDS) is sufficient to disassemble the nanocage, which quantitatively reassembles upon SDS removal. Electron microscopy and X-ray crystallography show that the reassembled protein is identical to the untreated one. The newly developed procedure was used to encapsulate two small molecules. When compared to the existing disassembly/reassembly procedures, our approach can be applied in a wide range of pH values and temperatures, is compatible with a larger number of cargos and allows a higher protein recovery.

Keywords: disassembly/reassembly nanocage protocols; drug encapsulation; ferritin nanocages; human ferritin nanocarriers; reversible ferritin disassembly.

FIGURE 1

Gel electrophoresis under native conditions of hHFt to evaluate the assembly state upon different treatments. Panel (A) lane 1 untreated hHFt; from lane 2 to lane 6 hHFt incubated with 1.0%, 1.5%, 2.0%, 2.5% and 3.0% SDS, respectively. Panel (B) lane 1 untreated hHFt; from lane 2 to lane 6 hHFt incubated with 1.0%, 1.5%, 2.0%, 2.5% and 3.0% SDS after SDS removal by dialysis against 20 mM Tris-HCl pH 7.4. Panel (C) lane 1 untreated hHFt; from lane 2 to lane 10 hHFt incubated with 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, and 0.09% SDS, respectively. Panel (D) lane 1 : untreated hHFt; lane 2 hHFt_pH12; lane 3 hHFt_{pH 12->7.4} (reassembled); lane 4 hHFt_{pH 13}; lanes 5 hHFt_{pH 13->7.4} (reassembled); lanes 6 hHFt_{pH 1.5}; lane 7 hHFt_{pH 1.5->7.4} (reassembled); lane 8 hHFt_{pH 2}; lane 9 hHFt_{pH 2->7.4} (reassembled); lane 10 hHFt_{SDS 0.1%} (reassembled).

FIGURE 2

CD spectra of hHFt during the SDS treatment. Black line : untreated hHFt in 20 mM Tris-HCl pH 7.4; Red line: hHFt treated with 0.1% SDS; Blue line : hHFt in 20 mM Tris-HCl pH 7.4 after SDS removal.

FIGURE 3

TEM bright field images ((scale bar = 50 nm, upper inset, 20 nm) of hHFt during the SDS treatment. (A) Reference (untreated) hHFt. (B) hHFt _{SDS 0.1%}; (C) hHFt _{SDS 0.1%} reassembled after SDS removal. Samples were stained with 1.5% phosphotungstic acid. Notably, despite the use of negative staining, some hHFt nanocages appear slightly dark because of PTA absorption.

FIGURE 4

X-ray structure of the hHFt reassembled after disassembly in SDS: (A) the 24-mer (hHFt_SDS); (B) the single chain of hHFt. The asymmetric unit of the crystal contains one single chain; the 24-mer is generated by crystal symmetry. In panel B, the four helices (A-D), the short helix at the C-terminal tail (helix E) and the BC loop are highlighted. The structure has been deposited with the PDB code 8A5N.

FIGURE 5

Electrophoretic and spectroscopic analysis of Ru1-hHFt. (A) CD spectra of reference hHFt (black line), and of Ru1-hHFt (red line). (B) electrophoresis under native conditions of hHFt, lane 1 reference hHFt, lane 2 disassembled hHFt incubated with 0.1% SDS, and lane 3 Ru1-hHFt after encapsulation. (C) UV-vis spectra of reference hHFt (black line), and of Ru1-hHFt (red line). All the spectra were acquired in 20 mM Tris-HCl pH 7.4.

FIGURE 6

Electrophoretic and spectroscopic analysis of TRIL-hHFt: (A) Gel electrophoresis under native conditions of hHFt, lane 1 reference hHFt and lane 2 TRIL-hHFt after the encapsulation. (B) 15% SDS-PAGE gel of and treated samples: M proteins marker, lane 1 reference hHFt, lane 2 TRIL, lane 3 TRIL-hHFt after the encapsulation. (C) CD spectra of native hHFt (black line) and of TRIL-hHFt (red line) in 20 mM Tris-HCl pH 7.4.