Development of Protein‐Cage‐Based Delivery Nanoplatforms by Polyvalently Displaying β‐Cyclodextrins on the Surface of Ferritins Through Copper(I)‐Catalyzed Azide/Alkyne Cycloaddition

Abstract

Protein cages are spherical hollow macromolecules that are attractive platforms for the construction of nanoscale cargo delivery vehicles. Human heavy-chain ferritin (HHFn) is modified genetically to control the number and position of functional groups per cage. 24 β-CDs are conjugated precisely to the modified HHFn in specific locations through thiol-maleimide Michael-type addition followed by copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC). The resulting human ferritins displaying β-CDs (β-CD-C90 HHFn) can form inclusion complexes with FITC-AD, which can slowly release the guest molecule reversibly in a buffer solution via non-covalent β-CD/AD interactions. β-CD-C90 HHFn can potentially be used as delivery vehicles for insoluble drugs.

Cyclodextrin conjugated ferritin nanocages reduce intracellular cholesterol level in foam cells

“…Eighteen molecules of cyclodextrin were conjugated to the cage using CuAAC reaction. Previous studies using CuAAC reaction method makes use of long linkers such as N-propargyl-3-maleimidopropionamide as an alternative to the propargyl maleimide linker used in this study [32]. Although resulting in slightly enhanced conjugation efficiency, additional reaction steps for synthesis of the long linker can be avoided while maintaining at least 75% conjugation using propargyl maleimide.…”

“…Cyclodextrin was conjugated to the protein cage using copper (I) catalysed azide/alkyne cycloaddtion reaction (CuAAC) following the methods described previously with slight modifications [32]. Cysteine mutations on the cage was reduced by the addition of 15 molar excess of tris(2-carboxyethyl)phosphine (TCEP) and incubated for 1 h at room temperature (RT) on a 3D shaker.…”

Polyvalent Display of Monosaccharides on Ferritin Protein Cage Nanoparticles for the Recognition and Binding of Cell‐Surface Lectins

“…Human ferritin has been extensively used as a template for the synthesis of biomimetic nanomaterials and as a protein‐based targeted‐delivery nanoplatform in biomedical research . To display monosaccharides on the surface of human ferritin in a controlled manner, we used previously generated HFPCNs, which have a single cysteine residue (C90) for every subunit on their surface (24 cysteines per cage), as polyvalent nanoplatforms . Previously, we demonstrated that these cysteines are exposed on the surface of the nanoplatforms and are therefore fully available for the attachment of functional moieties …”

“…The solution was centrifuged to remove E. coli debris and the supernatant was heated at 60 °C for 10 min, precipitating many of the E. coli proteins, which were removed by centrifugation. The supernatant was loaded onto a 10 mm × 300 mm Superose 6 (GE Healthcare) size exclusion column and eluted with buffer containing 50 m M phosphate, 100 m M NaCl (pH 6.5) at a rate of 0.5 mL min −1 …”

“…The aromatic nitro groups were reduced using stannous chloride in absolute ethanol heated at 70 °C, to generate p ‐anilinyl‐linked sugar derivatives 3, 7. The amine termini were treated with N ‐succinimidyl‐3‐maleimido‐propionoate, to generate maleimido group terminated per‐acetylated‐mannopyranoside (AMM) 4 or maleimido group terminated per‐acetylated‐galactopyranoside (AGM) 8 (see Supporting Information in detail).…”