Engineering protein nanocages as carriers for biomedical applications

Bhaskar, Sathyamoorthy; Lim, Sierin

iers were proven to decrease the interleukin-6 levels of macrophages, showing that the engineered viral scaffolds counteract the innate immune response. 1 PEG acts as a stealth layer shielding the immunogenic epitopes on the surface of the nanocage structure. 73 This layer prevents the protein opsonin from adsorbing onto the surface, thereby shunting the recognition of the nanocage by phagocytic cells (reticuloendothelial system). 42 The PEG modification also benefits the tuneable solubility and structural integrity of the protein nanocage. 73 The spatial control of multifunctional groups on protein nanocages equips them for both hierarchical self-assembly and display of distinct functional ligands. Display of multiple types of ligands in a precise arrangement on nanoparticles is challenging. A distinct advantage of protein nanocages over other nanoparticles is that the position of each amino acid is spatially defined, allowing for precise spatial control of the displayed ligands. The N or C termini of the nanocage subunits 22, 43, 68 Engineered protein cages in biomedical applications S Bhaskar and S Lim that face the external surface are a natural choice for displaying functional ligands. Fusion of short peptides to these termini has been achieved through genetic engineering. Modification of recombinant vault by fusing a cysteine-rich 12-amino-acid peptide to the N terminus of the MVP (CP-MVP) leads to increased particle stability. 67 Engineering vault MVP C-terminal regions with tags such as epitopes, 33-amino-acid immunoglobulin G-binding peptide (Z domain) or 55-amino-acid epidermal growth factors, which are displayed at the caps of the vault, were shown to facilitate cell-specific targeting. 27 In another approach, Domingo et al. 74 denatured, mixed and reassembled protein subunits carrying different chimeric peptides to display multiple types of ligands (i.e. green fluorescent protein and two types of malaria epitopes) on the surface of a single E2 protein nanocage. The display of two types of HIV-derived antigenic epitopes (pep23 and RT2) provokes specific antibodies and T-cell responses. 74 Despite the natural spatial control in protein nanocages, achieving the desired symmetry is still a great challenge. 64 Production of protein nanocages with a dual architecture was achieved by toposelectively modifying the exterior surfaces of DNA-binding protein from starved cell cages by a masking/unmasking method based on solid supports. The distribution of the two functional domains, fluorophore and affinity tag, was manipulated by using different materials for the solid supports ( Figure 12 ). 75 This toposelective modification provides more sophisticated designs of multifunctional nanoplatforms. By combining genetic fusion with toposelective attachment of streptavidin, the universal coupling protein to which a biotinylated molecule could be attached, Suci et al. 76 achieved asymmetrical placement of streptavidin. This asymmetrical placement provides further spatial control over the display, providing tools with which to explore the effects of the polarized orientation of conjugated functional molecules on interactions with specific cell surface receptors.