Engineering protein nanocages as carriers for biomedical applications

Bhaskar, Sathyamoorthy; Lim, Sierin

Interior modification of protein nanocages. The interior cavity of the protein nanocages provides an ideal containment for molecular cargos. Tailoring the cage interior increases the encapsulation efficiency, binding affinity and modulated release profile. Genetic or chemical alterations can be made at precise locations to manipulate the nucleation and attachment of molecules. Molecular cargos such as small molecules, peptides, protein drugs, RNA/DNA drugs, imaging agents and polymers have been encapsulated and released from the interior of protein nanocages. Antibodies are potential cargo for larger protein nanocages, such as vaults. Modification of the nanocage interior by introducing cysteine residues has been a widely used approach. The modification facilitates covalent attachment of dye molecules, drugs and other active molecules through disulfide bonds. 5 Other modifications include the introduction of phenylalanine and the attachment of lipid substances and polymers inside the cage architecture. 70 The attachment of polymer chains to the interior surface offers spatial control of reactive sites, which has been shown to stabilize cages. Abedin et al. 71 created a polymer network inside a protein cage by introducing cysteine reactive residues by genetic manipulation. The polymer network was created by the sequential conjugation of multifunctional monomeric units by click chemistry, allowing the free amines to be incorporated into the polymer for internal functionalization (Figure 11 ). The polymer network covalently crosslinks the subunits of the protein cage, increasing its thermal stability to at least 120°C. 71 Exterior modification of protein nanocages. The exterior surface of protein nanocages is engineered to impart increased circulatory halflife, local accumulation, cellular penetration and triggering of specific cellular responses. The unique geometry of protein nanocages allows for multiple ligands and functional molecules to be displayed on their surfaces. Modifying molecules include small molecules (e.g. folate), peptides (e.g. RGD, transactivator of transcription 47-57 peptide (cell penetrating peptide), immunoglobulin G binding peptide (Z domain)), antibodies (e.g. anti-epidermal growth factor receptor and anti-CD4), nucleotides (e.g. DNA, RNA, aptamers) and polymers (e.g. PEG, PGA, poly(lactic-co-glycolic acid), polymethacrylate, polyamidoamine). The exterior surfaces of CPMV, cowpea chlorotic mottle virus (CCMV) and bacteriophages M13, Qb and MS2 have been modified with antibodies, peptides, transferrin and cell transduction domains or cell-penetrating peptides. 58 The adenovirus-based peptide RGD has a natural affinity for integrin receptors that are upregulated in tumor vessels. Modification of protein carriers with this peptide has been proven to aid tumor targeting. 8 Short, singlestranded oligonucleotides called aptamers with secondary structures capable of recognizing specific molecules can be incorporated onto the nanoparticles to enhance their specificity. These aptamers can bind to both extracellular (membrane) and intracellular proteins, thus facilitating aptamer-nanoparticle conjugate therapy. 72 Other functionalities can be imparted to the nanocages by attaching functional ligands to extend their half-life (e.g. PEG) and enhance penetration into cells (e.g. cell penetrating peptides). Folate-PEG displayed on adenoviral nanoparticles has been delivered to folate receptoroverexpressing cells. These nanocarr