Engineering protein nanocages as carriers for biomedical applications

Bhaskar, Sathyamoorthy; Lim, Sierin

The basic requirements for in vitro and in vivo imaging is to achieve high local concentrations of imaging agents and suppression of the quenching of fluorescent probes. Various contrast agents for enhanced MRI, positron electron tomography and near infrared fluorescence imaging have been encapsulated in protein nanocages. Single or coencapsulation of the contrast agents will allow for the development of protein nanocages with multimodal imaging ability. Of all protein nanocages, ferritin has dominated bioimaging applications. The inherent ability of ferritins to store iron as ferric oxyhydroxide particles makes them attractive as an MRI contrast agent. The ferric oxyhydroxide nanoparticles are superparamagnetic; therefore, endogenous ferritin loaded with these particles can act as a natural T2 MRI contrast agent. 87 At clinically significant magnetic field strengths (i.e. 1.5 and 3 T), ferritin has 10-100-fold less relaxivity per iron than commercially produced iron oxide nanoparticles. Loading of superparamagnetic iron oxide nanoparticles in ferritin nanocages shows higher r 2 relaxivity than endogenous ferritin. 64 Protein nanocages can be engineered in a chemoselective manner to increase the loading capacity of fluorophores (e.g. Alexa Fluor or fluorescein dyes on CPMV) with precisely defined positions on the exterior and interior of the cage. The imaging agents are attached to the rigid and ordered protein structure at distinct positions. The spatial confinement of dye molecules prevents them from reacting or aggregating with each other and therefore reduces the quenching possibility. The inherent ability of some near-infrared fluorescence dyes to penetrate tissues with reduced background noise makes them attractive for in vivo imaging. 3 Attachment of these dyes on the protein nanocages increases the local concentration resulting in reduced dosing. A notable example is luminescent semiconductor nanocrystals (quantum dots), which were displayed on engineered CPMV nanocages. 3 Bacteriophage MS2 nanocages were tested for their ability to carry the positron electron tomography imaging agent [ 18 F] fluorobenzaldehyde. The radiolabel was attached to the interior of the cage by bioconjugation. Positron electron tomography imaging analysis in rats showed that conjugation with protein nanocages increases the blood circulation time of the imaging agent without affecting biodistribution. 88 Protein nanocages can also be used as contrast agents in ultrasound imaging. Gas-filled protein based nanostructures derived from cyanobacterium Anabaena flos-aquae has been genetically engineered for enhanced harmonic properties that is optimal for in vitro and in vivo ultrasound imaging. 87 It has been shown that these protein nanocages can also be engineered for multimodal and targeted ultrasound imaging.