P22 viral capsids as nanocomposite high-relaxivity MRI contrast agents.

Attachment of multiple chelated Gd(3+) ions to the interior of bacteriophage P22 viral capsids affords nanoscale MRI contrast agents with extremely high relaxivity values. Highly fenestrated "wiffleball" morphology is unique to P22 and assures water exchange between the environment and interior cavity of the capsid. The cavity of P22 "wiffleball" was functionalized with a branched oligomer comprising multiple DTPA-Gd complexes resulting in an impressive payload of 1,900 Gd(3+) ions inside each 64 nm capsid. High relaxivities of r(1,ionic) = 21.7 mM(-1) s(-1) and r(1,particle) = 41,300 mM(-1) s(-1) at 298 K, 0.65 T (28 MHz) are reported, with r(1)/r(2) ratio of 0.80 and optimized rotational correlation time for this system. Specific design modifications are suggested for future improvements of viral capsid-based MRI contrast agents directed toward clinical translation.

Site-directed coordination chemistry with P22 virus-like particles.

Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications.

Functional virus-based polymer-protein nanoparticles by atom transfer radical polymerization.

Viruses and virus-like particles (VLPs) are useful tools in biomedical research. Their defined structural attributes make them attractive platforms for engineered interactions over large molecular surface areas. In this report, we describe the use of VLPs as multivalent macroinitiators for atom transfer radical polymerization. The introduction of chemically reactive monomers during polymerization provides a robust platform for post-synthetic modification via the copper-catalyzed azide-alkyne cycloaddition reaction. These results provide the basis to construct nanoparticle delivery vehicles and imaging agents using protein-polymer conjugates.

Protein cage nanoparticles bearing the LyP-1 peptide for enhanced imaging of macrophage-rich vascular lesions.

Cage-like protein nanoparticles are promising platforms for cell- and tissue-specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heat shock protein from Methanococcus jannaschii (MjHsp) to detect atherosclerotic plaque lesions in a mouse model system. As macrophages are centrally involved in the initiation and progression of atherosclerosis, targeted imaging of macrophages is valuable to assess the biologic status of the blood vessel wall. LyP-1, a nine residue peptide, has been shown to target tumor-associated macrophages. Thus, LyP-1 was genetically incorporated onto the exterior surface of MjHsp, while a fluorescent molecule (Cy5.5) was conjugated on the interior cavity. This bioengineered protein cage, LyP-Hsp, exhibited enhanced affinity to macrophage in vitro. Furthermore, in vivo injection of LyP-Hsp allowed visualization of macrophage-rich murine carotid lesions by in situ and ex vivo fluorescence imaging. These results demonstrate the potential of LyP-1-conjugated protein cages as nanoscale platforms for delivery of imaging agents for the diagnosis of atherosclerosis.

Supramolecular protein cage composite MR contrast agents with extremely efficient relaxivity properties.

A DTPA-Gd containing polymer was grown in the interior of a heat shock protein cage resulting in T(1) particle relaxivities of 4200 mM(-1) sec(-1) for the 12 nm particle. Relaxivity parameters were determined, and this analysis suggests that the rotational correlation time has been optimized while the water exchange lifetime is longer than optimal. This synthetic approach holds much promise for the development of next generation contrast agents and this report will aid in their design.

From metal binding to nanoparticle formation: monitoring biomimetic iron oxide synthesis within protein cages using mass spectrometry.

Mass measurements of metal-mineralized protein cages allowed quantitative examination of the effects of metal-ion concentration on the final nanoparticle size. Modeling using a kinetic master equation suggests that particle growth involves both a binding phase and a growth phase (see picture; I: relative abundance; LiDps: a DNA binding protein; (n)Fe: number of Fe atoms).

Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture.

Protein cages, including viral capsids, ferritins, and heat shock proteins (Hsps), can serve as nanocontainers for biomedical applications. They are genetically and chemically malleable platforms, with potential as therapeutic and imaging agent delivery systems. Here, both genetic and chemical strategies were used to impart cell-specific targeting to the Hsp cage from Methanococcus jannaschii. A tumor vasculature targeting peptide was incorporated onto the exterior surface of the Hsp cage. This protein cage bound to alpha(v)beta(3) integrin-expressing cells. Cellular tropism was also imparted by conjugating anti-CD4 antibodies to the exterior of Hsp cages. These Ab-Hsp cage conjugates specifically bound to CD4(+) cells. Protein cages have the potential to simultaneously incorporate multiple functionalities, including cell-specific targeting, imaging, and therapeutic agent delivery. We demonstrate the simultaneous incorporation of two functionalities, imaging and cell-specific targeting, onto the Hsp protein cage.

Structural transitions in Cowpea chlorotic mottle virus (CCMV).

Viral capsids act as molecular containers for the encapsulation of genomic nucleic acid. These protein cages can also be used as constrained reaction vessels for packaging and entrapment of synthetic cargos. The icosahedral Cowpea chlorotic mottle virus (CCMV) is an excellent model for understanding the encapsulation and packaging of both genomic and synthetic materials. High-resolution structural information of the CCMV capsid has been invaluable for evaluating structure-function relationships in the assembled capsid but does not allow insight into the capsid dynamics. The dynamic nature of the CCMV capsid might play an important role in the biological function of the virus. The CCMV capsid undergoes a pH and metal ion dependent reversible structural transition where 60 separate pores in the capsid open or close, exposing the interior of the protein cage to the bulk medium. In addition, the highly basic N-terminal domain of the capsid, which is disordered in the crystal structure, plays a significant role in packaging the viral cargo. Interestingly, in limited proteolysis and mass spectrometry experiments the N-terminal domain is the first part of the subunit to be cleaved, confirming its dynamic nature. Based on our fundamental understanding of the capsid dynamics in CCMV, we have utilized these aspects to direct packaging of a range of synthetic materials including drugs and inorganic nanoparticles.

Selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture.

The antitumor agent doxorubicin was covalently bound and selectively released in a pH dependent manner from the interior surface of a genetically modified small heat shock protein (Hsp) cage.