Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading

Abstract

Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we have successfully demonstrated the site selective initiation of atom transfer radical polymerization (ATRP) reactions to form an addressable polymer constrained within the interior cavity of a VLP. This protein-polymer hybrid, of P22 and crosslinked poly(2-aminoethyl methacrylate), is potentially useful as a new high-density delivery vehicle for encapsulation and delivery of small molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of primary amine reactive molecules of interest, such as a fluorescein dye or a Gd-DTPA MRI contrast agent. Using this approach, a significant increase in labeling density of the VLP, compared to previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

X-ray spatial frequency heterodyne imaging of protein-based nanobubble contrast agents

“…The larger nanobubbles were prepared using two distinct morphologies of a viruslike particle (VLP) derived from the Salmonella typhimurium bacteriophage P22 capsid. This VLP is a protein cage composed of 420 subunits of a 46.6 kDa coat protein that assemble into an icosahedral capsid with the aid of a scaffolding protein [8][9][10][11]. The first VLP morphology used here is the empty shell formulation of the VLP (P22 ES), in which the scaffolding protein has been removed; the resulting protein cage has an outer diameter of approximately 58 nm and an inner diameter of approximately 48 nm.…”

“…P22 scaffolding protein was extracted to prepare the empty shell (ES) form of P22 VLP using a buffer (50mM sodium phosphate and 100 mM sodium chloride at pH 7.0) containing 0.5M of guanidineHCl followed by ultracentrifugation of the capsid [8] to pellet the capsid, which was subsequently resuspended in the same buffer. This extraction process was repeated four times.…”

“…Human heavy-chain ferritin (HFn) and the procapsid (PC) form of P22 were heterologously expressed in E. coli BL21 (DE3) and purified as previously described [8,29]. P22 scaffolding protein was extracted to prepare the empty shell (ES) form of P22 VLP using a buffer (50mM sodium phosphate and 100 mM sodium chloride at pH 7.0) containing 0.5M of guanidineHCl followed by ultracentrifugation of the capsid [8] to pellet the capsid, which was subsequently resuspended in the same buffer.…”

“…To this end, we present the development of a novel protein-based nanoparticle contrast agent comprised of nano-sized protein cage architectures that are filled with perfluoropropane (C 3 F 8 ) gas. The protein assemblies used to make these "nanobubble" contrast agents are apoferritin (approximately 12 nm outer diameter) [7] and a virus-like particle (VLP) derived from the Salmonella typhimurium bacteriophage P22 capsid (approximately 60 nm outer diameter) [8][9][10][11].…”

Genetic Assembly of Double‐Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging

“…Uncontrolled release of encapsulated fluorophores is another typical problem 6. None of the current methods provides precise control of the spatial distribution of the caged fluorophores, which makes it difficult to control the self‐quenching effect 7. Furthermore, hydrophilic fluorophores (e.g., fluorescent proteins) are typically net electrically charged, and therefore, encapsulating them inside small cage structures at high concentrations is difficult because of the repulsive electrostatic interactions 8…”