Tobacco mosaic virus rods and spheres as supramolecular high-relaxivity MRI contrast agents

Abstract

To compensate for the low sensitivity of magnetic resonance imaging (MRI), nanoparticles have been developed to deliver high payloads of contrast agents to sites of disease. Here, we report the development of supramolecular MRI contrast agents using the plant viral nanoparticle tobacco mosaic virus (TMV). Rod-shaped TMV nanoparticles measuring 300×18 nm were loaded with up to 3,500 or 2,000 chelated paramagnetic gadolinium (III) ions selectively at the interior (iGd-TMV) or exterior (eGd-TMV) surface, respectively. Spatial control is achieved through targeting either tyrosine or carboxylic acid side chains on the solvent exposed exterior or interior TMV surface. The ionic T₁ relaxivity per Gd ion (at 60 MHz) increases from 4.9 mM^-1s^-1 for free Gd(DOTA) to 18.4 mM^-1s^-1 for eGd-TMV and 10.7 mM^-1s^-1 for iGd-TMV. This equates to T₁ values of ~ 30,000 mM^-1s^-1 and ~ 35,000 mM^-1s^-1 per eGd-TMV and iGd-TMV nanoparticle. Further, we show that interior-labeled TMV rods can undergo thermal transition to form 170 nm-sized spherical nanoparticles containing ~ 25,000 Gd chelates and a per particle relaxivity of almost 400,000 mM^-1s^-1 (15.2 mM^-1s^-1 per Gd). This work lays the foundation for the use of TMV as a contrast agent for MRI.

Keywords: contrast agent; magnetic resonance imaging; tobacco mosaic virus; viral nanoparticle.

Figure 1

A PyMol image of tobacco mosaic virus highlighting the interior glutamic acids, GLU97 and GLU106 (orange), exterior tyrosine, TYR139 (yellow), for bioconjugation. Additional glutamic and aspartic acid residues (blue) and tyrosine residues (red) are highlighted for reference.

Figure 2

(A) Schematic illustration of the bioconjugation reactions used to incorporate terminal alkynes to the interior and exterior of TMV. (B) Schematic illustration of the CuAAC reaction to label TMV particles with Gd(DOTA). MALDI-TOF MS of (C) eGd-TMV and (D) iGd-TMV. In the MALDI-TOF MS, peaks labeled with eAlk and iAlk refer to the alkyne labeled proteins, 1-Gd, 2-Gd, and 3-Gd refer to coat proteins labeled with one, two, and three Gd(DOTA).

Figure 3

(A) Schematic illustration of the thermal transition from rod-shaped TMV to spherical nanoparticles. Representative TEM images of (B) iGd-TMV, (C) eGd-TMV to SNP, (D) iGd-TMV to SNP, 10 seconds, and (E) iGd-TMV to SNP 15 seconds. (F) SEM image of iGd-SNPs with (G) the corresponding DLS (Nanosight size analyzer). Scale bars = 500 nm.

Figure 4

(A) Phantom images of tubes containing eGd-TMV, iGd-TMV and Gd(DOTA) with the corresponding Gd concentrations in μM; measured using a clinical 1.5T MRI. Plot of 1/T₁ versus Gd concentration (mM) for eGd-TMV (B) and iGd-TMV (C) taken from three MR sources. The slopes of the plots correspond to the ionic relaxivity. Data were collected at varying field strengths (300 MHz, 64 MHz, and 60 MHz).

Figure 5

Graph comparing the nanoparticle relaxivities (left axis) and ionic relaxivities (right axis) of TMV particles formed in this paper (pattern) against other Gd-VNPs (solid) at 60 MHz (refs , and 13) or 64 MHz (refs , , and 14).