Vault Nanoparticles: Chemical Modifications for Imaging and Enhanced Delivery

Abstract

Vault nanoparticles represent promising vehicles for drug and probe delivery. Innately found within human cells, vaults are stable, biocompatible nanocapsules possessing an internal volume that can encapsulate hundreds to thousands of molecules. They can also be targeted. Unlike most nanoparticles, vaults are nonimmunogenic and monodispersed and can be rapidly produced in insect cells. Efforts to create vaults with modified properties have been, to date, almost entirely limited to recombinant bioengineering approaches. Here we report a systematic chemical study of covalent vault modifications, directed at tuning vault properties for research and clinical applications, such as imaging, targeted delivery, and enhanced cellular uptake. As supra-macromolecular structures, vaults contain thousands of derivatizable amino acid side chains. This study is focused on establishing the comparative selectivity and efficiency of chemically modifying vault lysine and cysteine residues, using Michael additions, nucleophilic substitutions, and disulfide exchange reactions. We also report a strategy that converts the more abundant vault lysine residues to readily functionalizable thiol terminated side chains through treatment with 2-iminothiolane (Traut's reagent). These studies provide a method to doubly modify vaults with cell penetrating peptides and imaging agents, allowing for in vitro studies on their enhanced uptake into cells.

Keywords: cell-penetrating transporter; chemical modification; imaging; nanoparticle; protein cage; vaults.

Figure 1

Covalent attachment of cell penetrating peptides and fluorescent probes to vault nanoparticles.

Figure 2

hMVP vault modification with Traut’s reagent followed by labeling with a fluorescent probe. Size measurements were determined by DLS. The number of probes attached to the vaults was determined using UV–vis at 494 nm with 70,000 M⁻¹ cm⁻¹ as the extinction coefficient. The labeling reaction was run in triplicate, and the reported values are of the average of the three trials.

Figure 3

Uptake of FITC, FITC vaults 1a, FITC-Vault-r8_Cleavable 8, and FITC-Vault-r8_Noncleavable 12 into (A) RAW264.7 cells, (B) HeLa cells, and (C) CHO-K1cells. Data represents mean ± SD, N ≥ 3 for all measurements.

Figure 4

Confocal images of live RAW264.7 (top), HeLa (middle), and CHO-K1 (bottom) cells incubated with 30 µg of modified vaults/500,000 cells.

Figure 5

Normalized viability of cells treated with modified vaults at a concentration of 30 µg vaults/500,000 cells, as determined by MTT assay over 16 h. Data represents mean ± SD, N ≥ 3 for all measurements.

Scheme 1

Chemoselective Fluorescein Labeling of Vaults.

Scheme 2

Synthesis of Doubly Modified hMVP Vaults with a Fluorescent Probe and CPP^{a a}(A) Activation of Ac-Cys-(dArg)₈-NH₂ using 2,2′-dipyridyl disulfide followed by attachment of the activated Cys-r8 CPP onto FITC vaults through a disulfide exchange with MVP cysteine residues. (B) Activation of CPP octaarginine using GMBS followed by the attachment of the activated octaarginine onto FITC vaults through a thiol-maleimide linkage.