Buckyballs meet viral nanoparticles: candidates for biomedicine

Abstract

Fullerenes such as C(60) show promise as functional components in several emerging technologies. For biomedical applications, C(60) has been used in gene- and drug-delivery vectors, as imaging agents, and as photosensitizers in cancer therapy. A major drawback of C(60) for bioapplications is its insolubility in water. To overcome this limitation, we covalently attached C(60) derivatives to Cowpea mosaic virus and bacteriophage Qbeta virus-like particles, which are examples of naturally occurring viral nanoparticle (VNP) structures that have been shown to be promising candidates for biomedicine. Two different labeling strategies were employed, giving rise to water-soluble, stable VNP-C(60) and VNP-PEG-C(60) conjugates. Samples were characterized using a combination of transmission electron microscopy, scanning transmission electron microscopy (STEM), gel electrophoresis, size-exclusion chromatography, dynamic light scattering, and Western blotting. "Click" chemistry bioconjugation using a poly(ethylene glycol) (PEG)-modified propargyl-O-PEG-C(60) derivative gave rise to high loadings of fullerene on the VNP surface, as indicated by the imaging of individual C(60) units using STEM. The cellular uptake of dye-labeled VNP-PEG-C(60) complexes in a human cancer cell line was found by confocal microscopy to be robust, showing that cell internalization was not inhibited by the attached C(60) units. These results open the door for the development of novel therapeutic devices with potential applications in photoactivated tumor therapy.

Figure 1

(A) Structure of CPMV and Qβ (reproduced from VIPERdb). (B) Derivatization of CPMV and Qβ with PCBA. (C) Derivatization of Qβ with propargyl-O-PEG-C₆₀; THPTA = tris(3-hydroxypropyl-4-triazolylmethyl)amine as an accelerating Cu-binding ligand, n = average of 20. (D, E) SEC using a Superose 6 column (black line = absorbance at 260 nm, grey line = absorbance at 280 nm). (F) SEC of Qβ mixed with propargyl-O-PEG-C₆₀ (dark blue line = 260 nm, light blue line = 280 nm) vs. Qβ reacted with propargyl-O-PEG-C₆₀ in presence of CuSO₄, THPTA, and Na ascorbate (red line = 260 nm, pink line = 280 nm). Peak a (19.6 min) = VNP aggregates, b (28.3 and 29.0 min) = intact Qβ-PEG-C₆₀ and Qβ, respectively, c (44.9 min) = broken VNPs, d (53 min) = click reaction reagents (ascorbate, ligand). (G) SEC of Qβ-PEG-C₆₀ after purification (red line = 260 nm, pink line = 280 nm). vs. Qβ (dark blue line = 260 nm, light blue line = 280 nm). (H, I) Coomasie gel showing coat proteins and western blot using anti-C₆₀ specific antibodies. 1 = CPMV-C₆₀, 2 = CPMV, 3 = Qβ-C₆₀, 4 = Qβ. (J) Hydrodynamic radius as determined by DLS. (K) Coomassie gel showing the coat proteins: 5 = Qβ, 6 = Qβ-azide, 7 = Qβ-PEG-C₆₀ (aggregate, peak a of Panel F), 8 = Qβ-PEG-C₆₀ (intact VNPs, panel G), (L) Hydrodynamic radius determined by DLS. * Differences were significant with p < 0.05. (M, N) TEM of uranyl acetate-stained VNP-C₆₀ conjugates. (O) STEM of osmium-tetroxide-stained Qβ-C₆₀ conjugates. Arrows indicate heavily stained C₆₀ nanoparticles bound around the equators of Qβ particles. (P) TEM of uranyl acetate-stained Qβ-PEG-C₆₀ conjugates. (Q) STEM of OsO₄-stained Qβ-PEG-C₆₀ conjugates. Inset reveals higher loading of VNPs with C₆₀ nanoparticles, indicated by dots of bright contrast.

Figure 2

Confocal microscopy. (A) HeLa cells only. (B–F) Cells treated with Qβ-PEG-C₆₀-A568 particles. Blue = nuclei (DAPI), red = Qβ-PEG-C₆₀-A568, green = WGA-A488. (D) Z-section image (1.2 µm deep) recorded along the line shown in C; step size 0.3 µm. (E,F) Same cell as shown in D, image reconstructions using Imaris software.