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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Am Chem Soc. Author manuscript; available in PMC 2010 December 2.
Published in final edited form as:
PMCID: PMC2797550
NIHMSID: NIHMS159006

Buckyballs meet Viral Nanoparticles – Candidates for Biomedicine

Abstract

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Fullerenes such as C60 show promise as functional components in several emerging technologies. For biomedical applications, C60 has been used in gene- and drug-delivery vectors, as imaging agents, and as photosensitizers in cancer therapy. A major drawback of C60 for bioapplications is its insolubility in water. To overcome this limitation, we covalently attached C60 derivatives to Cowpea mosaic virus and bacteriophage Qβ virus-like particles, as 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 and stable VNP-C60 and VNP-PEG-C60 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 PEG-modified propargyl-O-PEG-C60 derivative gave rise to high loadings of fullerene on the VNP surface, indicated by the imaging of individual C60 units by STEM. The cellular uptake of dye-labeled VNP-PEG-C60 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 C60 units. These results open the door for the development of novel therapeutic devices with potential applications in photo-activated tumor therapy.

The fullerene class of carbon allotropes shows promise as functional components in several emerging technologies. Properties such as high electron affinity and charge transport capabilities have made derivatives of C60 (aka Buckyball) and carbon nanotubes particularly attractive for next generation photovoltaic and electrical energy storage devices.1 More recently, there has been increased interest in studying fullerenes for use in biomedicine. C60 has been used in gene-delivery vectors, HIV-1 protease inhibitors, magnetic resonance imaging agents, and drug-delivery.2 Due to its exceptional radical-scavenging properties, C60 is a promising candidate for photosensitizers in cancer therapy and the treatment of inflammatory diseases.3

A major drawback of C60 for biological applications is its insolubility in water, and numerous modifications have been made to increase its aqueous biocompatibility.4 We describe the alternative approach of covalently attaching C60 derivatives to a larger biological structure, in this case viral nanoparticles (VNPs). VNPs are naturally occurring self-assembling protein structures with potential applications ranging from materials to biomedicine.5 Here we employ Cowpea mosaic virus (CPMV) and the capsid of bacteriophage Qβ (Fig. 1A), both of which are 30 nm in size and have icosahedral symmetry. Qβ is formed from 180 copies of a single coat protein subunit, while CPMV is composed of 60 copies of two different coat proteins, designated the large (L) and small (S) subunits. The capsid of each VNP offers multivalent attachment sites at solvent-exposed amino acids, on which diverse molecules such as redox-active moieties, imaging agents, and targeting ligands have been previously displayed.5 The generation of aggregates of single-walled carbon nanotubes and Flock House virus has been reported.6

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 ...

We envisioned that VNPs could serve both as hydrophilic “chaperones” for C60, making the fullerene water soluble, and as platforms for the organized assembly of multiple C60 units in combination with other functional molecules. The specific, localized binding of C60 to VNP scaffolds could resolve problems of aggregation and cluster formation common to unbound fullerene derivatives. The goal of this study was to determine whether the advantages of fullerenes and VNPs could be combined by covalently attaching C60 derivatives to CPMV and Qβ. To test the potential of the hybrid nanomaterials as candidates for biomedical applications such as photodynamic tumor therapy, the cellular uptake of VNP-C60 complexes in a human cancer cell lines was studied.

The VNPs were decorated with C60 in two ways. First, the well-characterized fullerene derivative 1-(3-carboxypropyl)-1-phenyl-[6,6]C61 (PCBA) was activated by carbodiimide-N-hydroxysuccinimide chemistry and coupled to solvent-exposed Lys residues on CPMV and Qβ (Fig. 1B and Supp. Info.). Second, we employed the copper-catalyzed azide-alkyne cycloaddition (CuCAAC) “click” reaction,7 which has found wide application due to its high rate and specificity. CuAAC protocols have been developed and improved in the past several years for the attachment of functional molecules to VNPs with high loadings.8 In the present case, a propargyl-O-PEG-C60 derivative was synthesized and conjugated to azide-modified Qβ particles using an optimized procedure8a (Fig. 1C and Supp. Info), resulting in significantly higher loading than the attachment of PCBA using activated ester chemistry. In both cases, the existence of only a single linker group on each C60 derivative prevented covalent aggregation of the nanoparticles. The resulting hybrid complexes (VNP-C60 and VNP-PEG-C60) were soluble and stable in aqueous buffer solutions for at least several months.

VNP-C60 and VNP-PEG-C60 samples were characterized by a combination of techniques. Size-exclusion chromatography (SEC, Fig. 1D,E), transmission electron microscopy (TEM, Figure 1M,N), scanning transmission electron microscopy (STEM, Fig. 1O), and native gel electrophoresis (Supp. Info.) of VNP-C60 particles confirmed their intact nature with no detectable decomposition. The covalent attachment of C60 was verified in both cases by western blotting using an anti-C60 antibody (Fig. 1H,I) and in case of Qβ also by STEM (Fig. 1O). Dynamic light scattering (DLS, Fig. 1J) showed no significant changes in the apparent size of CPMV and Qβ upon attachment of C60, consistent with low coverage (see below). SEC of Qβ and Qβ-C60 showed no change in retention time, whereas CPMV exhibited a change in retention time upon fullerene attachment (23.9 min to 26.0 min). These differing SEC comparisons may reflect differing chemical interactions with the chromatography stationary phase that complicate the correlation between retention time and size. C60 is perhaps unusual in this respect, since it is spherical and hydrophobic. Interestingly, C60 moieties were found attached only to the S protein of CPMV (Fig. 1I), presumably at the highly reactive K38 residue.9

Similarly, the click reaction to prepare Qβ-PEG-C60 gave a mixture of intact particles (peak b in Fig. 1F) plus aggregated material (peak a) and broken particles (peak c). No interaction was observed between Qβ-azide and propargyl-O-PEG-C60 in the absence of CuI, supporting the covalent nature of the derivatization. Intact Qβ-PEG-C60 particles were purified by SEC and reanalyzed (Fig. 1G), showing a shift to shorter retention time compared to the underivatized particle (28.9 min to 28.1 min), consistent with an increase in size upon C60 attachment. TEM (Fig. 1P), STEM (Fig. 1Q), and native gels (Supp. Info.) further confirmed the structural integrity of the Qβ-PEG-C60 conjugate. The hydrodynamic radius of the particle was found by DLS to increase from 13.1 nm for unmodified Qβ to 16.7 nm for Qβ-PEG-C60, in good agreement with the expected dimensions of the attached species (1 nm diameter for C60 plus approximately 2.3 nm length of the PEG-1000 chain, see Supporting Information).

Covalent attachment of PEG-C60 to Qβ was further verified by the appearance of two distinct bands on denaturing gel electrophoresis, corresponding to non-labeled and PEG-C60-conjugated coat proteins (Fig. 1K). Western blotting was attempted but was not successful, which may be due to blocking of the antibody-fullerene interaction by the PEG chains (not shown).

A combination of techniques was used to quantify the degree of C60 loading. STEM (Fig. 1O) and UV-vis absorbance (332 nm, Supp. Info.) indicated sparse decoration of Qβ (approximately 3 C60 molecules per particle) with PCBA using carbodiimide chemistry. This modest loading level derives from the relative aqueous insolubility of PCBA and the modest rates of amine-NHS ester reactions, and is presumably similar for the analogous CPMV reactions.

In contrast, Qβ-PEG-C60 showed a much higher level of coverage. Individual C60 particles were easily visualized in large numbers around each VLP as dots of bright contrast in STEM images after osmium tetroxide staining (Fig. 1Q). UV-vis absorbance spectroscopy (332 nm, Supp. Info.) indicated a loading of 45–50 C60 molecules per Qβ particle. Quantitative comparison of the intensities of the derivatized and unlabeled protein bands in denaturing protein gels after Coomassie staining (Fig. 1K) gave rise to a similar estimate of 30–40 C60 molecules per Qβ-PEG-C60 conjugate. The significantly greater loading is likely the result of better solubility of the PEGylated C60 reagent in the aqueous reaction mixture and the higher efficiency of the CuAAC reaction.

To evaluate the potential of the hybrid nanomaterials as candidates for biomedical applications, cellular uptake of dye-labeled VNP-C60 (not shown) and VNP-PEG-C60 complexes in the HeLa human cancer cell line was studied using confocal microscopy (Fig. 2 and Supp. Info.) Qβ-PEG-C60 was labeled with approximately 60 AlexaFluor568 (A568) fluorophores per particle in a second CuAAC reaction (Supp. Info.), with dye attachment being confirmed by UV-vis, SEC, and native and denaturing gel electrophoresis (Supp. Info.). Cellular uptake was revealed by the acquisition of Z-dimensional fluorescence data (Fig. 2), and was found to be the same as for analogous Qβ particles bearing only the dye (data not shown), showing that internalization was not inhibited by the attached C60 units.

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 conclusion, we have demonstrated that modified fullerene and protein nanoparticles can be covalently linked to each other with high efficiency by click chemistry, retaining the structural, spectroscopic, and biological properties of each. The hybrid VNP-C60/VNP-PEG-C60 complexes were water-soluble and biocompatible, and the VNPs serve as scaffolds and vehicles for detectable C60-delivery into cells. This opens the door for the development of novel therapeutic devices with potential applications in photo-activated tumor therapy. Studies along these lines are currently under investigation in our laboratories.

Supplementary Material

1_si_003

Acknowledgement

We thank Dr. So-Hye Cho and Dr. Rebecca Taurog for TEM studies. This work was supported by the NIH 1K99EB009105 (to N.F.S), R01CA112075 (to MM and MGF), RR021886 (to MGF), the American Heart Association (postdoctoral fellowship to NFS), and the W.M. Keck Foundation. Work at Sandia National Labs was funded by Sandia’s Laboratory Directed Research and Development Program and the Department of Energy Basic Energy Sciences Program. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000.

Footnotes

Supporting Information Available: Experimental details and supporting Figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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