Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells

Abstract

Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach.

Figure 1

Synthesis of DNA functionalized hollow SiO₂ particles using gold nanoparticles as sacrificial templates.

Figure 2

STEM images of Au@SiO₂ particles (A–C) and hollow SiO₂ particles (D–F) in scanning, z-contrast, and transmission modes, respectively. In A–C, the Au NP cores are visible. After the addition of I₂, the Au NP cores dissolve leaving a hollow interior (E–F). Scale bars are 100 nm.

Figure 3

(A) Extinction spectra of Au NP templates, silica-coated Au NPs (Au@SiO₂), and hollow SiO₂ particles resulting from the treatment of the Au@SiO₂ particles with I₂. The Au@SiO₂ particles exhibit a distinct absorption at ~530 nm that is characteristic of Au NP, albeit slightly red-shifted due to the silica shell. After treatment with I₂, the hollow SiO₂ particles do not contain this absorption band, confirming the dissolution of the Au NP core. (B) Melting analysis of DNA functionalized SiO₂ particles hybridized using a self-complementary linker. The sharp melting transition (full-width half-max = ~2°C) is indicative of cooperative binding.

Figure 4

Cellular uptake of the DNA functionalized Au@SiO₂ and DNA functionalized hollow SiO₂ particles into C166 cells. (A) C166 cells were treated with Au@SiO₂ particles (upper panel) and hollow SiO₂ particles (lower panel) functionalized with Cy5 dye-labeled anti-eGFP DNA oligonucleotides. Cy5 fluorescence is observed in the cytoplasm, but not in the nuclei, indicating the internalization of the particles into the cells. Scale bar = 20 μm. (B) eGFP knockdown at the mRNA level determined by qRT-PCR. mRNA levels of cells treated with anti-eGFP DNA oligonucleotide functionalized Au@SiO₂ and hollow SiO₂ particles were significantly reduced when compared with those treated with scrambled-sequence DNA particles. (C) Minimal cell toxicity of the Au@SiO₂ and hollow SiO₂ particles toward C166 cells was detected using the MTT assay. Cells treated with the eGFP-targeted and non-targeted DNA oligonucleotides showed up to 95% viability compared with non-treated cells.