Peptide antisense nanoparticles

Abstract

We have designed a heterofunctionalized nanoparticle conjugate consisting of a 13-nm gold nanoparticle (Au NP) containing both antisense oligonucleotides and synthetic peptides. The synthesis of this conjugate is accomplished by mixing thiolated oligonucleotides and cysteine-terminated peptides with gold nanoparticles in the presence of salt, which screens interactions between biomolecules, yielding a densely functionalized nanomaterial. By controlling the stoichiometry of the components in solution, we can control the surface loading of each biomolecule. The conjugates are prepared easily and show perinuclear localization and an enhanced gene regulation activity when tested in a cellular model. This heterofunctionalized structure represents a new strategy for preparing nanomaterials with potential therapeutic applications.

Fig. 1.

Preparation of peptide and oligonucleotide functionalized gold nanoparticles. (A) Oligonucleotides were modified with a 3′ thiol group, and peptides were synthesized with an N-terminal cysteine to facilitate attachment to the gold nanoparticle surface. The key to creating the peptide ASNPs is the addition of salt before the addition of biomolecules to screen the charges. (B) Oligonucleotide and peptide loading as a function of solution stoichiometry on 13-nm gold nanoparticles determined by fluorescent labeling.

Fig. 2.

Activity of peptide antisense nanoparticles. (A) Representative Western blots showing the expression of GAPDH in HeLa cells treated with various concentrations and compositions of nanoparticle conjugates. GAPDH expression is reduced in a dose- and sequence-dependent manner. α-Tubulin is shown as the loading control. (B) Relative decrease in GAPDH expression in HeLa cells. α-Tubulin was used as a loading control and for subsequent normalization of GAPDH knockdown. The error bars represent the standard deviation from at least 3 Western blots.

Fig. 3.

Confocal fluorescence microscopy images of HeLa cells treated with 13-nm (A–C) or 5-nm (D–F) antisense particles functionalized with Cy5-terminated oligonucleotides (red) (A and D), Tat peptides and Cy5-terminated oligonucleotides (B and E), or NLS peptides and Cy5-terminated oligonucleotides (C and F). Nucleus (blue) is stained with Hoechst 33342. Scale bar is 10 μm in each image.