Intracellular Fate of Spherical Nucleic Acid Nanoparticle Conjugates

Abstract

Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome.

Immunomodulatory spherical nucleic acids

“…Similarly to previous observations involving other cell lines (25,26), immunomodulatory SNAs (Fig. 1A) are taken up by RAW 264.7 macrophages, a model antigen-presenting cell (APC) line, via endocytosis (Fig.…”

“…Various SNAs have been evaluated for their potential to treat a wide variety of diseases and conditions that may not be easily addressable with conventional therapeutic strategies (23,24). SNAs, especially ones containing natural phosphodiester nucleic acids, are internalized into various cells, mainly via caveolin-mediated endocytosis, leading to endosomal localization (25,26). Once inside the cell, the nucleic acid components of SNAs resist nuclease degradation, leading to longer intracellular lifetimes.…”

“…Once inside the cell, the nucleic acid components of SNAs resist nuclease degradation, leading to longer intracellular lifetimes. Moreover, SNAs, due to their multifunctional chemical structures, have the ability to bind their targets in a high-affinity multivalent fashion (25,26). Therefore, in addition to demonstrating many of the characteristics that would be considered preferred for designing immunomodulatory therapies, SNAs also provide a chemical and structural scaffold for evaluating the structure-activity relationships for the two major components: the oligonucleotide shell and the nanoparticle core.…”

Investigating the stability of DNA-coated gold nanoparticles

“…18,[27][28][29] On the other hand, DNA-gold nanoparticles have also shown great potential and exciting opportunities for disease diagnostic and therapeutic treatment and used in gene regulation, drug delivery, cancer cell imaging and photothermal therapy. 2,18,30,31 One of the inherent properties of DNA-coated AuNPs is the ability to traverse the charged cell membrane and enter a large variety of different cell types despite their polyanionic nature. By contrast, single or double stranded oligonucleotides of the same sequence usually require the use of lipid or cationic transfection agents in order to overcome the Coulombic repulsive forces between the cell membrane and the DNA phosphate backbone.…”

“…32 However, one of the most important properties of DNA-coated AuNPs is there great intracellular stability and increased resistance towards degradation by nucleases. 30,31,33 Research that involves the use of nucleic acids in biological environments is often hindered by the process of enzymatic hydrolysis, which leads to their degradation and renders them inactive. 34 DNA-coated AuNPs have shown an increased resistance to nucleases such as DNaseI.…”