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Relationships between Poly(ethylene glycol) Modifications on RNA-Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time.

ABSTRACT: Two synthetic approaches that allow one to control PEG content within spherical nucleic acids (SNAs) have been developed. One approach begins with RNA-modified gold nanoparticles followed by a backfill of PEG 2K alkanethiols, and the other involves co-adsorption of the two entities on a gold nanoparticle template. These two methods have been used to explore the role of PEG density on the chemical and biological properties of RNA-SNAs. Such studies show that while increasing the extent of PEGylation within RNA-SNAs extends their blood circulation half-life in mice, it also results in decreased cellular uptake. Modified ELISA assays show that constructs, depending upon RNA and PEG content, have markedly different affinities for class A scavenger receptors, the entities responsible, in part, for cellular internalization of SNAs. In designing SNAs for therapeutic purposes, these competing factors must be considered and appropriately adjusted depending upon the desired use.

SUBMITTER: Chinen AB

PROVIDER: S-EPMC5439959 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Relationships between Poly(ethylene glycol) Modifications on RNA-Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time.

Chinen Alyssa B AB Ferrer Jennifer R JR Merkel Timothy J TJ Mirkin Chad A CA

Bioconjugate chemistry 20161020 11

Two synthetic approaches that allow one to control PEG content within spherical nucleic acids (SNAs) have been developed. One approach begins with RNA-modified gold nanoparticles followed by a backfill of PEG 2K alkanethiols, and the other involves co-adsorption of the two entities on a gold nanoparticle template. These two methods have been used to explore the role of PEG density on the chemical and biological properties of RNA-SNAs. Such studies show that while increasing the extent of PEGylat ...[more]

PMID: 27762539

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Project description:Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics for treating infections caused by drug-resistant bacteria; yet, many peptides are limited by toxicity to eukaryotic cells and instability in biological environments. Conjugation to linear polymers that reduce cytotoxicity and improve stability, however, often decreases antimicrobial activity. In this work, we combine the biocompatibility advantages of poly(ethylene glycol) (PEG) with the efficacy merits of nonlinear polymer architectures that accommodate multiple AMPs per molecule. By conjugating a chemokine-derived AMP, stapled Ac-P9, to linear and star-shaped PEG with various arm numbers and lengths, we investigated the role of molecular architecture in solution properties (i.e., ζ-potential, size, and morphology) and performance (i.e., antimicrobial activity, hemolysis, and protease resistance). Linear, 4-arm, and 8-arm conjugates with 2-2.5 kDa PEG arms were found to form nanoscale structures in solution with lower ζ-potentials relative to the unconjugated AMP, suggesting that the polymer partially shields the cationic AMP. Reducing the length of the PEG arms of the 8-arm conjugate to 1.25 kDa appeared to better reveal the peptide, seen by the increased ζ-potential, and promote assembly into particles with a larger size and defined spherical morphology. The antimicrobial effects exerted by the short 8-arm conjugate rivaled that of the unconjugated peptide, and the AMP constituents of the short 8-arm conjugate were protected from proteolytic degradation. All other conjugates examined also imparted a degree of protease resistance, but exhibited some reduced level of antimicrobial activity as compared to the AMP alone. None of the conjugates caused significant cytotoxic effects, which bodes well for their future potential to treat infections. While enhancing proteolytic stability often comes with the cost of lower antimicrobial activity, we have found that presenting AMPs at high density on a neutral nonlinear polymer strikes a favorable balance, exhibiting both enhanced stability and high antimicrobial activity.

Project description: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.

Dataset Information

Relationships between Poly(ethylene glycol) Modifications on RNA-Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time.

Publications

Relationships between Poly(ethylene glycol) Modifications on RNA-Spherical Nucleic Acid Conjugates and Cellular Uptake and Circulation Time.

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