ABSTRACT
The effects of spherical nucleic acid (SNA) gold nanoparticle conjugates on the activation of macrophages in vitro and release of cytokines in vivo were explored. Herein, we show that G-quadruplexes, the formation of which is enhanced on gold nanoparticle surfaces, elicit an increase in cytokine release from mouse and human macrophages and induce the upregulation of activation receptors as well as NO2 production in vitro. Moreover, these G-rich SNAs can induce cytokine release when injected intravenously, though there were no severe, long-term effects observed. These results further reinforce the notion that nucleic acid sequence and structure play an important role in how SNAs interact in biological milieu and highlight a key design parameter.
Subject(s)
G-Quadruplexes , Gold/chemistry , Macrophage Activation/drug effects , Macrophages/metabolism , Metal Nanoparticles/administration & dosage , Nucleic Acids/chemistry , Animals , Cells, Cultured , Cytokines/metabolism , Humans , In Vitro Techniques , Macrophages/drug effects , Metal Nanoparticles/chemistry , Mice , Mice, Inbred C57BL , Nitrogen Dioxide/metabolism , Nucleic Acids/metabolismABSTRACT
The effect of serum protein adsorption on the biological fate of Spherical Nucleic Acids (SNAs) is investigated. Through a proteomic analysis, it is shown that G-quadruplexes templated on the surface of a gold nanoparticle in the form of SNAs mediate the formation of a protein corona that is rich in complement proteins relative to SNAs composed of poly-thymine (poly-T) DNA. Cellular uptake studies show that complement receptors on macrophage cells recognize the SNA protein corona, facilitating their internalization, and causing G-rich SNAs to accumulate in the liver and spleen more than poly-T SNAs in vivo. These results support the conclusion that nucleic acid sequence and architecture can mediate nanoparticle-biomolecule interactions and alter their cellular uptake and biodistribution properties and illustrate that nucleic acid sequence is an important parameter in the design of SNA therapeutics.
Subject(s)
Endocytosis , Macrophages/metabolism , Nucleic Acids/metabolism , Protein Corona/metabolism , Animals , Base Sequence , Blood Proteins/metabolism , Cell Line , G-Quadruplexes , Humans , Liver/metabolism , Mice, Inbred C57BL , Receptors, Cell Surface/metabolism , Spleen/metabolism , Tissue DistributionABSTRACT
An allosterically regulated, asymmetric receptor featuring a binding cavity large enough to accommodate three-dimensional pharmaceutical guest molecules as opposed to planar, rigid aromatics, was synthesized via the Weak-Link Approach. This architecture is capable of switching between an expanded, flexible "open" configuration and a collapsed, rigid "closed" one. The structure of the molecular receptor can be completely modulated in situ through the use of simple ionic effectors, which reversibly control the coordination state of the Pt(II) metal hinges to open and close the molecular receptor. The substantial change in binding cavity size and electrostatic charge between the two configurations is used to explore the capture and release of two guest molecules, dextromethorphan and ß-estradiol, which are widely found as pollutants in groundwater.
Subject(s)
Coordination Complexes/chemistry , Dextromethorphan/chemistry , Estradiol/chemistry , Platinum/chemistry , Coordination Complexes/chemical synthesis , Molecular Structure , Quantum Theory , Static ElectricityABSTRACT
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.
Subject(s)
Polyethylene Glycols/chemistry , RNA/chemistry , RNA/metabolism , Adsorption , Animals , Biological Transport , Gold/chemistry , Half-Life , Metal Nanoparticles/chemistry , Mice , Models, Molecular , Nucleic Acid Conformation , RNA/bloodABSTRACT
Ribonucleic acids (RNAs) are key components in many cellular processes such as cell division, differentiation, growth, aging, and death. RNA spherical nucleic acids (RNA-SNAs), which consist of dense shells of double-stranded RNA on nanoparticle surfaces, are powerful and promising therapeutic modalities because they confer advantages over linear RNA such as high cellular uptake and enhanced stability. Due to their three-dimensional shell of oligonucleotides, SNAs, in comparison to linear nucleic acids, interact with the biological environment in unique ways. Herein, the modularity of the RNA-SNA is used to systematically study structure-function relationships in order to understand how the oligonucleotide shell affects interactions with a specific type of biological environment, namely, one that contains serum nucleases. We use a combination of experiment and theory to determine the key architectural properties (i.e., sequence, density, spacer moiety, and backfill molecule) that affect how RNA-SNAs interact with serum nucleases. These data establish a set of design parameters for SNA architectures that are optimized in terms of stability.
Subject(s)
Drug Design , RNA/chemistry , Base Sequence , Gold/chemistry , Metal Nanoparticles/chemistry , Models, Molecular , Nucleic Acid Conformation , RNA/genetics , RNA Stability , Surface PropertiesABSTRACT
Patients whose cancer is detected early are much more likely to have a positive prognosis and outcome. Nanoflares hold promise as a practical diagnostic platform for the early detection of cancer markers in living cells. These probes are based on spherical nucleic acid (SNAs) and are typically composed of gold nanoparticle cores and densely packed and highly oriented oligonucleotide shells; these sequences are complementary to specific mRNA targets and are hybridized to fluorophore-labeled reporter strands. Nanoflares take advantage of the highly efficient fluorescence quenching properties of gold, the rapid cellular uptake of SNAs that occurs without the use of transfection agents, and the enzymatic stability of such constructs to report a highly sensitive and specific signal in the presence of intracellular target mRNA. In this chapter, we will focus on the synthesis, characterization, and diagnostic applications of nanoflares as they relate to cancer markers.
Subject(s)
Immobilized Nucleic Acids , Metal Nanoparticles , Nanoconjugates , Nanomedicine/methods , Neoplasms/diagnosis , Animals , Fluorescent Dyes , Humans , Immobilized Nucleic Acids/chemical synthesis , Immobilized Nucleic Acids/chemistry , Metal Nanoparticles/chemistry , Nanoconjugates/chemistryABSTRACT
To understand the effect of three-dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence-specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid-based nanostructures, and SNAs in particular, function in complex biological milieu.
Subject(s)
Blood Proteins/chemistry , G-Quadruplexes , Nanoparticles , Nucleic Acids/chemistryABSTRACT
Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in bloodso-called circulating tumor cells (CTCs)may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.
