Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates

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Immunomodulatory nucleic acids have extraordinary promise for treating disease, yet clinical progress has been limited by a lack of tools to safely increase activity in patients. Immunomodulatory nucleic acids act by agonizing or antagonizing endosomal toll-like receptors (TLR3, TLR7/8, and TLR9), proteins involved in innate immune signaling. Immunomodulatory spherical nucleic acids (SNAs) that stimulate (immunostimulatory, IS-SNA) or regulate (immunoregulatory, IR-SNA) immunity by engaging TLRs have been designed, synthesized, and characterized. Compared with free oligonucleotides, IS-SNAs exhibit up to 80-fold increases in potency, 700-fold higher antibody titers, 400-fold higher cellular responses to a model antigen, and improved treatment of mice with lymphomas. IR-SNAs exhibit up to eightfold increases in potency and 30% greater reduction in fibrosis score in mice with nonalcoholic steatohepatitis (NASH). Given the clinical potential of SNAs due to their potency, defined chemical nature, and good tolerability, SNAs are attractive new modalities for developing immunotherapies.

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confidence: 99%

Immunomodulatory spherical nucleic acids

Radovic‐Moreno

Chernyak

Mader

et al. 2015

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233

“…These constructs have been shown to enter Significance To our knowledge, as the first genetic-based approach for the simultaneous isolation and intracellular genetic analysis of live circulating tumor cells, NanoFlares provide opportunities for invasive cancer study, diagnosis, prognosis, and personalized therapy. over 60 cell lines and primary cells tested to date, and they are believed to do so by engaging scavenger receptors that facilitate caveolin-mediated endocytosis (17). They are stable to nuclease degradation (21), lack cytotoxicity (22), and are effective for single-gene and multigene detection from a single nanoconstruct (18).…”

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confidence: 99%

NanoFlares for the detection, isolation, and culture of live tumor cells from human blood

Halo

McMahon

Angeloni

et al. 2014

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201

146

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 blood-so-called circulating tumor cells (CTCs)-may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable livecell 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.cancer metastasis | nanotechnology | diagnostic | mRNA | NanoFlares

“…These binding events displace the flares from the gold surface, resulting in a discrete "turning on" of fluorescence, the extent of which is quantifiably related to the expression level of the target RNA. Importantly, the Nanoflare enters live cells via receptormediated endocytosis without the need for harmful transfection techniques (19,(23)(24)(25)(26)(27)(28) and with negligible cytotoxicity (29) and immunogenicity (30). As a result, the Nanoflare has grown into a…”

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confidence: 99%

Quantification and real-time tracking of RNA in live cells using Sticky-flares

Briley

Bondy

Randeria

et al. 2015

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105

We report a novel spherical nucleic acid (SNA) gold nanoparticle conjugate, termed the Sticky-flare, which enables facile quantification of RNA expression in live cells and spatiotemporal analysis of RNA transport and localization. The Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner. On recognition, the Sticky-flare transfers a fluorophore-conjugated reporter to the transcript, resulting in a turning on of fluorescence in a quantifiable manner and the fluorescent labeling of targeted transcripts. The latter allows the RNA to be tracked via fluorescence microscopy as it is transported throughout the cell. We use this novel nanoconjugate to analyze the expression level and spatial distribution of β-actin mRNA in HeLa cells and to observe the real-time transport of β-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Stickyflares for tracking transcripts that undergo more extensive compartmentalization by fluorophore-labeling U1 small nuclear RNA and observing its distribution in the nucleus of live cells.he study of RNA is a critical component of biological research and in the diagnosis and treatment of disease. Recently, the localization of mRNA has been identified as an essential process for a number of cellular functions, including restricting the production of certain proteins to specific compartments within cells (1). For instance, synaptic potentiation, the basis of learning and memory, relies on the local translation of specific mRNAs in pre-and postsynaptic compartments (2). Likewise, the misregulation of RNA distribution is associated with many disorders, including mental retardation, autism, and cancer metastasis (3-5). However, despite the significant role of mRNA transport and localization in cellular function, the available methods to visualize these phenomena are severely limited. For example, FISH, the most commonly used technique to analyze spatial distribution of RNA, requires fixation and permeabilization of cells before analysis (6). As a result, analysis of dynamic RNA distribution is restricted to a single snapshot in time (7,8). With such a limitation, understanding the translocation of RNA with respect to time, cell cycle, or external stimulus is difficult if not impossible. Furthermore, fixed cell analysis is a lengthy and highly specialized procedure due to the number of steps necessary to prepare a sample. Fixation, permeabilization, blocking, and staining processes each require optimization and vary based on cell type and treatment conditions, rendering FISH prohibitively complicated in many cases. Likewise, live cell analysis platforms such as molecular beacons require toxic transfection techniques, such as microinjection or lipid transfection, and are rapidly sequestered to the nucleus on cellular entry (9, 10). Recently more sophisticated live cell analyses have been developed that use genetic engineering to introduce exogenous hybrid gene...