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Review

Detection of nanoparticles’ ability to stimulate toll-like receptors using HEK-Blue reporter cell lines: Version 2

Edward Cedrone  1 , Marina A. Dobrovolskaia  1
In: National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May. NCL Method ITA-29.
2023 Jan.
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Review

Detection of nanoparticles’ ability to stimulate toll-like receptors using HEK-Blue reporter cell lines: Version 2

Edward Cedrone et al.
Free Books & Documents

Excerpt

This document describes a protocol for analyzing nanoparticles’ ability to activate innate pattern recognition receptors of the toll-like receptor (TLR) family. TLRs play a vital role in the innate immune response; activation of these receptors leads to cytokine secretion that guides communication between innate and adaptive immunity [1]. For this reason, TLR agonists are explored as adjuvants for vaccines and immunotherapies [2, 3]. Often, such agonists are formulated using nanoparticles to achieve delivery to a specific subset of immune cells. Via TLRs, the immune cells sense and respond to microbes and their components, such as flagellin, CpG DNA, lipopolysaccharide, beta-glucans, single- and double-stranded RNA, to name a few [3]. Endogenous TLR ligands such as heat shock proteins, fibrinogen and monosodium urate crystals have also been described [4]. Nanoparticles may activate TLRs directly due to either their payloads such as immunostimulatory TLR agonists or the carrier itself. Nanoparticles may also indirectly activate TLRs by inducing immunogenic cell death and the host cells’ release of endogenous TLR ligands. The indirect activation could also be due to the nanoparticle carrier or the drug payload. Therefore, understanding nanoparticles’ ability to activate TLRs may help verify the activity of TLR agonists formulated using nanoparticles and get an insight into mechanisms of the innate immune responses to nanoparticle formulations, APIs and carriers, should such responses occur. This protocol complements NCL protocols ITA-10, STE-1, and STE-4, used for the detection of nanoparticles’ ability to induce cytokine responses, quantification of endotoxins, and quantification of beta-glucans, respectively.

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References

    1. Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Microbes Infect. 2004. Dec;6(15):1382–7. - PubMed
    1. Gnjatic, S., Sawhney, N. B., & Bhardwaj, N. (2010). Toll-like receptor agonists: are they good adjuvants? Cancer journal (Sudbury, Mass.), 16(4), 382–391. - PMC - PubMed
    1. Luchner M, Reinke S, Milicic A. TLR Agonists as Vaccine Adjuvants Targeting Cancer and Infectious Diseases. Pharmaceutics. 2021. Jan 22;13(2):142. - PMC - PubMed
    1. Yu L, Wang L, Chen S. Endogenous toll-like receptor ligands and their biological significance. J Cell Mol Med. 2010. Nov;14(11):2592–603. - PMC - PubMed
    1. Invivogen product sheets for HEK Blue TLR cell lines, HEK Blue Detection, SEAP Reporter Assay Kit and SEAP Protein. (https://www.invivogen.com/hek).

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