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 . 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 . Endogenous TLR ligands such as heat shock proteins, fibrinogen and monosodium urate crystals have also been described . 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.
- NCL_Method_ITA-29_Aug2021.pdf(PDF | 499 KB)
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