An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition

Take a multiwell plate with different reporter macrophages harboring the secreted embryonic alkaline phosphatase, or SEAP, gene or a secreted luciferase gene under inducible promoters.

Add media containing different peptide-gold nanoparticle hybrids mixed with lipopolysaccharides or LPS to designated wells.

LPS binds to macrophage toll-like receptors, or TLR, triggering internalization into the endosome.

The endosomal proton pump transports protons inside. The acidification triggers TLR signaling, activating transcription factors.

These factors induce the expression of the reporter genes, producing SEAP or luciferase.

Alternatively, internalization of LPS along with the hybrids causes the negatively charged peptide groups to sequester luminal protons, preventing endosomal acidification and inhibiting TLR signaling.

Collect the media, centrifuge to separate the supernatant, and add substrates for SEAP and luciferase.

SEAP converts its substrate to a colored product, while luciferase catalyzes its substrate to produce luminescence.

A reduction in reporter signals indicates the effectiveness of the hybrids in TLR signaling inhibition.