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.

Centrifuge 20 volumes of the peptide-GNP hybrid solution in 1.5-milliliter eppendorf tubes at 18,000 times g for 30 minutes. Carefully discard the supernatants. Transfer the hybrids to a single fresh tube, and wash them twice with 1 milliliter of PBS. After this, resuspend the washed hybrids in one volume of R10 medium. Mix equal volumes of the concentrated hybrids and the LPS-containing R10 medium such that the final concentration of the hybrids and LPS are 100 hundred nanomolar and 10 nanograms per milliliter respectively.

Next, remove the culture medium from the macrophage culture plate. Add 100 microliters of the hybrid-LPS mixture into each well with three replicates for each condition. Include a negative control and an LPS control. Incubate at 37 degrees Celsius for 24 hours, then, transfer the medium of each well to a separate centrifuge tube. Centrifuge the tubes at 18,000 times g in 4 degrees Celsius for 30 minutes. Transfer the supernatants into a fresh 96-well round bottom plate.

To begin the reporter assay on these samples, add 180 microliters of pre-warmed QUANTI-Blue solution to each well of a new 96-well flat bottom plate, then transfer 20 microliters of the supernatants from each sample into this plate. Incubate at 37 degrees Celsius for one to two hours until the dark blue color develops to an optical density over 1. After this, use a plate reader to record the absorption at 655 nanometers. Next, transfer 10 microliters of fresh supernatant from each sample into a 96-well clear flat-bottom white plate.

Using an automatic injector, add 50 microliters of the luciferase solution to each well and immediately record the luminescence well by well. Then, validate the inhibitory effect of the potential candidates and evaluate the TLR specificity as outlined in the text protocol.