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

1. Screening for Potential TLR4 Nano-inhibitors Using the Reporter Cells

NOTE: Since TLR4 signaling utilizes both MyD88-dependent land TRIF-dependent pathways, it is selected as the primary target to encompass a wide range of TLR signaling pathways. THP-1-XBlue reporter cells are used to mainly examine the NF-κB/AP-1 activation while THP-1-Dual cells are for IRF activation from the TRIF-dependent signal transduction.

1. Identify the optimal LPS dose by generating a dose-response curve prior to the screening.​
   1. Dilute the working LPS solution to a final concentration of 0.01 – 100 ng/mL in R10 medium (make dilution in log10 scale). Layout the plate design and make dilution in a 96-well round-bottom culture plate. Make sure that each well has a minimum of 110 μL of solution after dilution.
   2. Gently remove the culture medium from the cell-seeded plate using a vacuum aspirator.
   3. Transfer the LPS containing R10 medium prepared in the dilution plate (1.1.1) into the culture plate according to the layout of the samples. Incubate the plate at 37 °C (in a cell culture incubator) for 24 h.
   4. At 24 h, carefully transfer the supernatants (80 μL/well) into a new 96-well round-bottom plate. Conduct the colorimetric and/or luciferase luminescence assay on these solutions immediately or store them at 4 °C (several hours) or -20 °C (days) prior to the assay development.
      NOTE: The design of the plate layout should be simple and clear for experiment and data analysis. For each condition, 2-4 replicates should be considered. Always include a negative control group (LPS null). It is recommended to use THP-1-XBlue cells for NF-κB/AP-1 activation as the Dual cells have a relatively high background of NF-κB/AP-1 activation after being differentiated into macrophages. However, it is feasible to use the Dual cells for reporting both NF-κB/AP-1 and IRF activation.
2. Develop the colorimetric assay for the NF-κB/AP-1 activation and the luciferase luminescence assay for IRF activation.
   1. To assess the NF-κB/AP-1 activation, transfer 20 μL of supernatant of each sample into a new 96-well flat-bottom culture plate, and add 180 μL of pre-warmed SEAP substrate solution into each well. Incubate the plate at 37 °C for 1 – 2 h to allow the color development (pink to dark blue). Collect the absorption at 655 nm on a plate reader.
      CAUTION: The incubation time can be varied based on the color development (30 min up to overnight incubation). It is recommended to wait until the optical density (O.D.) of the darkest color reaches above 1 while avoiding the saturation of the color development (O.D >3).
   2. For the analysis of IRF activation, transfer 10 μL of supernatant of each sample into a 96-well clear flat-bottom white plate. Add the luciferase solution (50 μL) and immediately collect the luminescence well by well.
      NOTE: It is highly recommended to use a luminescence plate reader with an auto-injection function to ensure consistency in the luminescence reading from well to well and from plate to plate. If the substrate solutions are manually injected, please ensure a consistent incubation time and reading set-up for each well.
3. Conduct the screening assay on various peptide-GNP hybrids with 10 ng/mL LPS stimulation (based on 1.1). Follow the same procedure in 1.2 for the reporter assay development.
   1. Concentrate the peptide-GNP hybrids to 200 nM in an R10 medium using the centrifugation method. Centrifuge 20 volumes of the hybrid solution (10 nM) at 18,000 x g for 30 min, and carefully discard the supernatants. Collect the hybrids (at the bottom of the tube) into a tube, wash them with PBS twice, and re-suspend the hybrids in one volume of the R10 medium.
   2. Mix equal volumes of the concentrated hybrids and the LPS (20 ng/mL) containing R10 medium to have a final concentration of the hybrids and LPS to be 100 nM and 10 ng/mL, respectively.
   3. Remove the culture medium from the cultured plate and add 100 μL of the mixed solution into each well (3 replicates for each condition); include a negative control (medium only) and a LPS control (10 ng/mL LPS without hybrids).
   4. After 24 h incubation at 37 °C, transfer the medium of each well into a tube and centrifuge the tubes at 18,000 x g, 4 °C for 30 min. Collect the supernatants (50-80 μL/tube) into a 96-well round-bottom plate, and perform the reporter assay as described in 3.2.
      CAUTION: When discarding the supernatants after centrifugation, do not agitate the hybrids at the bottom of the tube.
      NOTE: The centrifugation in step 1.3.4 is very important to remove the non-internalized nanoparticle hybrids from the culture medium, and can avoid the interference of the hybrids with the colorimetric/luminescence readings. This is because the gold nanoparticles can absorb wide wavelengths of light depending on their size and agglomeration.

2. Validating the Inhibitory Effect of the Potential Candidates

NOTE: To confirm the inhibitory effect of the potential candidates from the screening, two approaches are employed. One is to examine the dose responses of the stimulants (LPS) at a fixed hybrid concentration (or the other way around); the other is to directly look at the inhibition of the NF-κB/AP-1 and IRF3 signals via immunoblotting.

1. In approach one, perform the reporter assay with 100 nM of the lead hybrids and two LPS concentrations at 1 ng/mL and 10 ng/mL following the same procedures described in 1.3. Include an inactive hybrid (based on the screening results) as a hybrid control for comparison.
2. For the immunoblotting approach, please follow the standard experimental procedures.
   1. Culture THP-1 cells in R10 medium, seed the cells into a 12-well culture plate (2 x 10⁶ cells/well), and differentiate them into macrophages by treating the cells with 50 ng/mL PMA for 24 h followed by resting for 2 days.
   2. After cell differentiation, stimulate cells with 10 ng/mL LPS with/without the hybrids (100 nM) over time (up to 4 h). At various time points (0, 5, 15, 30, 60, 120, and 240 min), prepare the cell lysates for immunoblotting. Include an inactive hybrid as a control.
   3. Probe the signals of IκBα, phosphorylated p65, and phosphorylated IRF3 to examine the inhibitory effect of the lead hybrids on the activation of NF-κB and IRF3. Probe the β-actin and total IRF3 signals as internal controls.
      NOTE: The signal transduction often occurs much faster (in a few hours) than the expression of the reporter enzymes SEAP and luciferase (24 h). It is highly recommended to also perform a viability assay of the tested hybrids at 24 h as another validation method.

3. Evaluating the TLR Specificity

NOTE: To investigate the TLR specificity of the lead peptide-GNP hybrid, other TLR signaling pathways are tested, including TLR2, TLR3, and TLR5. TLR7, 8, and 9 are excluded because the THP-1-derived macrophages do not respond well to the stimulation of these TLRs due to the lack of TLR7, 8, and 9 expression in macrophages.

1. Test various concentrations (1 ng/mL to 25 μg/mL) of the ligands specific to TLR2 (Pam3CSK4), TLR3 (poly I:C), and TLR5 (flagellin) on both reporter cells derived macrophages to obtain the optimal concentration following the same procedure in 1.1 and 1.2.
2. Treat the cells with the mixtures of the lead hybrid (100 nM) and each TLR ligand at the concentration obtained from 3.1 to evaluate the inhibitory specificity of the lead hybrid according to the experimental procedure described in 1.3. Include the inactive hybrid as a control for comparison.