The overall goal of this screening procedure is to identify novel bioactive nanoparticles that can inhibit toll-like receptor signaling. This method can help you physically identify novel nanoinhibitors targeting toll-like receptor signaling in innate immune cells. The main advantage of this technique is that it is fast and robust and easy to perform using reporter cell systems.
The applications of this technique extend toward the therapy of many inflammatory diseases because toll-like receptor signaling is found to be involved in the pathogenesis of these diseases. Though this method can provided high screening on and gold nanoparticles, it can also be applied to other nanoparticle systems as well as therapeutic small molecules and the biologies. To begin this procedure, prepare the culture mediums as outlined in the text protocol.
Next, add 100 milliliters of ultra-pure endotoxin-free water to a clean 125 milliliter glass flask. Add one pouch of secreted embryonic alkaline phosphatase substrate powder, and swirl the solution gently until it is fully dissolved. Incubate at 37 degrees Celsius for one hour to ensure complete dissolution of the substrates.
Using a 0.2 micrometer membrane, filter the solution. Store at four degrees Celsius until ready to use. Next, add one pouch of luciferase substrate powder to a 50 milliliter centrifuge tube containing 25 milliliters of ultra pure endotoxin-free water.
After the powder has dissolved completely, store at four degrees Celsius for up to a week until ready to use. Then, prepare the stock solutions of all other reagents as outlined in the text protocol. After culturing and preparing the cells, transfer them from the culture flask to a centrifuge tube.
Spin down the cells at 300 times G for five minutes. Then, resuspend them in the R-10 medium at a concentration of 1 X 10 to the 6 cells per milliliter. Add aliquots of the PMA solution to the cell suspension such that the final concentration is 50 nanograms per milliliter.
Pour the cell suspension into a sterile reagent reservoir. Using a multi-channel pipette, transfer 100 microliters of the cell suspension into each well of a 96-well flat bottom culture plate. Incubate in a cell culture incubator for 24 hours at 37 degrees Celsius.
After this, use a vacuum aspirator to carefully remove the culture medium. Using an inverted microscope, check the cells to ensure that the derived macrophages are adhered at the bottom of the well. Gently wash the cells twice using 100 microliters of PBS per well for each wash.
Then, add 100 microliters of fresh R-10 medium to each well. Let the cells rest for two days in an incubator at 37 degress Celsius before conducting the reporter assay. First, determine the optimal LPS dose as outlined in the text protocol.
Next, 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 supernatant from each sample into the plate. Incubate at 37 degrees Celsius for one to two hours to allow for the color to develop from pink to dark blue.
After the optical density is above one, use a plate reader to determine the absorption at 655 nanometers. To begin analysis of IRF activation, transfer 10 microliters of fresh supernatant from each sample into a fresh 96-well clear flat bottom white plate. Using auto-injection, add 50 microliters of the luciferase solution to each well.
Then immediately collect the luminescence well by well to produce a dose response curve to determine the optimal LPS concentration for nanoparticle screening. Next, centrifuge 20 volumes of the peptide-GMP 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 one milliliter of PBS. After this, resuspend the washed hybrids in one volume of R-10 medium. Mix equal volumes of the concentrated hybrids and the LPS containing R-10 medium such that the final concentration of the hybrids and LPS are 100 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 and four 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 one. 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. In this study, the activation of NF-Kappa B AP1 is detected by the SEAP colorimetric assay, in which TLR-4 activation by LPS results in the activation of NF-Kappa B AP1 and the production of SEAP.
The released SEAP converts the substrate, leading to a color change that is proportional to the amount of SEAP released upon stimulation. This is quantified by measuring the absorbance at 655 nanometers. Next, LPS-mediated activation of IRF led to the expression of luciferase, catalyzing the substrate to produce luminescence.
An optimal LPS concentration of 10 nanograms per milliliter is determined from these dose responses to screen a previously established library of peptide-GMP hybrids. From this, a group of hybrids is identified for their potent ability to inhibit LPS-triggered activation for both NF-Kappa B AP1 and IRF. Validation of this inhibitory activity shows that as the concentration of LPS increases, the inhibitory effects of the hybrid reduce, as expected.
Immunoblotting is then conducted to confirm inhibition activity. The lead hybrid is seen to reduce p65 phosphorylation, inhibit I-Kappa B alpha degradation, and delay IRF3 phosphorylation, confirming that the lead hybrid, P12, is able to inhibit LPS-mediating TLR-4 signaling. Additional investigation reveals that P12 also reduces TLR-2 and TLR-5-mediated NF-Kappa B AP1 signaling, as well as TLR-3-mediated IRF activation, suggesting that P12 has potent inhibitory activity on multiple TLR pathways.
Following this procedure, other measures like immunoblotting and the cytotoxicity assay is to be performed in order to validate the screening results and to avoid false positive discovery. After its development, this technique paved the way for other researchers in the field of nanomedicine to explore nanoinhibitors in toll-like receptor signaling as next-generation and informatory therapeutics.
