Peptide-poloxamine nanoparticles is one of the potential pulmonary mRNA delivery systems. Our protocol makes the peptide-poloxamine nanoparticles with higher productivity and efficiency, which facilitates the development of the mucosal mRNA vaccine. The materials required by the technique are low-cost, and the protocol can be completed in a short time.
The peptide-poloxamine nanoparticles have the ability to mediate efficient mRNA transfection. Peptide-poloxamine nanoparticles exhibited successful mRNA transfection in human bronchial epithelial cells, which implicates additional applications for mucosal COVID-19 vaccines, treatment of lung cancers, and cystic fibrosis gene therapy. I expect the individual to have some basic knowledge in a related field and with the protocol and a lot of the techniques as well, before he or she starts performing the experiments.
After synthesizing the open reading frame of Metridia luciferase and cloning it into the pUC57 vector, prepare a linearized DNA template using BamHI and KpnI at 37 degrees Celsius for one hour. Prepare a reaction of 20-microliters volume for mRNA synthesis using a T7 transcription kit and pseudouridine as described in the manuscript. Mix the components thoroughly and incubate at 37 degrees Celsius for three hours.
Then, remove the secondary structure of 50 micrograms of uncapped IVT mRNA by heating it at 65 degrees Celsius for 10 minutes. Use the Cap 1 capping system for the IVT mRNA capping, and prepare Cap 1 by modifying the 7-methylguanosine cap structure using S-adenosylmethionine and 2'O-methyltransferases at 37 degrees Celsius for 30 to 60 minutes. Measure the concentration of capped IVT mRNA with a UV-visible spectrophotometer.
Using an RNA marker, analyze the molecular size of capped IVT mRNA in a 1%formaldehyde denaturing agarose gel containing 18%formaldehyde. Prepare 10 milligrams per milliliter stock solution of poloxamine 704 by solubilizing the T704 in nuclease-free water. Prepare 2 milligrams per milliliter stock solution of the peptide by solubilizing the peptide in nuclease-free water.
Store the prepared solution at 4 degrees Celsius. Thaw the IVT mRNA on ice. Before opening the tube, centrifuge the tube at 300 times G for three seconds at room temperature, and dilute the IVT mRNA solution with nuclease-free water.
Prepare T704 and synthetic peptide mix solution by diluting T704 solution to eight micrograms per microliter concentration, and synthetic peptide solution to 0.555 micrograms per microliter concentration, with nuclease-free water, respectively. Incubate the mixed solution for 15 minutes at room temperature before further use. Draw the IVT mRNA solution into a one-milliliter syringe, avoiding air gaps or bubbles in the syringe tip.
Load the syringe into one side of the cartridge next to the rotating block. Then, fill a one-milliliter syringe with T704 and synthetic peptide mix solution, and remove any bubbles or air gaps at the syringe tip. Place the syringe into the other inlet of the pump, and set the pump with a total flow rate of 4 to 10 milliliters per minute.
Place a ten-milliliter RNase-free conical tube to collect the mixed IVT mRNA/PP-sNp solution at the end of the flow path of the mixing device. Run the pump to start the mixing, ensuring the parameters are input correctly. After running the pump for six seconds, collect the IVT mRNA/PP-sNp sample from the conical tube.
Plate human bronchial epithelial cells and a dendritic cell line in 96-well plates 24 hours before transfection. Grow the cells in a culture medium supplemented with 10%heat-inactivated fetal bovine serum and 1%penicillin streptomycin. After removing the growth medium, wash the plated cells with 0.2 milliliters per well PBS.
Add 170 microliters of serum-free culture medium to each well containing the plated cell cultures. Then, add 30 microliters of IVT mRNA/PP-sNp formulation containing 0.4 micrograms of Metridia luciferase mRNA drop wise into each well. Incubate the cells with the IVT mRNA/PP-sNp formulation in a humidified 5%carbon dioxide-enriched atmosphere at 37 degrees Celsius for four hours.
Aspirate the transfection medium and add 0.2 milliliters of fresh culture medium supplemented with 10%heat-inactivated fetal bovine serum and 1%penicillin streptomycin. Incubate the transfected cells for 24 hours as demonstrated earlier and collect the supernatants to be detected from each well. Prepare a fresh assay solution by adding PBS to the coelenterazine substrate.
Mix the solution thoroughly by vortexing for 10 seconds. Add 50 microliters of the collected supernatant to a 96-well plate. Set up the microplate reader with a reading time of 1, 000 milliseconds and add 30 microliters of coelenterazine solution to each well manually or by automated injection.
Immediately after adding the coelenterazine solution to the supernatant, click on Start to measure the luminescence signal. The recombinant plasmid was isolated and digested to produce the linearized DNA template. The in vitro transcription produced upcapped and capped MetLuc mRNA.
Cells transfected with Metridia luciferase mRNA/PP-sNp displayed significantly higher expression of luciferase as compared to those transfected with commercially-available lipid-based transfection reagent, naked Metridia luciferase mRNA, PBS, or T7044 synthetic peptides, mixed solution in human bronchial epithelial cells, 16 HBE, and dendritic cell line DC2.4 Metridia luciferase mRNA/PP-sNp showed higher luciferase expression than LP.suggesting that the PP-sNp delivery system is important for protecting the MetLuc mRNA against degradation and for promoting the transfection efficiency of the exogenous Metridia luciferase mRNA. Please ensure that the synthetic structure of uncapped Met-mRNA is successfully removed. Calculate and download the Met-mRNA T704 and peptide compound carefully to prepare and compact the poloxamine nanoparticles.
Transmission electron microscopy can be performed to observe the morphology of peptide-poloxamine nanoparticles. This technique helps researchers to develop mucosal mRNA vaccines and enable high-throughput screening of gene therapy formulations for every related cells.
