This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 82041045 and 82173764), the major project of Study on Pathogenesis and Epidemic Prevention Technology System (2021YFC2302500) by the Ministry of Science and Technology of China, the Chongqing Talents: Exceptional Young Talents Project (CQYC202005027), and the Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0136). The authors are grateful to Dr. Xiaoyan Ding for measuring the hydrodynamic diameter (nm) and polydispersity index (PDI).

1. In vitro transcription of chemically modified mRNA
NOTE: It is required to use nuclease-free tubes, reagents, glassware, pipette tips, etc., because RNases are ubiquitous in the environment, such as laboratory solutions, instrument surfaces, hair, skin, dust, etc. Clean the bench surfaces and pipettes thoroughly before use, and wear gloves to avoid RNase contamination.
<ol>
	<li>Perform linearization of the DNA template.
	<ol>
		<li>Synthesize the open reading frame (ORF) of Metridia luciferase (MetLuc) flanked by a T7 polymerase promoter, 5&#39; untranslated region (UTR), 3&#39;UTR, and poly (A) tails and clone it into the PUC57 vector, which is expressed in bacteria (see Table of Materials). 
		NOTE: The coding sequence of the DNA template is provided in Supplementary File 1.</li>
		<li>Culture bacteria, lyse the bacteria, and purify the plasmid DNA by the corresponding column (see plasmid DNA extraction kit&#160;in the Table of Materials).</li>
		<li>Perform a single 50 &#181;L reaction that contains 2 &#181;L of BamHI, 2 &#181;L of KpnI (see Table of Materials), and 2 &#181;g of plasmid DNA at 37 &#176;C for 1 h, and achieve linearization of the DNA template.</li>
	</ol>
	</li>
	<li>Perform in vitro transcription to generate uncapped-IVT mRNA.
	<ol>
		<li>Perform mRNA synthesis by a single 20 &#181;L reaction using a T7 transcription kit and pseudouridine (see Table of Materials): 10 &#181;L (0.8-1 &#181;g) of linearized DNA template, 1.5 &#181;L (150 mM) of ATP, pseudo-UTP, GTP, and CTP, 2 &#181;L of 10x transcription buffer, 1 &#181;L of T7 enzyme, and 1 &#181;L of nuclease-free water (NF-water). Mix the abovementioned components thoroughly and incubate at 37 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Remove the template DNA.
	<ol>
		<li>Add 1 &#181;L (1 U) of DNase (RNase-free) after the transcription process and incubate at 37 &#176;C for 15 min.</li>
	</ol>
	</li>
	<li>Perform uncapped-IVT mRNA purification using lithium chloride precipitation.
	<ol>
		<li>Purify the uncapped-IVT mRNA using lithium chloride (see Table of Materials) with a working concentration of 10 mM. Add 50 &#181;L of 10 mM lithium chloride to 20 &#181;L of uncapped-IVT mRNA solution.</li>
		<li>Mix the complete volume thoroughly and chill at &#8722;20 &#176;C for 1 h. Collect the uncapped-IVT mRNA for 12 min at 12,000 x g, which routinely produces 80-120 &#181;g of uncapped-IVT mRNA from each single 20 &#181;L reaction.</li>
	</ol>
	</li>
	<li>Perform IVT mRNA capping.
	<ol>
		<li>Perform the IVT mRNA capping using the cap 1 capping system (see Table of Materials). Briefly, remove the secondary structure of 50 &#181;g of uncapped-IVT mRNA by heating at 65 &#176;C for 10 min, then link the 5&#39; end of uncapped-IVT mRNA with cap 1, which is prepared by modifying the m7G cap structure using 2.5 &#181;L of s-adenosylmethionine (SAM) (20 mM) and 4 &#181;L of 2&#39;-O-methyltransferase (100 U) in a 100 &#181;L reaction at 37 &#176;C for 30-60 min.</li>
		<li>Purify 100 &#181;L of the capped IVT mRNA using 250 &#181;L of 10 mM&#160;lithium chloride and dilute in 50 &#181;L of NF-water.</li>
	</ol>
	</li>
	<li>Determine the purity and molecular size of capped-IVT mRNA.
	<ol>
		<li>Measure the concentration of capped-IVT mRNA with a UV-visible spectrophotometer. Using an RNA marker (see Table of Materials), analyze the molecular size of capped-IVT mRNA in a 1% formaldehyde denaturing agarose gel containing 18% formaldehyde (voltage is 120 V). Ensure the uncapped-IVT mRNA and capped-IVT mRNA are stored at &#8722;80 &#176;C. 
		&#8203;NOTE: The IVT mRNA was considered to have good purity in an A260/A280 ratio of 1.8-2.1 and an A260/A230 ratio of 2.0 or slightly higher.</li>
	</ol>
	</li>
</ol>
2. Generation of IVT mRNA/PP-sNp
<ol>
	<li>Prepare poloxamine 704 (T704) stock solution by solubilizing the T704 (see Table of Materials) in NF-water to obtain a 10 mg/mL stock solution. Store the prepared solution at 4 &#176;C. 
	NOTE: T704 contains an X-shaped structure made of an ethylenediamine central group bonded to four chains of poly(propylene oxide) (PPO) blocks and poly(ethylene oxide) (PEO) blocks24. The molecular weight (Mw) of T704 is 5500.</li>
	<li>Prepare the synthetic peptide (sPep, see Table of Materials) stock solution by solubilizing the sPep (sequence: KETWWETWWTEWWTEWKKKKRRRRRKKKKGACSE 
	RSMNFCG) in NF-water to obtain a 2 mg/mL stock solution and store at 4 &#176;C.</li>
	<li>Prepare the IVT mRNA solution by thawing the IVT mRNA (step 1) on ice and centrifuging shortly for 3 s at 300 x g at room temperature before opening the tube. Dilute the IVT mRNA solution to 0.04 &#181;g/&#181;L with NF-water. 
	NOTE: It is recommended to work in a biosafety cabinet whenever possible with the IVT mRNA.</li>
	<li>Prepare T704 and sPep mix solution by diluting the sPep solution to 0.555 &#181;g/&#181;L and the T704 solution to 8 &#181;g/&#181;L with NF-water. Incubate the mixed solution for 15 min at room temperature prior to further use. 
	NOTE: Calculate the required sPep component based on the desired N/P ratio. The N/P ratio is the total number of nitrogen residues (N) within the sPep to the total number of negatively charged phosphate groups (P) within the IVT mRNA. Calculate the required T704 based on the weight/weight (w/w) ratio between T704 and IVT mRNA. It is necessary that the N/P ratio is 5 and the w/w ratio is 100.</li>
	<li>Prepare the IVT mRNA/PP-sNp formulation following the steps below.
	<ol>
		<li>Draw the IVT mRNA solution (step 3) into a 1 mL syringe, ensuring no air gaps or bubbles in the syringe tip. Load the syringe into one side of the cartridge next to the rotating block.</li>
		<li>Fill a 1 mL syringe with the T704 and sPep mix solution (step 4). Remove any bubbles or air gaps at the syringe tip and put the syringe into the other inlet of the pump (see Table of Materials).</li>
		<li>Set the pump with a flow ratio of 1:1 and a total flow rate of 4-10 mL/min. 
		NOTE: A total flow rate of 6 mL/min is optimal in the studies presented here.</li>
		<li>Place a 10 mL RNase-free conical tube (see Table of Materials) to collect the mixed IVT-mRNA/PP-sNp solution at the end of the flow path of the mixing device.</li>
		<li>Run the pump to start the mixing, ensuring the parameters are input correctly. After the pump is finished running for 6 s, collect the IVT-mRNA/PP-sNp sample from the conical tube. 
		NOTE: PP-sNp&#160;were generated using a microfluidic mixer (Supplementary Figure 1). The device comprises a constant flow pump, link device, chip, and fixed device. In the mixing process, the constant flow pump connected to the chip delivers liquid to the chip according to the preset flow rate. The connected constant flow pump can be connected to multiple input channels of the chip with a single output channel. The components of the device, including the chip, were obtained from commercial sources and rationally assembled (see Table of Materials). The parameters, such as flow rate and volume of the IVT mRNA solution and T704-sPep mixed solution, may differ from those displayed in the current protocol if some different setup is used and must be optimized accordingly.</li>
	</ol>
	</li>
</ol>
3. Measurement of the hydrodynamic diameter and polydispersity of IVT-mRNA/PP-sNp
<ol>
	<li>Dilute an aliquot of the IVT mRNA/PP-sNp solution (step 2) with NF-water to obtain a final volume of 1 mL.</li>
	<li>Measure the hydrodynamic size and polydispersity index (PDI) using a semi-micro cuvette25. Add the IVT mRNA/PP-sNp solution into the cuvette and place it into the particle size meter (see Table of Materials). Set up a standard operating procedure and click on Start to begin the data acquisition.</li>
</ol>
4. Preparation of the cells for transfection
<ol>
	<li>Plate human bronchial epithelial cells (16HBE) and a dendritic cell line (DC2.4) in 96-well-plates at a density of 3.5 x 104 cells/well 24 h prior to transfection. Grow the cells in a culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. 
	NOTE: The cells were obtained from a commercial source (see Table of Materials).</li>
	<li>Incubate the cells in an incubator (37 &#176;C and 5% CO2 atmosphere) for 24 h to ensure the cells are 60%-80% confluent before the transfection.</li>
</ol>
5. Transfection of the cultured cells
<ol>
	<li>After removing the growth medium, wash the plated cells with 0.2 mL/well 1x PBS.</li>
	<li>Add 170 &#181;L of serum-free culture medium to each well containing the plated cell cultures.</li>
	<li>Add the IVT mRNA/PP-sNp formulation containing 0.4 &#181;g of MetLuc mRNA (prepared in step 2) dropwise with an amount of 30 &#181;L/well.</li>
	<li>Incubate the cells with the IVT mRNA/PP-sNp formulation at 37 &#176;C in a humidified 5% CO2-enriched atmosphere for 4 h.</li>
	<li>Replace the transfection medium with 0.2 mL of fresh culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% (v/v) penicillin/streptomycin. 
	NOTE: The fetal bovine serum was heated at 56 &#176;C for 30 min for inactivation.</li>
	<li>Incubate the transfected cells at 37 &#176;C and in a 5% CO2-enriched atmosphere for 24 h and collect the supernatants to be detected from each well.</li>
</ol>
6. Analysis of cell transfection efficacy using Metridia luciferase (MetLuc) assay
<ol>
	<li>Assay the supernatant from each well for MetLuc activity using coelenterazine substrate (see Table of Materials) following the steps below.
	<ol>
		<li>Prepare a fresh assay solution by adding 1x PBS to the coelenterazine substrate (the concentration of coelenterazine is 15 mM).</li>
		<li>Vortex the coelenterazine solution for 10 s for thorough mixing.</li>
		<li>Add 50 &#181;L of supernatant (collected in step 5) to a 96-well plate.</li>
		<li>Set up the microplate reader (see Table of Materials) with a 1,000 ms reading time before adding 30 &#181;L of coelenterazine solution (15 mM) to each well manually or by automated injection.</li>
		<li>Click on Start to measure the luminescence signal immediately after adding the coelenterazine solution to the supernatant. 
		NOTE: The luminescence signal is measured using a microplate reader, and its activity is expressed in relative light units. The values obtained from PBS-transfected wells will be used as blank controls.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Nature milestones in vaccines. Springer Nature Available from: <a target="_blank" href="https://www.nature.com/collections/hcajdiajij">https://www.nature.com/collections/hcajdiajij</a> (2020)</li><li>Barbier, A. J., Jiang, A. Y., Zhang, P., Wooster, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+progress+of+mRNA+vaccines+and+immunotherapies.">The clinical progress of mRNA vaccines and immunotherapies.</a> Nature Biotechnology. 40 (6), 840-854 (2022).</li><li>Mulligan, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I/II+study+of+COVID-19+RNA+vaccine+BNT162b1+in+adults.">Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults.</a> Nature. 586 (7830), 589-593 (2020).</li><li>Tang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnologies+in+delivery+of+DNA+and+mRNA+vaccines+to+the+nasal+and+pulmonary+mucosa.">Nanotechnologies in delivery of DNA and mRNA vaccines to the nasal and pulmonary mucosa.</a> Nanomaterials. 12 (2), 226 (2022).</li><li>Freyn, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-targeting,+nucleoside-modified+mRNA+influenza+virus+vaccine+provides+broad+protection+in+mice.">A multi-targeting, nucleoside-modified mRNA influenza virus vaccine provides broad protection in mice.</a> Molecular Therapy. 28 (7), 1569-1584 (2020).</li><li>Wu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+SARS-CoV-2+mRNA+vaccines+confer+broad+neutralization+as+primary+or+booster+series+in+mice.">Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice.</a> Vaccine. 39 (51), 7394-7400 (2021).</li><li>Chaudhary, N., Weissman, D., Whitehead, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+vaccines+for+infectious+diseases:+Principles,+delivery+and+clinical+translation.">mRNA vaccines for infectious diseases: Principles, delivery and clinical translation.</a> Nature Reviews Drug Discovery. 20, 817-838 (2021).</li><li>Kormann, M. S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+therapeutic+proteins+after+delivery+of+chemically+modified+mRNA+in+mice.">Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.</a> Nature Biotechnology. 29 (2), 154-157 (2011).</li><li>Tavernier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+as+gene+therapeutic:+How+to+control+protein+expression.">mRNA as gene therapeutic: How to control protein expression.</a> Journal of Controlled Release. 150 (3), 238-247 (2011).</li><li>Walsh, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+two+RNA-based+Covid-19+vaccine+candidates.">Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates.</a> The New England Journal of Medicine. 383 (25), 2439-2450 (2020).</li><li>Baden, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+the+mRNA-1273+SARS-CoV-2+vaccine.">Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.</a> The New England Journal of Medicine. 384 (5), 403-416 (2021).</li><li>Guan, S., Rosenecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnologies+in+delivery+of+mRNA+therapeutics+using+nonviral+vector-based+delivery+systems.">Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems.</a> Gene Therapy. 24 (3), 133-143 (2017).</li><li>Sharova, L. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Database+for+mRNA+half-life+of+19+977+genes+obtained+by+DNA+microarray+analysis+of+pluripotent+and+differentiating+mouse+embryonic+stem+cells.">Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells.</a> DNA Research. 16 (1), 45-58 (2009).</li><li>Houseley, J., Tollervey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+pathways+of+RNA+degradation.">The many pathways of RNA degradation.</a> Cell. 136 (4), 763-776 (2009).</li><li>Kowalski, P. S., Rudra, A., Miao, L., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivering+the+messenger:+Advances+in+technologies+for+therapeutic+mRNA+delivery.">Delivering the messenger: Advances in technologies for therapeutic mRNA delivery.</a> Molecular Therapy. 27 (4), 710-728 (2019).</li><li>Sahin, U., Karik&#243;, K., T&#252;reci, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRNA-based+therapeutics-developing+a+new+class+of+drugs.">MRNA-based therapeutics-developing a new class of drugs.</a> Nature Reviews Drug Discovery. 13, 359-380 (2014).</li><li>Hajj, K. A., Whitehead, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+translation:+Non-viral+materials+for+therapeutic+mRNA+delivery.">Tools for translation: Non-viral materials for therapeutic mRNA delivery.</a> Nature Reviews Materials. 2, 17056 (2017).</li><li>Deering, R. P., Kommareddy, S., Ulmer, J. B., Brito, L. A., Geall, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+vaccines:+Prospects+for+non-viral+delivery+of+mRNA+vaccines.">Nucleic acid vaccines: Prospects for non-viral delivery of mRNA vaccines.</a> Expert Opinion on Drug Delivery. 11 (6), 885-899 (2014).</li><li>Wadhwa, A., Aljabbari, A., Lokras, A., Foged, C., Thakur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+in+the+delivery+of+mRNA-based+vaccines.">Opportunities and challenges in the delivery of mRNA-based vaccines.</a> Pharmaceutics. 12 (2), 102 (2020).</li><li>Hou, X., Zaks, T., Langer, R., Dong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanoparticles+for+mRNA+delivery.">Lipid nanoparticles for mRNA delivery.</a> Nature Reviews Materials. 6 (12), 1078-1094 (2021).</li><li>Sahin, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID-19+vaccine+BNT162b1+elicits+human+antibody+and+T(H)1+T+cell+responses.">COVID-19 vaccine BNT162b1 elicits human antibody and T(H)1 T cell responses.</a> Nature. 586 (7830), 594-599 (2020).</li><li>Azzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immune+response+in+BNT162b2+COVID-19+vaccine+recipients.">Mucosal immune response in BNT162b2 COVID-19 vaccine recipients.</a> EBioMedicine. 75, 103788 (2022).</li><li>Guan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+peptide-poloxamine+nanoparticles+enable+in+vitro+and+in+vivo+genome+restoration+for+cystic+fibrosis.">Self-assembled peptide-poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis.</a> Nature Nanotechnology. 14 (3), 287-297 (2019).</li><li>Pitard, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+self-assembling+DNA/poloxamine+nanospheres+for+in+vivo+gene+transfer.">Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer.</a> Nucleic Acids Research. 32 (20), 159 (2004).</li><li>Hiroi, T., Shibayama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+particle+size+distribution+in+turbid+solutions+by+dynamic+light+scattering+microscopy.">Measurement of particle size distribution in turbid solutions by dynamic light scattering microscopy.</a> Journal of Visualized Experiments. (119), e54885 (2017).</li><li>Hassett, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+lipid+nanoparticle+size+on+mRNA+vaccine+immunogenicity.">Impact of lipid nanoparticle size on mRNA vaccine immunogenicity.</a> Journal of Controlled Release. 335, 237-246 (2021).</li><li>Suk, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+penetration+of+fresh+undiluted+sputum+expectorated+by+cystic+fibrosis+patients+by+non-adhesive+polymer+nanoparticles.">The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles.</a> Biomaterials. 30 (13), 2591-2597 (2009).</li><li>Kim, N., Duncan, G. A., Hanes, J., Suk, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barriers+to+inhaled+gene+therapy+of+obstructive+lung+diseases:+A+review.">Barriers to inhaled gene therapy of obstructive lung diseases: A review.</a> Journal of Controlled Release. 240, 465-488 (2016).</li><li>Liu, Z., Fontana, F., Python, A., Hirvonen, J. T., Santos, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+production+of+particles:+Mechanism,+methodology,+and+applications.">Microfluidics for production of particles: Mechanism, methodology, and applications.</a> Small. 16 (9), 1904673 (2020).</li><li>Guan, S., Darmst&#228;dter, M., Xu, C., Rosenecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+investigations+on+optimizing+and+nebulization+of+IVT-mRNA+formulations+for+potential+pulmonary-based+alpha-1-antitrypsin+deficiency+treatment.">In vitro investigations on optimizing and nebulization of IVT-mRNA formulations for potential pulmonary-based alpha-1-antitrypsin deficiency treatment.</a> Pharmaceutics. 13 (8), 1281 (2021).</li><li>Shepherd, S. J., Issadore, D., Mitchell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+formulation+of+nanoparticles+for+biomedical+applications.">Microfluidic formulation of nanoparticles for biomedical applications.</a> Biomaterials. 274, 120826 (2021).</li><li>Han, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+confined+impingement+jets+mixer+for+flash+nanoprecipitation.">A simple confined impingement jets mixer for flash nanoprecipitation.</a> Journal of Pharmaceutical Sciences. 101 (10), 4018-4023 (2012).</li></ol>

The authors have nothing to disclose.

The protocol described here not only allows the cost-effective and rapid production of IVT mRNA vaccine formulations with defined properties, but it also offers the possibility to customize the PP-sNp formulation according to specific therapeutic purposes, such as gene therapy. In order to ensure the successful generation of IVT mRNA/PP-sNp, it is suggested to pay extra attention to some critical steps. When working with mRNA, always remember that RNase-free conditions should be maintained throughout the process because ...

Vaccination has been heralded as one of the most efficient medical interventions for reducing the morbidity and mortality caused by infectious diseases1. The importance of vaccines has been demonstrated since the outbreak of coronavirus disease 2019 (COVID-19). As opposed to the traditional concept of injecting inactivated or live-attenuated pathogens, state-of-the-art vaccine approaches, such as nucleic acid-based vaccines, concentrate on preserving the immune-stimulatory properties of the target pathogens while avoiding the potential safety issues associated with the conventional whole-microbial virus- or in bacteria-based vaccines. Both DNA- and RNA (i.e., in vitro transcribed messenger RNA, IVT mRNA)-based vaccines exhibit prophylactic to therapeutic potential against a variety of diseases, including infectious diseases and cancers2,3. In principle, the potential of nucleic acid-based vaccines relates to their production, efficacy, and safety4. These vaccines can be manufactured in a cell-free manner to allow cost-effective, scalable, and rapid production.
A single nucleic acid-based vaccine can encode multiple antigens, enabling the target of numerous viral variants or bacteria with a reduced number of inoculations and strengthening the immune response against resilient pathogens5,6.&#160;Besides, nucleic acid-based vaccines could mimic the natural invasion process of virus or bacterial infection, bringing both B cell- and T cell-mediated immune responses. Unlike some virus- or in DNA-based vaccines, IVT mRNA-based vaccines offer a huge advantage in terms of safety. They can rapidly express the desired antigen in the cytosol and are not integrated into the host genome, obviating concerns about insertional mutagenesis7. IVT-mRNA is automatically degraded after successful translation, so its protein expression kinetics can be easily controlled8,9. Catalyzed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, efforts from companies/institutions worldwide have enabled the release to the market of many types of vaccines. IVT mRNA-based vaccine technology shows great potential and, for the first time, has demonstrated its previously anticipated success, owing to its rapid design and flexible capacity to adapt to any target antigens within several months. The success of IVT mRNA vaccines against COVID-19 in clinical applications not only opened up a new era of IVT mRNA vaccine research and development but also accumulated valuable experience for the rapid development of effective vaccines for dealing with outbreaks of infectious diseases10,11.
Despite the promising potential of IVT mRNA vaccines, the efficient intracellular delivery of IVT mRNA to the site of action (i.e., cytoplasm) continues to pose a major hurdle12, especially for those administered via the airways4. IVT mRNA is inherently an unstable molecule with an extremely short half-life (~7 h)13, which renders IVT mRNA highly prone to degradation by the ubiquitous RNase14. The lymphocytes of the innate immune system tend to engulf the recognized IVT mRNA in cases of in vivo application. Moreover, the high negative charge density and large molecular weight (1 x 104-1 x 106 Da) of IVT mRNA impair its effective permeation across the anionic lipid bilayer of cellular membranes15. Therefore, a delivery system with certain bio-functional materials is required to inhibit the degradation of the IVT mRNA molecules and facilitate cellular uptake16.
Apart from a few exceptional cases in which naked IVT mRNA was directly utilized for in vivo investigations, various delivery systems are used to carry IVT mRNA to the therapeutic site of action17,18. Previous studies have revealed that only a few IVT mRNAs are detected in cytosol without the assistance of a delivery system19. Numerous strategies have been developed to improve RNA delivery with continuous efforts in the field, ranging from protamine condensation to lipid encapsulation20. Lipid nanoparticles (LNPs) are the most clinically advanced among the mRNA delivery vehicles, as proved by the fact that all the approved mRNA COVID-19 vaccines for clinical use employ LNP-based delivery systems21. However, LNPs cannot mediate effective mRNA transfection when the formulations are delivered via the respiratory route22, which remarkably limits the application of these formulations in inducing mucosal immune responses or addressing pulmonary-related diseases such as cystic fibrosis or &#945;1-antitrypsin deficiency. Therefore, developing a novel delivery system to facilitate the efficient delivery and transfection of IVT mRNA in airway-related cells is required to solve this unmet need.
It has been confirmed that the peptide-poloxamine self-assembled nanoparticle (PP-sNp) delivery system can mediate the efficient transfection of nucleic acids in the respiratory tract of mice23. The PP-sNp adopts a multifunctional modular design approach, which can integrate different functional modules into the nanoparticles for rapid screening and optimization23. The synthetic peptides and electrically neutral amphiphilic block copolymers (poloxamine) within the PP-sNp can spontaneously interact with IVT mRNA to generate uniformly distributed nanoparticles with a compact structure and smooth surface23. PP-sNp&#160;can improve the gene transfection effect of IVT mRNA molecules in cultured cells and the respiratory tract of mice23. The present study describes a protocol for generating PP-sNp&#160;containing IVT mRNA that encodes Metridia luciferase (MetLuc-mRNA) (Figure 1). Controlled and rapid mixing via a microfluidic mixing device, which employs the staggered herringbone mixing design, is utilized in this protocol. The procedure is easy to execute and allows the generation of PP-sNp&#160;with more uniform sizes. The general goal of PP-sNp production using the microfluidic mixer is to create PP-sNp&#160;for mRNA complexation in a well-controlled manner, thus allowing efficient and reproducible cell transfection in vitro. The present protocol describes the preparation, assembly, and characterization of PP-sNp&#160;containing MetLuc-mRNA.

<table><tbody><tr><td>BamHI</td><td>Takara</td><td>1010</td><td></td></tr><tr><td>cap 1 capping system</td><td>Jinan</td><td>M082</td><td></td></tr><tr><td>Dendritic cell-line</td><td>Sigma</td><td>SCC142</td><td></td></tr><tr><td>DNA sequence</td><td>Genescript</td><td></td><td></td></tr><tr><td>Human bronchial epithelial cells</td><td>Sigma</td><td>SCC150</td><td></td></tr><tr><td>KpnI</td><td>Takara</td><td>1068</td><td></td></tr><tr><td>LP</td><td>Beyotime</td><td>C0533</td><td></td></tr><tr><td>Lithium chloride</td><td>APEXBio</td><td>B6083</td><td></td></tr><tr><td>Malvern Zetasizer Nano ZS90</td><td>Malvern</td><td>NB007605</td><td></td></tr><tr><td>Microfluidic chip</td><td>ZHONGXINQIHENG</td><td>Standard PDMS chip</td><td></td></tr><tr><td>Microplate readers</td><td>ThermoFisher</td><td>Varioskan lux</td><td></td></tr><tr><td>NanoDrop One</td><td>ThermoFisher</td><td>ND-ONE-W (A30221)</td><td></td></tr><tr><td>Nuclease-free water</td><td>ThermoFisher</td><td>AM9932</td><td></td></tr><tr><td>OptiMEM</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Pseudouridine</td><td>APE&amp;times;Bio</td><td>B7972</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Quanti-Luc</td><td>InvivoGen</td><td>Rep-qlc2</td><td></td></tr><tr><td>RiboRuler High Range RNA Ladder</td><td>ThermoFisher</td><td>SM1821</td><td></td></tr><tr><td>RNase-free conical tube</td><td>Biosharp</td><td>BS-100-M</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>ThermoFisher</td><td>C11875500BT</td><td></td></tr><tr><td>Syringe pump</td><td>Chemyx</td><td>Fusion 101</td><td></td></tr><tr><td>T7 transcription Kit</td><td>Jinan</td><td>E131</td><td></td></tr></tbody></table>

efficient transfection of in vitro transcribed mrna in cultured cells using peptide-poloxamine nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp&#160;encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp&#160;is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp&#160;is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp&#160;nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

The recombinant plasmid was digested to produce the linearized DNA template (Figure 2A). Using the protocol described, The T7 in vitro transcription kit can produce up to 80-120 &#181;g of uncapped MetLuc-mRNA per 20 &#181;L reaction and 50-60 &#181;g of capped MetLuc-mRNA per 100 &#181;L reaction. When analyzed with electrophoresis, intact MetLuc-mRNA with high quality should show a single and clear band, as displayed in Figure 2B. Contaminants introdu...

Watch this Scientific Journal Video about Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles at JoVE.com

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

efficient-transfection-vitro-transcribed-mrna-cultured-cells-using

Research

JoVE Journal

Immunology and Infection

3.2K Views.  Third Military Medical University. Peptide-poloxamine nanodeeltjes is een van de potentiële pulmonale mRNA-afgiftesystemen. Ons protocol maakt de peptide-poloxamine nanodeeltjes met een hogere productiviteit en efficiëntie, wat de ontwikkeling van het mucosale mRNA-vaccin vergemakkelijkt. De materialen die nodig zijn voor de techniek zijn goedkoop en het protocol kan in korte tijd worden voltooid.De peptide-poloxamine nanodeeltjes hebben het vermogen om efficiënte mRNA-transfectie te bemiddelen. Peptide-poloxamine na...

Video: Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Bioengineering

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

Biochemistry

Comparative Analysis of Forward and Reverse Transfection Methods on mPSCs

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Author Spotlight: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical ...

Developmental Biology

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.

Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes

Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Samenvatting

Meer video's verkennen