Name: efficient transfection of in vitro transcribed mrna in cultured cells using peptide-poloxamine nanoparticles
Uploaded: 2022-08-17T14:20:00
Description: Watch this Scientific Journal Video about Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles at JoVE.com

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 fr...

<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 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#215;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. ...

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.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...