Name: synthesis and characterization of mrna-loaded poly(beta aminoesters) nanoparticles for vaccination purposes
Uploaded: 2021-08-13T14:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes at JoVE.com

Financial support from MINECO/FEDER (grants SAF2015-64927-C2-2-R, RTI2018-094734-B-C22, and COV20/01100) is acknowledged. CGF acknowledged her IQS PhD Fellowship.

1. Synthesis of pBAE polymer with end oligopeptides (OM-pBAE)
<ol>
	<li>Polymerization of C6-pBAE
	<ol>
		<li>Add 5-amino-1-pentanol (38 mmol; MW = 103.16 Da) 1-hexylamine (38 mmol; MW = 101.19 Da) into a round-bottom glass flask (100 mL). Then, add 1,4-butanediol diacrylate (82 mmol; MW = 198.22 Da).</li>
		<li>Pre-heat the silicone oil bath at 90 &#176;C, place the round-bottom flask into the oil bath and stir the mixture with the aid of a magnetic stir bar overnight (~18 h). Then, take the product from the round-bot...

<ol>
	<li>Chumakov, K., Benn, C. S., Aaby, P., Kottilil, S., Gallo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+existing+live+vaccines+prevent+COVID-19.">Can existing live vaccines prevent COVID-19.</a> Science. 368 (6496), 1187-1188 (2020).</li><li>Zhang, C., Maruggi, G., Shan, H., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+mRNA+vaccines+for+infectious+diseases.">Advances in mRNA vaccines for infectious diseases.</a> Frontiers in Immunology. 10, 1-13 (2019).</li><li>Wherry, E. J., Jaffee, E. M., Warren, N., D'Souza, G., Ribas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+did+we+get+a+COVID-19+vaccine+in+less+than+1+year.">How did we get a COVID-19 vaccine in less than 1 year.</a> Clinical Cancer Research. 27 (8), 2136-2138 (2021).</li><li>Folegatti, P. M., 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+the+ChAdOx1+nCoV-19+vaccine+against+SARS-CoV-2:+a+preliminary+report+of+a+phase+1/2,+single-blind,+randomised+controlled+trial.">Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial.</a> The Lancet. 396 (10249), 467-478 (2020).</li><li>Geall, A. J., Mandl, C. W., Ulmer, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA:+The+new+revolution+in+nucleic+acid+vaccines.">RNA: The new revolution in nucleic acid vaccines.</a> Seminars in Immunology. 25 (2), 152-159 (2013).</li><li>Ulmer, J. B., 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=Recent+innovations+in+mRNA+vaccines.">Recent innovations in mRNA vaccines.</a> Current Opinion in Immunology. 41, 18-22 (2016).</li><li>Kranz, L. M., 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=Systemic+RNA+delivery+to+dendritic+cells+exploits+antiviral+defence+for+cancer+immunotherapy.">Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy.</a> Nature. 534 (7607), 396-401 (2016).</li><li>Milane, L., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+approval+of+nanotechnology-based+SARS-CoV-2+mRNA+vaccines:+impact+on+translational+nanomedicine.">Clinical approval of nanotechnology-based SARS-CoV-2 mRNA vaccines: impact on translational nanomedicine.</a> Drug Delivery and Translational Research. 1 (4), 3 (2020).</li><li>Green, J. J., Langer, 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=A+combinatorial+polymer+library+approach+yields+insight+into+nonviral+gene+delivery.">A combinatorial polymer library approach yields insight into nonviral gene delivery.</a> Accounts of Chemical Research. 41 (6), 749-759 (2008).</li><li>Guerrero-C&#225;zares, H., 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=Biodegradable+polymeric+nanoparticles+show+high+efficacy+and+specificity+at+DNA+delivery+to+human+glioblastoma+in+vitro+and+in+vivo.">Biodegradable polymeric nanoparticles show high efficacy and specificity at DNA delivery to human glioblastoma in vitro and in vivo.</a> ACS Nano. 8 (5), 5141-5153 (2014).</li><li>Kozielski, K. L., Tzeng, S. Y., Hurtado De Mendoza, B. A., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreducible+cationic+polymer-based+nanoparticles+for+efficient+and+environmentally+triggered+cytoplasmic+siRNA+delivery+to+primary+human+brain+cancer+cells.">Bioreducible cationic polymer-based nanoparticles for efficient and environmentally triggered cytoplasmic siRNA delivery to primary human brain cancer cells.</a> ACS Nano. 8 (4), 3232-3241 (2014).</li><li>Segovia, N., Dosta, P., Cascante, A., Ramos, V., Borr&#243;s, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligopeptide-terminated+poly(&#946;-amino+ester)s+for+highly+efficient+gene+delivery+and+intracellular+localization.">Oligopeptide-terminated poly(&#946;-amino ester)s for highly efficient gene delivery and intracellular localization.</a> Acta Biomaterialia. 10 (5), 2147-2158 (2014).</li><li>Dosta, P., Segovia, N., Cascante, A., Ramos, V., Borr&#243;s, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+charge+tunability+as+a+powerful+strategy+to+control+electrostatic+interaction+for+high+efficiency+silencing,+using+tailored+oligopeptide-+modified+poly+(beta-amino+ester)s+(PBAEs).">Surface charge tunability as a powerful strategy to control electrostatic interaction for high efficiency silencing, using tailored oligopeptide- modified poly (beta-amino ester)s (PBAEs).</a> Acta Biomaterialia. 20, 82-93 (2015).</li><li>Fornaguera, C., 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+delivery+system+for+targeting+antigen-presenting+cells+in+vivo.">mRNA delivery system for targeting antigen-presenting cells in vivo.</a> Advanced Healthcare Materials. 7 (17), 180033 (2018).</li><li>Fornaguera, C., Castells-Sala, C., L&#225;zaro, M. &. #. 1. 9. 3. ;., Cascante, A., Borr&#243;s, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+optimized+freeze-drying+protocol+for+OM-PBAE+nucleic+acid+polyplexes.">Development of an optimized freeze-drying protocol for OM-PBAE nucleic acid polyplexes.</a> International Journal Pharmaceutics. 569, (2019).</li><li>Fornaguera, C., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+to+characterize+and+purify+polymeric+nanoparticles.">Analytical methods to characterize and purify polymeric nanoparticles.</a> International Journal of Polymer Science. , (2018).</li><li>Fornaguera, C., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+polymeric+nanoparticle+dispersions+for+biomedical+applications:+size,+surface+charge+and+stability.">Characterization of polymeric nanoparticle dispersions for biomedical applications: size, surface charge and stability.</a> Pharmaceutical Nanotechnology. 6 (3), 147-164 (2018).</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 (10), 759-780 (2014).</li><li>Fan, Y. N., 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=Cationic+lipid-assisted+nanoparticles+for+delivery+of+mRNA+cancer+vaccine.">Cationic lipid-assisted nanoparticles for delivery of mRNA cancer vaccine.</a> Biomaterials Science. 6 (11), 3009-3018 (2018).</li><li>Le Moignic, A., 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=Preclinical+evaluation+of+mRNA+trimannosylated+lipopolyplexes+as+therapeutic+cancer+vaccines+targeting+dendritic+cells.">Preclinical evaluation of mRNA trimannosylated lipopolyplexes as therapeutic cancer vaccines targeting dendritic cells.</a> Journal of Controlled Release. 278, 110-121 (2018).</li><li>Banerji, A., 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+vaccines+to+prevent+COVID-19+disease+and+reported+allergic+reactions:+Current+evidence+and+approach.">mRNA vaccines to prevent COVID-19 disease and reported allergic reactions: Current evidence and approach.</a> Journal of Allergy and Clinical Immunology: In Practice. 9 (4), 1423-1437 (2021).</li><li>Kaczmarek, J. C., Kowalski, P. S., 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=Advances+in+the+delivery+of+RNA+therapeutics:+from+concept+to+clinical+reality.">Advances in the delivery of RNA therapeutics: from concept to clinical reality.</a> Genome Medicine. 9 (1), 1-16 (2017).</li><li>Dosta, P., 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=Delivery+of+anti-microRNA-712+to+inflamed+endothelial+cells+using+poly(&#946;-amino+ester)+nanoparticles+conjugated+with+VCAM-1+targeting+peptide.">Delivery of anti-microRNA-712 to inflamed endothelial cells using poly(&#946;-amino ester) nanoparticles conjugated with VCAM-1 targeting peptide.</a> Advanced Healthcare Materials. , 1-11 (2021).</li><li>Segovia, N., 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=Hydrogel+doped+with+nanoparticles+for+local+sustained+release+of+siRNA+in+breast+cancer.">Hydrogel doped with nanoparticles for local sustained release of siRNA in breast cancer.</a> Advanced Healthcare Materials. 4 (2), 271-280 (2015).</li></ol>

After the outbreak of the Covid-19 pandemic last year, the importance of vaccines in terms of infectious disease control has manifested as a critical component8. Efforts from scientists worldwide have enabled the release to the market of many vaccines. For the first time in history, mRNA vaccines have demonstrated their previously hypothesized success, thanks to their rapid design because of their capacity to adapt to any novel antigen within some months5,<sup cl...

Infectious diseases have represented a severe threat to millions of human beings around the globe and are still one of the leading causes of death in some developing countries. Prophylactic vaccination has been one of the most effective interventions of modern society to prevent and control infectious diseases1,2. These critical milestones of science in 20th-century relevance have been remarked by the recent worldwide Covid-19 pandemic caused by the SARS-CoV-2 virus3. Recognizing the importance of having efficient vaccines to curtail the dissemination of the disease, cooperative efforts from all biomedical communities have successfully resulted in many prophylactic vaccines in the market in less than a year4.
Traditionally, vaccines were composed of attenuate (live, reduced virulence) or inactivated (death particles) viruses. However, for some diseases with no margin for safety errors, viral particles are not possible, and protein subunits are used instead. Nevertheless, subunits usually do not enable the combination of more than one epitope/antigen, and adjuvants are required to enhance vaccination potency5,6. Therefore, the need for novel vaccine types stands clear.
As demonstrated during the current pandemic, novel vaccine candidates based on nucleic acids can be advantageous in terms of avoiding long development processes and providing high versatility while producing, at the same time, a vital patient immunization. This is the case of mRNA vaccines, which were initially designed as experimental cancer vaccines. Thanks to their natural capacity to produce antigen-specific T-cell responses3,5,6,7. Being mRNA the molecule that encodes the antigenic protein, only changing the same, the vaccine can be rapidly tailored to immunize other variants of the same microorganism, different strains, other infectious microorganisms, or even become a cancer immunotherapeutic treatment. In addition, they are advantageous in terms of large-scale production costs. However, mRNA has a significant hurdle that hampers their naked administration: its stability and integrity are compromised in physiological media, full of nucleases. For this reason, the use of a nanometric carrier that protects it and vectorizes mRNA to the antigen-presenting cells is required2,8.
In this context, poly(beta aminoesters) (pBAE) are a class of biocompatible and biodegradable polymers that demonstrated a remarkable ability to complex mRNA in nanometric particles, thanks to their cationic charges9,10,11. These polymers are composed of ester bonds, which makes their degradation easy by esterases in physiological conditions. Among the pBAE library candidates, those functionalized with end cationic oligopeptides showed a higher capacity to form small nanoparticles to efficiently penetrate cells through endocytosis and transfect the encapsulated gene material. Furthermore, thanks to their buffering capacity, the acidification of the endosome compartment allows endosomal escape12,13. Namely, a specific kind of pBAE, including hydrophobic moieties on their backbone (the so-named C6 pBAE) to enhance their stability and end-oligopeptide combination (60% of polymer modified with a tri-lysine and 40% of the polymer with a tri-histidine) that selectively transfects antigen-presenting cells after parenteral administration and produce the mRNA encoded antigen presentation followed by mice immunization has been recently published14. In addition, it has also been demonstrated that these formulations could circumvent one of the main bottleneck steps of nanomedicine formulations: the possibility to freeze-dry them without losing their functionality, which enables long-term stability in soft dry environments15.
In this context, the objective of the current protocol is to make the procedure for the formation of the mRNA nanoparticles available to the scientific community by giving a description of the critical steps in the protocol and enabling the production of efficient vaccines for infectious diseases prevention and tumor treatment applications.
The following protocol describes the complete workout to synthesize oligopeptide end-modified poly(beta aminoesters) - OM-pBAE polymers that will further be used for nanoparticle synthesis. In the protocol, nanoparticles formulation is also included. In addition, critical steps for the success of the procedure and representative results are also provided to ensure that the resulting formulations accomplish the required quality control characterization features to define a positive or negative result. This protocol is summarized in Figure 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-butanediol diacrylate</td>
			<td>Sigma Aldrich</td>
			<td>123048</td>
			<td></td>
		</tr>
		<tr>
			<td>1-hexylamine</td>
			<td>Sigma Aldrich</td>
			<td>219703</td>
			<td></td>
		</tr>
		<tr>
			<td>5-amino-1-pentanol</td>
			<td>Sigma Aldrich</td>
			<td>411744</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Panreac</td>
			<td>141007</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b antibody</td>
			<td>BD</td>
			<td>550993</td>
			<td></td>
		</tr>
		<tr>
			<td>CD86 antibody</td>
			<td>Bioligend</td>
			<td>105007</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlor hydroxhyde</td>
			<td>Panreac</td>
			<td>181023</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform-d</td>
			<td>Sigma Aldrich</td>
			<td>151823</td>
			<td></td>
		</tr>
		<tr>
			<td>Cys-His-His-His peptide</td>
			<td>Ontores</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Cys-Lys-Lys-Lys peptide</td>
			<td>Ontores</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>Sigma Aldrich</td>
			<td>151882</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC reagent for Rnase free water</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td>This reagent is important to treat MilliQ water to remove any RNases of the buffers</td>
		</tr>
		<tr>
			<td>Diethyl eter</td>
			<td>Panreac</td>
			<td>212770</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>mRNA EGFP</td>
			<td>TriLink Technologies</td>
			<td>L-7601</td>
			<td></td>
		</tr>
		<tr>
			<td>mRNA OVA</td>
			<td>TriLink Technologies</td>
			<td>L-7610</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboGreen kit</td>
			<td>ThermoFisher</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>Sigma Aldrich</td>
			<td>71196</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Fisher Scientific</td>
			<td>11570626</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-mouse AlexaFluor488 antibody</td>
			<td>Abcam</td>
			<td>Ab450105</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle Tracking Analyzer</td>
			<td>Malvern Panalytical</td>
			<td>NanoSight NS300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Magnetic Ressonance Spectrometer</td>
			<td>Varian</td>
			<td>400 MHz</td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer</td>
			<td>Malvern Panalytical</td>
			<td>Nano ZS</td>
			<td>For zeta potential and hydrodynamic size determination</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NTA software</td>
			<td>Malvern Panalytical</td>
			<td>MAN0515-02-EN-00</td>
			<td></td>
		</tr>
		<tr>
			<td>NovoExpress Software</td>
			<td>Agilent</td>
			<td>Not specified</td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer software</td>
			<td>Malvern Panalytical</td>
			<td>DTS Application</td>
			<td>To analyze surface charge and hydrodynamic sizes</td>
		</tr>
	</tbody>
</table>

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.

Polymer synthesis and characterization 
The OM-pBAE synthesis procedure&#160;is given in Figure 2. As Figure 2A shows, the first step to obtain the OM-pBAE is to synthesize the C6-pBAE by adding the amines (1-hexylamine and 5-amino-1-pentanol, ratio 1:1) to the diacrylate (1,4-butanediol diacrylate). This reaction is carried out at 90 &#176;C for 20 h and with constant stirring. Afterward, a solution of oligopeptides is added to a solution ...

Watch this Scientific Journal Video about Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes at JoVE.com

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.

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 ...

This protocol is significant because it demonstrates, how to prepare Polymeric nanoparticles, encapsulated nucleic acids in one simple step. The main advantages of this technique, are versatility, as it can be applied to different nucleic acids and cell types. The use of simple procedures for particle preparation, such as pipetting up and down, and the possibility to extend the stability of the nanoparticles and drum conditions by lyophilization.Demonstrating the procedure will be Laura Olmo, Coral Garcia-Fernandez, Maria Stampa Lopez-Pinto and Maria Navalon-Lopez, phd student of our laboratory and Marta Diaz-Caballero, a post doc for our laboratory. For polyplexes formation, thaw the previously prepared polymers, C6 peptide, poly beta amino esters, and vortex the solution. After pipetting the polymer mix up and down, prepare a 12.5 millimolar solution in sodium acetate.Vortex the solution and wait for 10 minutes. Next, prepare mRNA at 0.5 milligrams per milliliter and mixed by pipetting. Vortex the polymer solution again, then mix the genetic material solution in the polymer solution in a one-to-one ratio in a micro centrifuge tube and incubate the tube at 25 degrees Celsius for 30 minutes in a Thermoblock.After incubation precipitate the mixture with one to two RNase free water by adding the sample to a micro centrifuge tube containing the water, then to include the excipients, add a 20 millimolar heaps and 4%sucrose solution in the same volume as the mixture of mRNA and poly beta amino esters, and mixed by pipetting. For Lyophilization after freezing the polyplex solution at minus 80 degrees Celsius for one hour, perform the primary drying, then immediately store the polyplexes at minus 20 degrees Celsius to avoid rehydration. For polyplex resuspension, remove the lyophilized nano-particles from the minus 20 degrees Celsius freezer and rapidly add the corresponding amount of deep hydrogenated water to re-disperse the solid and achieved the desired concentration.Pipette gently until total resuspension and once dissolved, pipette up and down vigorously avoiding bubbles. After 24 hours of transfection for qualitative assessment by fluorescence microscopy, place the 96 well plate containing the transfected Hela cells onto the microscope and start visualizing using the 10 times objective. First, apply a white balance to create a background reference for the software, then acquire an image to overlay it with the fluorescence image during the analysis.Change the microscope mode to reflection mode and move the filter wheel to the blue laser to visualize EGFP. Then acquire images for all the conditions or wells employing the same exposure time. For quantitative assessment by flow cytometry, wash the Hela cells transfected in a 96 well plate using 100 microliters of PBS per well.After aspirating the PBS at 25 microliters of trypsin per well and incubate at 37 degrees Celsius for five minutes. Once the cells are detached, add the previously recovered media to inactivate the trypsin, then fix the cells by adding 31.25 microliters of 10%formalin per well and incubating for 20 minutes. Next turn on the flow cytometer and the software, then set up the proper conditions for the experiment, including the type of plate sample volume and other parameters such as shaking and rinsing between samples.Set up the appropriate parameters to quantify the percentage of positively transfected cells by first viewing the flow data on forward scattered light versus side scattered light to distinguish the cells from the debris. And then plotting another scatterplot, comparing the amplitude versus height to gate and discriminate individual cells. One day before transfection, plate 10, 000 JAWS II cells per well in a 96 well plate for overnight incubation.The following day transfect the cells with 0.6 micrograms mRNA per well and incubate the plate for 24 hours in a dry air incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, wash the cells remaining in the well with 25 microliters of PBS. After aspirating the PBS at 25 microliters of trypsin and incubate the plate for five minutes at 37 degrees Celsius to detach the cells.Stop the trypsin reaction by adding the previously recovered media onto the correspondent well. Then centrifuge the plate, aspirate the media, and fix the cells by adding 50 microliters of PBS and 2.5%formalin per well followed by incubating the plate at four degrees Celsius for 20 minutes, centrifuge the plate. Then add 50 microliters of PBS and 3%BSA per well and incubator for 30 minutes at four degrees Celsius.After incubation centrifuge the plate again, then add 50 microliters of the mouse ovalbumin antibody in PBS and 3%BSA and incubator for 30 minutes at four degrees Celsius. Repeat centrifugation step, then wash the cells with 50 microliters of PBS. After aspirating the PBS, add the secondary antibodies and incubate for one hour at four degrees Celsius.After incubation, repeat the centrifugation and washing steps. Then resuspend the cells and 100 microliters of PBS and 2.5%formalin for flow cytometry analysis. Physiochemical characterization of fresh and lyophilized GFP mRNA encapsulating nanoparticles show no significant differences in size and poly dispersity index.Transmission electron microscopy of the polyplexes confirms the formation of spherical and mono dispersed nanoparticles of approximately 50 nanometers in diameter. Encapsulation efficiency data of a Lego peptide and modified poly beta amino ester polyplexes encapsulating GFP, or ovalbumin show no significant differences depending on the type of mRNA. A qualitative analysis of EGFP expression, 24 hours post-transaction in the JAWS II cell line is shown here.Quantitatively compared to the negative control. The percent GFP positive cells is significantly higher and comparable to a positive control performed with a classical transfection reagent. The ovalbumin mRNA loaded oligopeptide and modified poly beta amino ester nanoparticles can activate dendritic cells as seen by the significantly higher expression of CD11b and CD86 membrane markers in transfected cells compared to non-transfected cells.Our vaccination platform based on the use of mRNA as the active principle, as well as our proprietary polymers can be adapted to be used in prophylaxis as well as for cancer therapeutics. We can use our technology to make a highlight that contribution in cancer immunotherapeutics. since we can encapsulate tumor-associated antigens in our nanoparticles and make a shift in cancer treatments.

synthesis-characterization-mrna-loaded-polybeta-aminoesters

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...