Name: formulating and characterizing lipid nanoparticles for gene delivery using a microfluidic mixing platform
Uploaded: 2021-02-25T13:00:00
Description: Watch this Scientific Journal Video about Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform at JoVE.com

Thank you to Atul Saluja, Yatin Gokarn, Maria-Teresa Peracchia, Walter Schwenger, and Philip Zakas for their guidance and contributions towards LNP development.

A schematic of the overall process is provided in Figure 1.
1. Preparation of buffers
NOTE: Sterile filtering of the buffers is highly suggested here to remove any particulates which may impact the nucleic acid and LNP quality.
<ol>
	<li>Phosphate Buffered Saline (PBS)
	<ol>
		<li>Prepare 1x PBS using 8 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl in nuclease free water and adjust th...

<ol>
	<li>Mitchell, M. J., Billingsley, M. M., Haley, R. M., Wechsler, M. E., Peppas, N. A., Langer, 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=Engineering+precision+nanoparticles+for+drug+delivery.">Engineering precision nanoparticles for drug delivery.</a> Nature Reviews Drug Discovery. , 1-24 (2020).</li><li>Davis, M. E., Chen, Z., Shin, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+therapeutics:+an+emerging+treatment+modality+for+cancer.">Nanoparticle therapeutics: an emerging treatment modality for cancer.</a> Nanoscience and technology: A collection of reviews from nature journals. (239), 250 (2010).</li><li>Patra, J. K., Das, G., Fraceto, L. F., 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=Nano+based+drug+delivery+systems:+recent+developments+and+future+prospects.">Nano based drug delivery systems: recent developments and future prospects.</a> J Nanobiotechnol. 16 (71), (2018).</li><li>Rai, R., Alwani, S., Badea, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+nanoparticles+in+gene+therapy:+New+avenues+of+design+and+optimization+for+delivery+applications.">Polymeric nanoparticles in gene therapy: New avenues of design and optimization for delivery applications.</a> Polymers. 11 (4), 745 (2019).</li><li>Bailey, C. M., Nagarajan, R., Camesano, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+polymer+micelles+of+controlled+size,+stability,+and+functionality+for+siRNA+delivery.">Designing polymer micelles of controlled size, stability, and functionality for siRNA delivery.</a> ACS Symposium Series. 1271, 35-70 (2017).</li><li>Yin, 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=Non-viral+vectors+for+gene-based+therapy.">Non-viral vectors for gene-based therapy.</a> Nature Reviews Genetics. 15 (8), 541-555 (2014).</li><li>Bailey-Hytholt, C. M., Nagarajan, R., Camesano, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F&#246;rster+resonance+energy+transfer+probing+of+assembly+and+disassembly+of+short+interfering+RNA/Poly(ethylene+glycol)-Poly-L-Lysine+polyion+complex+micelles.">F&#246;rster resonance energy transfer probing of assembly and disassembly of short interfering RNA/Poly(ethylene glycol)-Poly-L-Lysine polyion complex micelles.</a> Molecular Assemblies: Characterization and Applications. , 47-60 (2020).</li><li>Puri, A., Loomis, K., Smith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-based+nanoparticles+as+pharmaceutical+drug+carriers:+from+concepts+to+clinic.">Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic.</a> Crit Rev Ther Drug Carrier Syst. 26 (6), 523-580 (2009).</li><li>Cullis, P. R., Hope, 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=Lipid+nanoparticle+systems+for+enabling+gene+therapies.">Lipid nanoparticle systems for enabling gene therapies.</a> Molecular Therapy. 25 (7), 1467-1475 (2017).</li><li>Munsell, E. V., Ross, N. L., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+to+the+center+of+the+cell:+Current+Nanocarrier+design+strategies+targeting+biopharmaceuticals+to+the+cytoplasm+an+nucleus.">Journey to the center of the cell: Current Nanocarrier design strategies targeting biopharmaceuticals to the cytoplasm an nucleus.</a> Current Pharmaceutical Design. 22 (9), 1227-1244 (2016).</li><li>Zhao, Y., Huang, L. <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+gene+delivery.">Lipid nanoparticles for gene delivery.</a> Advances in Genetics. 88, 13-36 (2014).</li><li>Chen, 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=Influence+of+particle+size+on+the+in+vivo+potency+of+lipid+nanoparticle+formulations+of+siRNA.">Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA.</a> Journal of Controlled Release. 235, 236-244 (2016).</li><li>Wan, C., Allen, T. M., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanoparticle+delivery+systems+for+siRNA-based+therapeutics.">Lipid nanoparticle delivery systems for siRNA-based therapeutics.</a> Drug Delivery and Translational Research. 4 (1), 74-83 (2014).</li><li>Kulkarni, J. A., Cullis, P. R., Van Der Meel, R. <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+enabling+gene+therapies:+From+concepts+to+clinical+utility.">Lipid nanoparticles enabling gene therapies: From concepts to clinical utility.</a> Nucleic Acid Therapeutics. 28 (3), 146-157 (2018).</li><li>Shin, M. 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=COVID-19+vaccine+development+and+a+potential+nanomaterial+path+forward.">COVID-19 vaccine development and a potential nanomaterial path forward.</a> Nature Nanotechnology. 15 (8), 646-655 (2020).</li><li>Thanh Le, T., 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+COVID-19+vaccine+development+landscape.">The COVID-19 vaccine development landscape.</a> Nature Reviews. Drug Discovery. 19 (5), 305-306 (2020).</li><li>Tam, Y. Y. C., Chen, S., Cullis, P. R. <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+lipid+nanoparticles+for+siRNA+delivery.">Advances in lipid nanoparticles for siRNA delivery.</a> Pharmaceutics. 5 (3), 498-507 (2013).</li><li>Cayabyab, C., Brown, A., Tharmarajah, G., Thomas, 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+lipid+nanoparticles.">mRNA lipid nanoparticles.</a> Precision Nanosystems Application Note. , (2019).</li><li>Gilleron, 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=Image-based+analysis+of+lipid+nanoparticle-mediated+siRNA+delivery,+intracellular+trafficking+and+endosomal+escape.">Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.</a> Nature Biotechnology. 31 (7), 638-646 (2013).</li><li>Suzuki, Y., Ishihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+activity+and+uptake+mechanism+of+siRNA-lipid+nanoparticles+with+an+asymmetric+ionizable+lipid.">Structure, activity and uptake mechanism of siRNA-lipid nanoparticles with an asymmetric ionizable lipid.</a> International Journal of Pharmaceutics. 510 (1), 350-358 (2016).</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>Schmid, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acidic+environment+in+endocytic+compartments.">The acidic environment in endocytic compartments.</a> Biochemical Journal. 303 (2), 679-680 (1994).</li><li>Maugeri, 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=Linkage+between+endosomal+escape+of+LNP-mRNA+and+loading+into+EVs+for+transport+to+other+cells.">Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.</a> Nature Communications. 10 (1), (2019).</li><li>Kulkarni, J. A., Witzigmann, D., Leung, J., Tam, Y., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+role+of+helper+lipids+in+lipid+nanoparticle+formulations+of+siRNA.">On the role of helper lipids in lipid nanoparticle formulations of siRNA.</a> Nanoscale. (45), (2019).</li><li>Hafez, I. M., Maurer, N., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+whereby+cationic+lipids+promote+intracellular+delivery+of+polynucleic+acids.">On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids.</a> Gene Therapy. 8 (15), 1188-1196 (2001).</li><li>Hafez, I. M., Culis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+lipid+polymorphism+in+intracellular+delivery.">Roles of lipid polymorphism in intracellular delivery.</a> Advanced Drug Delivery Reviews. 47 (2-3), 139-148 (2001).</li><li>Evers, M. J. 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=State-of-the-art+design+and+rapid-mixing+production+techniques+of+lipid+nanoparticles+for+nucleic+acid+delivery.">State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery.</a> Small Methods. 2 (9), 1700375 (2018).</li><li>Mui, B. 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=Influence+of+polyethylene+glycol+lipid+desorption+rates+on+pharmacokinetics+and+pharmacodynamics+of+siRNA+lipid+nanoparticles.">Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles.</a> Molecular Therapy - Nucleic Acids. 2 (139), (2013).</li><li>Zukancic, 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=The+importance+of+poly(Ethylene+glycol)+and+lipid+structure+in+targeted+gene+delivery+to+lymph+nodes+by+lipid+nanoparticles.">The importance of poly(Ethylene glycol) and lipid structure in targeted gene delivery to lymph nodes by lipid nanoparticles.</a> Pharmaceutics. 12 (11), 1-16 (2020).</li><li>NEBioCalculator. New England BioLabs Inc Available from: <a target="_blank" href="https://nebiocalculator.neb.com/#!/formulas">https://nebiocalculator.neb.com/#!/formulas</a> (2020)</li><li>Kastner, 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=High-throughput+manufacturing+of+size-tuned+liposomes+by+a+new+microfluidics+method+using+enhanced+statistical+tools+for+characterization.">High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization.</a> International Journal of Pharmaceutics. 477 (1-2), 361-368 (2014).</li><li>Zhigaltsev, I. 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=Bottom-up+design+and+synthesis+of+limit+size+lipid+nanoparticle+systems+with+aqueous+and+triglyceride+cores+using+millisecond+microfluidic+mixing.">Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing.</a> Langmuir. 28 (7), 3633-3640 (2012).</li><li>Belliveau, N. 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=Microfluidic+synthesis+of+highly+potent+limit-size+lipid+nanoparticles+for+in+vivo+delivery+of+siRNA.">Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA.</a> Molecular Therapy - Nucleic Acids. 1 (8), 37 (2012).</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=Optimization+of+lipid+nanoparticles+for+intramuscular+administration+of+mRNA+vaccines.">Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines.</a> Molecular Therapy - Nucleic Acids. 15, 1-11 (2019).</li><li>Tanaka, 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=The+delivery+of+mRNA+to+colon+inflammatory+lesions+by+lipid-nano-particles+containing+environmentally-sensitive+lipid-like+materials+with+oleic+acid+scaffolds.">The delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid-like materials with oleic acid scaffolds.</a> Heliyon. 4 (12), 00959 (2018).</li><li>Singh, 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=Nucleic+acid+lipid+nanoparticles.">Nucleic acid lipid nanoparticles.</a> Precision Nanosystems Application Note. , (2018).</li></ol>

Reproducibility, speed, and low volume screening are significant advantages of using microfluidic mixing to form LNPs compared to other existing methods (e.g., lipid film hydration and ethanol injection). We have demonstrated the reproducibility of this method with no impact on encapsulation efficiency or particle size observed with different LNP batches. This is an essential criterion for any therapeutic, including LNPs, to become clinically available.
The technique described here employs sta...

Drug carriers are used to protect and deliver a therapeutic with typical favorable properties including low cytotoxicity, increased bioavailability, and improved stability1,2,3. Polymeric nanoparticles, micelles, and lipid-based particles have previously been explored for nucleic acid encapsulation and delivery4,5,6,7. Lipids have been used in different types of nanocarrier systems, including liposomes, and lipid nanoparticles, as they are biocompatible with high stability8. LNPs can readily encapsulate nucleic acids for gene delivery9,10. They protect the nucleic acid from degradation by serum proteases during systemic circulation11 and can improve delivery to specific sites, as the surface topography and physical properties of LNPs influence their biodistribution12. LNPs also improve tissue penetration and cellular uptake9. Previous studies have demonstrated the success of siRNA encapsulation within an LNP13, including the first commercially available LNP containing siRNA therapeutic for the treatment polyneuropathy of hereditary transthyretin-mediated amyloidosis14&#160;treatment that was approved by United States Food and Drug Administration (FDA) and European Medicines Agency in 2018. More recently, LNPs are being studied for the delivery of larger nucleic acid moieties, namely mRNA and DNA9. As of 2018, there were ~ 22 lipid-based nucleic acid delivery systems undergoing clinical trials14. Additionally, mRNA containing LNPs are currently leading candidates and have been employed for a COVID-19 vaccine15,16. The potential success for these non-viral gene therapies requires forming small (~100 nm), stable, and uniform particles with high encapsulation of the nucleic acid.
Use of an ionizable lipid as a main component in the LNP formulation has shown advantages for complexation, encapsulation, and delivery effciciency14. Ionizable lipids typically have an acid dissociation constant (pKa) &#60; 7; for example, dilinoleylmethyl-4-dimethylaminobutyrate (D-Lin-MC3-DMA), the ionizable lipid used in the FDA approved LNP formulation, has a pKa of&#160;6.4417. At low pH, the amine groups on the ionizable lipid become protonated and positively charged, allowing for the assembly with negatively charged phosphate groups on mRNA and DNA. The ratio of amine, &#34;N&#34;, groups to phosphate, &#34;P&#34;, groups is used to optimize the assembly. The N/P ratio is dependent on the lipids and nucleic acids used, which varies depending on the formulation18. After formation, the pH can be adjusted to a neutral or physiological pH to allow for therapeutic administration. At these pH values, the ionizable lipid is also deprotonated which imparts neutral surface charge to the LNP.
The ionizable lipid also aids in endosomal escape19,20. LNPs undergo endocytosis during cellular uptake and must be released from the endosome in order to deliver the mRNA cargo into the cell cytoplasm or DNA cargo to the nucleus21. Inside the endosome is typically a more acidic environment than the extracellular medium, which renders the ionizable lipid positively charged22,23. The positively charged ionizable lipid can interact with negative charges on the endosomal lipid membrane, which can cause destabilization of the endosome allowing for the release of the LNP and nucleic acid. Different ionizable lipids are currently being studied for improving efficacy of both LNP distribution, as well as endosomal escape14.
Other typical components of an LNP include helper lipids, such as a phosphatidylcholine (PC) or phosphoethanolamine (PE) lipid. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) are commonly used helper lipids24,25. DOPE has been shown to form an inverted hexagonal II (HII) phase and enhance transfection by membrane fusion26, while DSPC has been thought to stabilize LNPs with its cylindrical geometry27. Cholesterol is also incorporated in the formulation in order to increase membrane rigidity, subsequently aiding in the stability of the LNP. Finally, lipid-conjugated polyethylene glycol (PEG) is included in the formulation to provide the necessary steric barrier to aid in particle self-assembly27. PEG also improves the storage stability of LNPs by preventing aggregation. Furthermore, PEG is often used as a stealth component and can increase the circulation time for the LNPs. However, this attribute can also pose challenges for recruitment of LNPs to hepatocytes through an endogenous targeting mechanism driven by apolipoprotein E (ApoE)28. Thus, studies have investigated the acyl chain length for diffusion of PEG from the LNP, finding that short lengths (C8-14) dissociate from the LNP and are more amenable to ApoE recruitment compared to longer acyl lengths28. Further, the degree of saturation of the lipid tail that PEG is conjugated to has been shown to influence the tissue distribution of LNPs29. Recently, Tween 20, which is a commonly used surfactant in biological drug product formulations and has a long unsaturated lipid tail, was shown to have high transfection in draining lymph nodes compared to PEG-DSPE, which largely transfected the muscle at the injection site29. This parameter can be optimized to achieve the desired LNP biodistribution.
Conventional methods of forming LNPs include the thin-film hydration method and ethanol-injection method27. While these are readily available techniques, they are also labor intensive, can result in low encapsulation efficiency, and are challenging to scale up27. Advancements in mixing techniques have resulted in methods more amenable to scale up, while developing more uniform particles27. These methods include T-junction mixing, staggered herringbone mixing, and microfluidic hydrodynamic focusing27. Each method has a unique structure, but all allow for rapid mixing of an aqueous phase containing the nucleic acid with an organic phase containing the lipid components, resulting in high encapsulation of the nucleic acid27. In this protocol, rapid and controlled mixing through a microfluidic cartridge is utilized, which employs the staggered herringbone mixing design. This protocol outlines the preparation, assembly, and characterization of nucleic acid containing LNPs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (C-14 PEG)</td>
			<td>Avanti Polar Lipids</td>
			<td>880151P</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;l Graduated Filter Tips&#160; (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1121-3810</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;l Graduated Filter Tips (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1111-2831</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l Beveled Filter Tips (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l Graudated Filter Tips (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#946;-Hydroxy-5-cholestene, 5-Cholesten-3&#946;-ol (Cholesterol)</td>
			<td>Sigma-Aldrich</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes (1 ml syringe)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes (3 ml syringe)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-436</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer General Use Syringe Needles (BD Blunt Fill Needle 18G)</td>
			<td>Thermo Fisher Scientific</td>
			<td>23-021-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Beckman coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black 96 well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-245-177</td>
			<td></td>
		</tr>
		<tr>
			<td>BrandTech BRAND BIO-CERT RNase-, DNase-, DNA-free microcentrifuge tubes (1.5mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-380-813</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Fisher Scientific</td>
			<td>02-002-611</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 500ml Vacuum Filter/Storage Bottle System, 0.22 um pore</td>
			<td>Corning</td>
			<td>430769</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable folded capillary cells</td>
			<td>Malvern</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, Pure 200 proof</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Brand Semi-Micro Cuvette</td>
			<td>Thermo Fisher Scientific</td>
			<td>14955127</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Conical Tubes (15 mL) (DNase-RNase-free)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12500</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Amicon Ultra Centrifugal Filter Units</td>
			<td>Thermo Fisher Scientific</td>
			<td>UFC901024</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAssemblr Benchtop</td>
			<td>Precision Nanyosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9930</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;P7589</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT RiboGreen RNA Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>02-004-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate, Dihydrate, granular</td>
			<td>Fisher Scientific</td>
			<td>02-004-056</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax i3x</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

formulating and characterizing lipid nanoparticles for gene delivery using a microfluidic mixing platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Multiple batches of LNPs with the same lipid formulation and N/P ratio of 6 were developed on separate days to demonstrate reproducibility of the technique. Batch 1 and 2 resulted in overlapping size distributions with similar polydispersity (Figure 2A) No significant difference was observed in the size or encapsulation efficiency between the two different batches (Figure 2B). The encapsulation efficiency was high for each batch ...

Watch this Scientific Journal Video about Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform at JoVE.com

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

This protocol can be used to produce nucleic acid encapsulated lipid nanoparticles with high reproducibility and speed and low volumes. The formulation parameters can be tuned to achieve a desired bio distribution based on the clinical application. The standard herringbone design of the microfluidic cartridge allows a rapid, precise and controlled laminar flow during mixing.The method is amenable to scaling up and generates uniform, highly encapsulated particles. With most commercially approved gene therapy products relying on viral methods, this procedure can provide insight into gene delivery, as its non-viral approach allows applications for which repeat dosing is necessary. Begin by adding the appropriate amount of each liquid stock solution to a glass vial with intermittent vortexing.as indicated in the table, followed by the addition of 200 proof ethanol to a final volume of 533 microliters. Then add the appropriate amount of mRNA stock and dilute with citrate buffer to achieve a final volume of 1.5 milliliters at the desired concentration. To prime the microfluidic channels, first enter the priming parameter into the instrument software as outlined in the table.Next, open the instrument lid and place a microfluidic cartridge into the rotating block. Load at least 500 microliters of ethanol into a 1 milliliter syringe, taking care of that there are no bubbles or air gaps at the syringe tip and insert the syringe into the right inlet of the cartridge. Load a five milliliter syringe with 1.5 milliliters of citrate buffer, taking care that there are no air bubbles or gaps, and insert the syringe into the left inlet of the cartridge.Place two 15 milliliter conical tubes into the clip holders to serve as waste containers and click Go"to mix the solutions. When the instrument stops priming as indicated by the bottom blue light shutting off, open the late and properly dispose of the conical tubes and syringes. For lipid nanoparticle formation, set the appropriate formulation parameters as indicated in the table and load a 1 milliliter syringe with a previously prepared lipid mix.Remove any air gaps or bubbles in the syringe tip and insert the syringe into the right side of the cartridge. Load the previously prepared nucleic acid solution into a 3 milliliter syringe, taking care of that there are no bubbles or air gaps in the syringe tip, and insert the syringe into the left inlet of the cartridge. Place a 15 milliliter RNAase-free conical tube labeled with the sample name into the left tube clip and place a 15 milliliter waste conical in the right tube clip.Close the instrument lid and click Go.The lipid and mRNA solutions will flow through the microfluidic cartridge and the lipid nanoparticle solution will be collected in the conical tubes. Retain the conical tube at the end of the formulation process, then dilute the lipid nanoparticles with 5 milliliters of PBS and a biological safety cabinet. To perform a buffer exchange, first prewash a 100 kilodalton pore size ultracentrifuge filter with 2 milliliters of PBS, centrifuge, and then empty the PBS from the bottom compartment.Add the diluted lipid nanoparticles to the top compartment of the pre-washed ultracentrifuge filter for three centrifuge washes, discarding the flow-through and adding 5 milliliters of PBS to the ultracentrifuge filter after the first two washes. After the last wash, pipette the lipid nanoparticle solution against the walls of the ultracentrifuge filter a few times to minimize the nanoparticle loss before transferring the nanoparticle solution to a nuclease-free vial. Then add PBS to bring the nanoparticle suspension to the appropriate experimental concentration and volume as necessary.To assess the encapsulation efficiency of the lipid nanoparticles, first prepare two-fold serial dilutions of working nucleic acid solution in PBS to generate a standard curve, starting from 500 nanograms per milliliter and making at least five dilutions. Next, prepare the nanoparticle sample dilutions in PBS to achieve an appropriate theoretical concentration that lies around the midpoint of the standard curve. Then prepare the ribo green RNA reagent to detect presence of RNA both inside and outside of the lipid nanoparticle by mixing the appropriate amounts of RNA reagent, Triton X100 and PBS.Then, to detect the presence of RNA outside of the liquid nanoparticle, mix the appropriate amounts of RNA reagents and PBS only. Load replicates of the nucleic acid standard and lipid nanoparticle solutions into a black fluorescence-capable 96 well plate, then add an equal volume of RNA quantification reagent with, and without Triton X100 to the standard and sample replicates. Shake the plate in the plate reader for five minutes at room temperature protected from light to obtain a thorough mixing of the samples, then measure the fluorescence on a microplate reader at an excitation wavelength of 480 nanometers and an emission wavelength of 520 nanometers.To measure the hydrodynamic size and poly dispersity of the lipid nanoparticles, first dilute the nanoparticle solution 40 times in PBS and add the solution to a semi micro cuvette. Load the cuvette onto the Zetasizer and select a standard operating procedure to set the instrument to measure the nanoparticles according to their material, dispersant, temperature and cell type. Then click Start"to measure the hydrodynamic size and poly dispersity of the lipid nanoparticles.To measure the Zeta potential of lipid nanoparticles, dilute the nanoparticle solution 40 times in nuclease-free water and load the solution into a cuvette to the fill line. Load the cuvette into a Zeta potential analyzer, taking care that the electrodes make contact with the instrument, and set the instrument to measure the Zeta potential according to the specific makeup of the lipid nanoparticles. Then click Start"to measure the Zeta lipid nanoparticle potential.In this analysis, multiple batches of lipid nanoparticles with the same lipid formulation and amine to phosphate ratio were developed on separate days to demonstrate the reproducibility of the technique. As observed, batches one and two exhibited overlapping size distributions with a similar poly dispersity, with no significant differences observed in the size or encapsulation efficiency between the batches. Typically, changes in the formulation parameters induce some small yet statistically significant differences.For example, decreasing the amine to phosphate ratio results in a 4%decrease in the encapsulation efficiency, with a concomitant increase in the hydrodynamic diameter of the nanoparticles. Lipid nanoparticles formulated with different ionizable lipids but the same amine to phosphate ratio, exhibit a significant change in the encapsulation efficiency as well as slight differences in the particle diameter. The encapsulation of plasmid DNA results in larger particles compared to mRA encapsulating lipid nanoparticles, although both types of nanoparticle demonstrate a similar encapsulation efficiency.Changes in the flow rate process parameter, however, do not impact the lipid nanoparticle development. Lipid nanoparticles have great applications, the perfect example being the recently approved COVID-19 vaccines. This technique serves as a great tool set and paves the way for future applications by allowing lower limb formulation screening.

Research

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