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 the pH to 7.4.</li>
		<li>Sterilize by vacuum filtration using a 0.22 &#181;m pore-size filter.</li>
	</ol>
	</li>
	<li>Citrate Buffer
	<ol>
		<li>Prepare citrate buffer using 5 mM sodium citrate, 5 mM citric acid, and 150 mM sodium chloride in nuclease free water and adjust to pH 4.5.</li>
		<li>Sterilize by vacuum filtration using a 0.22 &#181;m pore-size filter. 
		&#8203;NOTE: Citrate buffer only needs to be prepared if mRNA is the nucleic acid that will be encapsulated in the LNP. If DNA will be encapsulated skip 1.2 and proceed to 1.3.</li>
	</ol>
	</li>
	<li>Malic Acid Buffer
	<ol>
		<li>Prepare malic acid buffer using 20 mM malic acid and 30 mM sodium chloride in nuclease free water and adjust to pH 3.0.</li>
		<li>Sterilize by vacuum filtration using a 0.22 &#181;m pore-size filter. 
		&#8203;NOTE: Malic acid buffer only needs to be prepared if DNA is the nucleic acid that will be encapsulated in the LNP. Skip 1.3 if mRNA is to be encapsulated. Citrate buffer is used for mRNA encapsulation, as the lower pH of 3.0 with malic acid buffer may lead to an increased likelihood of mRNA degradation. The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
2. Preparation of lipid mix
<ol>
	<li>If stock lipids are in powder form, solubilize in pure 200 proof ethanol.</li>
	<li>Calculate the required mix of lipid components based on the desired molar ratio. A molar ratio of 50:10:39:1 (ionizable lipid:helper lipid:cholesterol:PEG) will be used here as an example for a total lipid concentration of 10 mM. Table 1 shows the concentrations and volumes needed for each of these components. 
	NOTE: When calculating the volume needed to achieve the lipid mix concentration in ethanol (EtOH) for the microfluidic mixer, the total volume is accounted for to ensure that the addition of EtOH does not influence the lipid concentrations. For example, an ionizable lipid volume of 68.5 &#181;L is calculated by multiplying the 5 mM concentration in ethanol by a total lipid mix volume of 533 &#181;L and then dividing by the stock lipid concentration of 38.9 mM.</li>
	<li>Add the appropriate amount of each lipid stock solution to a glass vial to allow components to mix with intermittent vortexing. Add 200 proof ethanol for a total mixture of 533 &#181;L. For the example in Table 1, this is 254 &#181;L of ethanol. 
	&#8203;NOTE: For a single run to produce 1 mL of LNPs, 342.5 &#181;L of lipid solution is needed. This is due to a 3:1 mix of aqueous nucleic acid to organic lipid solution with some volume discarded before and after sample collection. A mix of 533 &#181;L is made to compensate as overage.</li>
</ol>
3. Preparation of nucleic acid solution
NOTE: Preparation and handling of nucleic acid solutions is to be performed in a sterile and RNase-free environment wherever possible. Work in a biosafety cabinet whenever possible with the nucleic acid.
<ol>
	<li>Calculate N/P ratio. The N/P ratio is the total number of ionizable lipid amine groups (N) to the total number of negatively charged nucleic acid phosphate groups (P). N/P ratio is often a parameter that can be optimized during LNP formation. Follow the steps below.
	<ol>
		<li>Calculate the number of N units using the below formula: 
		<img alt="Equation 1" src="/files/ftp_upload/62226/62226eq01.jpg" /> 
		NOTE: The ionizable lipid concentration (Table 1) is 5 mM, which is equivalent to 5 x 10-6 mol/mL. The required lipid injection volume is 0.3425 mL. For example, if the number of N units per molecule is 1, using the above equation, there are 1.03 x 1018 N units in the lipid mix.</li>
		<li>Calculate the P units for the desired N/P ratio. Here an N/P = 36 is used for example. 
		<img alt="Equation 2" src="/files/ftp_upload/62226/62226eq02.jpg" /></li>
	</ol>
	</li>
	<li>Calculate the necessary nucleic acid concentration to obtain 2.86 x 1016 P units using the below equation. 
	<img alt="Equation 3" src="/files/ftp_upload/62226/62226eq03.jpg" /> 
	Where, the number of P units per base pair for mRNA is 1 and DNA is 2. For an mRNA with 1,200 bases, the amount of mRNA required for a N/P = 36 is 3.96 x 10-11 moles.</li>
	<li>Calculate the mass concentration of mRNA required for N/P = 36 using the below equation. 
	<img alt="Equation 4" src="/files/ftp_upload/62226/62226eq04.jpg" /> 
	The average molecular weight of a ribonucleotide monophosphate unit is 322 g/mol30. With 1,200-base mRNA, the molecular weight of the mRNA is 386,400 g/mol. The required injection volume of nucleic acid solution is 1.028 mL. Thus, the concentration of mRNA needed is 1.488x10-5 g/mL, which is 14.88 &#181;g/mL.</li>
	<li>Make up 1.5 mL of 14.88 &#181;g/mL of mRNA in citrate buffer. 
	&#8203;NOTE: When DNA will be the nucleic acid encapsulated, use malic acid buffer to make up the nucleic acid solution.</li>
</ol>
4. Priming the microfluidic channels
NOTE: This protocol is adapted from the instrument manufacturer&#39;s guidelines.
<ol>
	<li>Input the priming parameters into the instrument software by clicking on the appropriate fields (Table 2). 
	NOTE: A flow ratio of 3:1 and flow rate of 4-12 mL/min is recommended27,31&#160;. This has been shown to be optimal in the studies presented here, as well as by the manufacturer. This can be varied if it is of interest towards the application.</li>
	<li>Open the instrument lid and place a microfluidic cartridge into the rotating block.</li>
	<li>Draw at least 0.5 mL of ethanol into a 1 mL syringe, ensuring there are no bubbles or air gaps at the syringe tip. Load this syringe into the right inlet of the cartridge.</li>
	<li>Fill a 3 mL syringe with 1.5 mL of aqueous buffer (citrate for RNA and malic acid for DNA), ensuring there are no air bubbles or gaps. Load this syringe into the left inlet of the cartridge.</li>
	<li>Insert two 15 mL conical tubes in the clip holders to serve as waste containers.</li>
	<li>Click on Run in the instrument software to begin the mixing, ensuring that the parameters are input correctly.</li>
	<li>When the instrument stops priming, indicated by the bottom blue light shutting off, open the lid and properly dispose of the conical tubes and syringes.</li>
</ol>
5. LNP formation
NOTE: This protocol is adapted from the instrument manufacturer&#39;s guidelines.
<ol>
	<li>Update the software with the formulation parameters by clicking on the appropriate fields (Table 2).</li>
	<li>Fill a 1 mL syringe with the lipid mix (prepared in step 2). Remove any air gaps or bubbles at the syringe tip and insert the syringe into the right side of the cartridge.</li>
	<li>Draw the nucleic acid solution (prepared in step 3) into a 3 mL syringe, ensuring there are no bubbles or air gaps in the syringe tip. Insert the syringe into the left inlet of the cartridge. 
	NOTE: Volumes are provided to make a 1 mL solution of LNPs. This instrument can incorporate syringe sizes up to 10 mL, and volumes can be scaled accordingly with no influence on the outcome. The maximum volume of LNPs that can be prepared in one preparation is 12 mL.</li>
	<li>Label a 15 mL RNase-free conical tube with the sample name and insert into the left tube clip. Place a 15 mL waste conical in the right tube clip.</li>
	<li>Close the instrument lid and click Run, after confirming correct input of parameters.</li>
	<li>After the instrument is finished running, properly discard the waste container and cartridge. Retain the conical tube with the LNP sample.</li>
	<li>Dilute the LNP 5x with PBS to minimize the ethanol to &#60;5% (v/v). 
	&#8203;NOTE: It is important to dilute the LNPs in PBS as soon as possible after microfluidic mixing to prevent degradation. Always perform the dilution in a biosafety cabinet and continuing to work in the biosafety cabinet throughout the buffer exchanges.</li>
</ol>
6. Buffer exchange
NOTE: Protocol for using ultra-centrifuge filters is provided. While this method results in a more time efficient exchange of buffers, dialysis may be substituted here.
<ol>
	<li>Pre-wash an ultra-centrifuge filter (100 kDa pore size) with 2 mL of PBS by centrifuging at 1000 x g for 5 min. Empty the PBS from the bottom compartment. 
	NOTE: PBS is chosen to increase the pH to 7.4 &#177; 0.2, which is physiologically relevant and will result in the ionizable lipid having a neutral charge.</li>
	<li>Add diluted LNPs to the top compartment of the pre-washed ultra-centrifuge filter and centrifuge at 1000 x g for 12 min.</li>
	<li>Discard the flow-through from the bottom compartment. Perform two more washes by adding 5 mL of PBS to the ultra-centrifuge filter each time. Centrifuge at the same parameters. There is no maximum volume that needs to be maintained. 
	NOTE: If a scaled-up volume of LNPs were prepared, increase the volume of PBS for each wash accordingly. For example, if 2 mL of LNPs were prepared in a single run, then 10 mL PBS per wash is suggested.</li>
	<li>Pipette the LNP solution against the walls of the ultra-centrifuge filter a few times to minimize LNP loss. Remove the LNP solution from the ultra-centrifuge filter and store in a nuclease free vial. Add PBS if needed to achieve a final volume of the LNP solution of 1 mL.</li>
	<li>Filter through a pre-wet 0.2 &#181;m syringe filter, if needed. 
	&#8203;NOTE: The protocol can be paused here.</li>
</ol>
7. Measure encapsulation efficiency
<ol>
	<li>Prepare a standard curve by making 2-fold serial dilutions of working nucleic acid solution in PBS, starting with a highest concentration of 500 ng/mL, and making at least five dilutions. Use PBS as a blank.</li>
	<li>Prepare the LNP sample dilutions. Dilute LNP samples with PBS, to achieve an approximate theoretical concentration that lies around the mid-point of the standard curve (eg. ~ 250 ng/mL of nucleic acid estimated from the initial concentration).</li>
	<li>Prepare a solution of the RNA quantification reagent (for mRNA measurements) with TritonX-100 to disrupt the LNPs and measure the total amount of nucleic acid inside and outside of the LNP. This solution contains 0.5% (v/v) RNA reagent, 0.4% (v/v) TritonX-100, and 99.1% (v/v) PBS.</li>
	<li>Prepare a solution of the reagent without TritonX-100 to measure the amount of nucleic acid not encapsulated in the LNPs. This solution contains 0.5% (v/v) RNA reagent and 99.5% (v/v) PBS. 
	NOTE: If LNPs encapsulate double stranded DNA (dsDNA), such as plasmid DNA, use the dsDNA reagent in 7.3 and 7.4 instead, following the same procedure.</li>
	<li>In a 96-well black fluorescence capable plate, load at least four replicates of each of the LNP and nucleic acid standard solutions prepared in 7.1 and 7.2.</li>
	<li>To half of the replicates of standards and samples, add an equal volume of the reagent containing TritonX-100. This will quantify the total amount of nucleic acid.</li>
	<li>To the remaining wells of standards and samples, add an equal volume of the reagent without TritonX-100. This will quantify the amount of nucleic acid not encapsulated inside the LNP.</li>
	<li>Shake the plate for 5 min at room temperature to ensure thorough mixing of standards and samples with the added reagent, taking precautions to avoid light exposure.</li>
	<li>Measure the fluorescence using a microplate reader, with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.</li>
	<li>Calculate the concentration of nucleic acid outside of the LNP using the standard curve made with the addition of the reagent without TritonX-100. Multiply by the dilution factor used in 7.2.</li>
	<li>Calculate the concentration of nucleic acid both inside and outside of the LNP using the standard curve made with the addition of the reagent containing TritonX-100. Multiply by the dilution factor used in 7.2.</li>
	<li>Calculate the concentration of nucleic acid inside by subtracting the concentration of nucleic acid outside (calculated from step 7.10) from the total concentration of nucleic acid both inside and outside (calculated from step 7.11)</li>
	<li>Quantify the encapsulation efficiency from the ratio of the concentration of nucleic acid inside the LNP (calculated from step 7.12) and the total concentration of nucleic acid (calculated from step 7.11). 
	&#8203;NOTE: The protocol can be paused here.</li>
</ol>
8. Concentration adjustments
<ol>
	<li>If needed, adjust the nucleic acid concentration within the LNP solution using the results from the encapsulation efficiency.</li>
	<li>If a less concentrated solution is desired, dilute the solution with PBS to achieve the desired concentration.</li>
	<li>If a more concentrated solution is desired, perform additional centrifugation runs using an ultra-centrifuge filter. 
	&#8203;NOTE: The protocol can be paused here.</li>
</ol>
9. Measure LNP hydrodynamic size and polydispersity
<ol>
	<li>Dilute an aliquot of the LNP solution 40x with PBS to obtain a final volume of 1 mL. 
	NOTE: This dilution may be changed if required. This dilution value is suggested as it uses a small volume of the LNP stock solution while providing quality results.</li>
	<li>Using a semi-micro cuvette, measure the hydrodynamic diameter and polydispersity index. Add the LNP solution into the cuvette and insert into the instrument. Set up an operating procedure in the instrument software to include the measurement type, sample details (material, dispersant, temperature, and cell type), and measurement instructions (number of runs). Click Start when ready to begin the measurement acquisition.</li>
</ol>
10. Measure LNP zeta potential
<ol>
	<li>Dilute an aliquot of the LNP solution 40x with nuclease free water to obtain a final volume of 1 mL. 
	&#8203;NOTE: Nuclease free water is used as the solvent for zeta potential measurements to minimize the influence of high salt buffers on conductivity.</li>
	<li>Using a folded capillary zeta cell, measure the zeta potential.
	<ol>
		<li>Add the LNP solution into the cuvette up to the fill line. Insert into the instrument ensuring that the electrodes are making contact with the instrument.</li>
		<li>Set up an operating procedure in the instrument software to include the measurement type, sample details (material, dispersant, temperature, and cell type), and measurement instructions (number of runs). Click Start when ready to begin the measurement acquisition.</li>
	</ol>
	</li>
</ol>

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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>

All authors are employees of Sanofi. The authors declare that they have no conflict of interest or competing financial interests.

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 ...

formulating-characterizing-lipid-nanoparticles-for-gene-delivery

Research

JoVE Journal

Bioengineering

22.2K Views.  Sanofi, Framingham, Massachusetts. Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

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

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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

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

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

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

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...