M.J.M. acknowledges support from a US National Institutes of Health (NIH) Director&#8217;s New Innovator Award (DP2 TR002776), a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a US National Science Foundation CAREER award (CBET-2145491), and additional funding from the National Institutes of Health (NCI R01 CA241661, NCI R37 CA244911, and NIDDK R01 DK123049).

NOTE: Always maintain RNase-free conditions when formulating mRNA-LNPs by wiping the surfaces and equipment with a surface decontaminant for RNases and DNA. Use only RNase-free tips and reagents.
All the animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at the University of Pennsylvania and a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania.
1. Pre-formulation preparation
<ol>
	<li>Fill a clean 4 L beaker with 200-300 mL of fresh 10x phosphate-buffered saline (PBS).
	<ol>
		<li>Dilute the 10x PBS in the beaker 10-fold with ultrapure water to obtain a beaker of 1x PBS. Ensure that the final volume of the 1x PBS is between 2-3 L.</li>
		<li>Place dialysis cassettes (20 kDa molecular weight cutoff [MWCO]) of the appropriate capacity for the LNP formulation into the filled beaker to hydrate them. 
		NOTE: Dialysis cassettes require a minimum of 2 min to hydrate before use.</li>
		<li>Place a stir bar into the beaker, and cover the beaker with aluminum foil.</li>
		<li>Place the covered beaker onto a magnetic stir plate. Turn on the stir plate, and set it to spin at 300-400 rpm.</li>
	</ol>
	</li>
	<li>Dilute a 100 mM citric acid buffer stock (pH 3) 10-fold with ultrapure water to create the 10 mM citric acid buffer used for mRNA dilution.</li>
	<li>Make C12-200 ionizable lipid, helper lipid, cholesterol, and lipid-anchored PEG (lipid-PEG) stock solutions by dissolving each lipid in 100% ethanol. The concentrations of the lipid components are detailed in Table 1.
	<ol>
		<li>Heat and mix the stock solutions at 37 &#176;C to ensure that they are fully dissolved.</li>
	</ol>
	</li>
</ol>
2. Preparation of the lipid and nucleic acid mixes
<ol>
	<li>Calculate the required volumes of ionizable lipid, helper lipid, cholesterol, and lipid-PEG based on the desired molar ratio and ionizable lipid to mRNA weight ratio (Table 1). Prepare 10% extra volume of the organic phase to account for dead volume in the syringes during formulation.
	<ol>
		<li>Combine the calculated volumes of the different lipid components in a conical tube.</li>
		<li>Dilute the lipids with 100% ethanol to a final volume, V, which represents 25% of the final volume of LNP.</li>
	</ol>
	</li>
	<li>Calculate the amount of mRNA required for formulation based on the ionizable lipid to mRNA weight ratio and the total volume of mRNA-LNP needed (Table 1).
	<ol>
		<li>Thaw the firefly luciferase-encoding mRNA on ice.</li>
		<li>Dilute the mRNA to a final volume, 3V, three times the volume of the organic phase, in 10 mM citric acid buffer. 
		&#8203;NOTE: Every lipid volume, V, must contain enough ionizable lipid for the mRNA diluted in 3V. This corresponds to the desired ionizable lipid to mRNA weight ratio.</li>
	</ol>
	</li>
</ol>
3. Microfluidic formulation of mRNA-LNPs
<ol>
	<li>Spray a delicate task wipe with a surface decontaminant for RNases and DNA, and wipe the interior of the microfluidic instrument thoroughly.
	<ol>
		<li>Open a new microfluidic cartridge, and insert it with the inlet channels facing down and away.</li>
		<li>Ensure that the conical tube arm holds two 15 mL conical tubes and is in the position furthest to the right.</li>
		<li>Configure two sterile 15 mL conical tubes on the holder with the caps off.</li>
	</ol>
	</li>
	<li>Fill a 5 mL syringe with the mRNA solution that contains mRNA diluted in 10 mM citric acid buffer.
	<ol>
		<li>Fill a 3 mL syringe with the lipid solution that contains the lipids diluted in pure 100% ethanol.</li>
		<li>Flip up the cartridge holder on the microfluidic device.</li>
		<li>Insert the 5 mL syringe, without the needle, that contains mRNA into the left inlet of the cartridge.</li>
		<li>Insert the 3 mL syringe, without the needle, that contains lipids into the right inlet of the cartridge.</li>
		<li>Flip the cartridge holder back down, and close the lid of the microfluidic instrument.</li>
	</ol>
	</li>
	<li>Turn on the microfluidic instrument by using the switch located on the back of the instrument.
	<ol>
		<li>Select Quick Run.</li>
		<li>Select the correct syringe types for the 5 mL and 3 mL syringes inserted.</li>
		<li>Input the parameters for mRNA-LNP formulation at a 3:1 aqueous to organic flow rate ratio as described in Table 2.</li>
		<li>Press the Next button to go to the next screen.</li>
		<li>Confirm that the correct parameters have been inputted.</li>
		<li>Press the Start button to formulate the mRNA-LNPs.</li>
	</ol>
	</li>
</ol>
4. Post-formulation processing and characterization of the mRNA-LNPs
<ol>
	<li>Load the mRNA-LNPs collected in the right conical tube into the previously hydrated dialysis cassettes. 
	NOTE: Do not puncture or damage the membrane of the cassette when loading the mRNA-LNPs. If the membrane is damaged, mRNA-LNPs will be lost upon loading.
	<ol>
		<li>Place the dialysis cassettes containing the mRNA-LNPs back into the beaker containing 1x PBS, and leave them to dialyze for a minimum of 2 h.</li>
		<li>After dialysis, bring the dialysis cassettes containing the mRNA-LNPs into a sterile biosafety cabinet, and remove the mRNA-LNPs using a syringe with a needle.</li>
		<li>Sterile-filter the mRNA-LNPs using a 0.22 &#181;m syringe filter, and collect them in a sterile conical tube.</li>
		<li>Place 65 &#181;L of the mRNA-LNP solution into a 1.5 mL microtube for characterization purposes.</li>
	</ol>
	</li>
	<li>Dilute 10 &#181;L of mRNA-LNPs 1:100 in 990 &#181;L of 1x PBS in a cuvette for the measurement of the hydrodynamic size and polydispersity index using dynamic light scattering.</li>
	<li>Assess the concentration and encapsulation efficiency of the mRNA-LNPs using a fluorescence assay (i.e., RiboGreen).
	<ol>
		<li>Prepare a stock solution of 1x TE buffer by diluting 20x TE buffer with molecular biology water.</li>
		<li>Prepare a stock solution of 1% Triton X-100 buffer by diluting Triton X-100 with 1x TE buffer.</li>
		<li>Prepare the fluorescent reagent by diluting the reagent stock 1:200 with 1x TE buffer.</li>
		<li>Dilute the mRNA-LNPs 1:100 in 1x TE buffer and 1% Triton X-100 buffer in a black-wall black-bottom 96-well plate in 100 &#181;L.</li>
		<li>Prepare a low-range standard curve by diluting the RNA standard as per the manufacturer&#39;s instructions, and plate the standard curve in the 96-well plate.</li>
		<li>Add 100 &#181;L of diluted fluorescent reagent to each well containing diluted mRNA-LNP or diluted standard.</li>
		<li>Place the 96-well plate inside a plate reader, and shake for 1 min, followed by a 4 min wait.</li>
		<li>Measure the fluorescence intensity of each well according to the manufacturer&#39;s protocol at an excitation/emission of 480 nm/520 nm.</li>
	</ol>
	</li>
	<li>Use the standard curve to convert the fluorescence values to concentrations.
	<ol>
		<li>Calculate the encapsulation efficiency by subtracting the value obtained from diluting LNPs in TE buffer (unencapsulated mRNA) from the value obtained from diluting LNPs in 1% Triton X-100 (total mRNA) and dividing by the value obtained from diluting LNPs in 1% Triton X-100, according to equation 4.4.</li>
		<li>Calculate the mRNA-LNP concentration used for cell and animal studies by subtracting the value obtained from diluting LNPs in TE buffer (unencapsulated mRNA) from the value obtained from diluting LNPs in 1% Triton X-100 (total mRNA).</li>
	</ol>
	</li>
	<li>Measure the pKa of the mRNA-LNPs by performing a 6-(p-toluidino)-2-naphthalenesulfonyl chloride (TNS) assay.
	<ol>
		<li>Prepare the TNS reagent by creating a 0.16 mM solution of TNS in ultrapure water. 
		NOTE: When using the reagent, be sure to keep it on ice and away from light to avoid degradation.</li>
		<li>Prepare buffered solutions of 150 mM sodium chloride, 20 mM sodium phosphate, 25 mM ammonium citrate, and 20 mM ammonium acetate adjusted to different pH values ranging from 2 to 12 in increments of 0.5.</li>
		<li>Add 2.5 &#181;L of the LNP to each pH-adjusted buffer solution in a black-wall black-bottom 96-well plate.</li>
		<li>Add TNS reagent to each well so that the final TNS concentration is 6 &#181;M.</li>
		<li>Incubate the 96-well plate in a dark location for 5 min.</li>
		<li>Measure the fluorescence at an excitation/emission of 322 nm/431 nm using a fluorescence plate reader.</li>
		<li>Fit the data to a sigmoidal curve, and calculate the pKa by using the fitted equation to find the pH at which 50% of the maximum intensity was observed.</li>
	</ol>
	</li>
	<li>Representative results for the mRNA-LNP hydrodynamic size, PDI, encapsulation efficiency, and pKa are shown in Table 3.</li>
</ol>
5. In vitro transfection of HepG2 cells
NOTE: Various other cell lines, such as HeLa cells or HEK-293T cells, may be used for assessing the transfection efficiency of LNPs in vitro. All cells should test negative for mycoplasma prior to the LNP transfection studies.
<ol>
	<li>Plate 5,000 HepG2 cells in each well of a white-wall clear-bottom 96-well plate in 100 &#181;L of complete cell culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin)
	<ol>
		<li>Leave the cells to adhere overnight in a controlled CO2 incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Dilute the mRNA-LNPs in complete cell culture medium so that the total dose is delivered in 100 &#181;L.
	<ol>
		<li>Remove the complete medium from the wells.</li>
		<li>Treat the cells with either mRNA-LNPs diluted in complete cell culture medium at the desired doses or complete medium (control).</li>
		<li>Place the 96-well plate containing the treated cells back into the controlled CO2 incubator for 24 hours.</li>
	</ol>
	</li>
	<li>Assess the transfection efficiency by performing a luciferase assay.
	<ol>
		<li>Prepare the luciferase reagent and 1x cell lysis buffer as per the manufacturer&#39;s instructions.</li>
		<li>Take out the 96-well plate containing the cells from the incubator, and bring it into a biosafety cabinet.</li>
		<li>Remove the cell culture medium from every well of the 96-well plate.</li>
		<li>Add 20 &#181;L of 1x lysis buffer to each well, followed by 100 &#181;L of the previously prepared luciferase assay reagent.</li>
		<li>Protect the plate from light, and place the plate into a plate reader to measure the bioluminescent signal of each well. 
		&#8203;NOTE: Representative results demonstrating the treatment of HepG2 cells with previously formulated C12-200 mRNA-LNPs are shown in Figure 1.</li>
	</ol>
	</li>
</ol>
6. In vivo evaluation of mRNA-LNPs in mice following tail vein injection
<ol>
	<li>Dilute the mRNA-LNP solution with sterile 1x PBS so that 2 &#181;g of total mRNA is present in a 100 &#181;L tail vein injection volume, which corresponds to a body weight dose of approximately 0.1 mg/kg for a 20 g mouse.</li>
	<li>Restrain each mouse using an approved method, and wipe the tail with a pad moistened with 70% alcohol.
	<ol>
		<li>Fill a 29 G insulin syringe with either 100 &#181;L of mRNA-LNP (2 &#181;g) or 100 &#181;L of 1x PBS. Remove any bubbles from the syringe.</li>
		<li>Insert the needle with the bevel facing up into the mouse&#39;s lateral tail vein, and slowly inject the 100 &#181;L of mRNA-LNP or 1x PBS.</li>
		<li>Remove the needle and apply pressure until hemostasis is achieved.</li>
	</ol>
	</li>
	<li>Prepare a stock solution of 15 mg/mL d-luciferin potassium salt in sterile 1x PBS 6 hours post injection.
	<ol>
		<li>Turn on the in vivo imaging system (IVIS), and open the imaging software.</li>
		<li>Press Initialize to prepare the instrument for data acquisition.</li>
		<li>Check the box next to luminescence, and set the exposure time to auto. Ensure that the correct field of view is selected for the number of mice being imaged.</li>
		<li>Administer 200 &#181;L of d-luciferin intraperitoneally (150 mg of luciferin per kg body weight) to the previously treated mice.</li>
		<li>Place the mice into an anesthesia chamber set to 2.5% isoflurane and an oxygen flowrate of 2.0 L/min.</li>
		<li>Wait 10 min for the luciferase signal to stabilize before imaging the mRNA-LNP-treated mice.</li>
		<li>Ensure that the mice are fully anesthetized, and redirect the anesthetic line to administer isoflurane via nose cones inside the imaging chamber.</li>
		<li>Transfer the mice to the nose cones, and ensure that they are on their backs with their abdomens exposed.</li>
		<li>Close the chamber door, and click the Acquire button in the software to obtain bioluminescence images. 
		NOTE: Representative results for mouse whole-body imaging following mRNA-LNP or 1X PBS treatment are shown in Figure 2.</li>
	</ol>
	</li>
</ol>

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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=A+single+administration+of+CRISPR/Cas9+lipid+nanoparticles+achieves+robust+and+persistent+in+vivo+genome+editing.">A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing.</a> Cell Reports. 22 (9), 2227-2235 (2018).</li><li>Truong, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanoparticle-targeted+mRNA+therapy+as+a+treatment+for+the+inherited+metabolic+liver+disorder+arginase+deficiency.">Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (42), 21150-21159 (2019).</li><li>Cheng, Q., 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=Dendrimer-based+lipid+nanoparticles+deliver+therapeutic+FAH+mRNA+to+normalize+liver+function+and+extend+survival+in+a+mouse+model+of+hepatorenal+tyrosinemia+type+I.">Dendrimer-based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I.</a> Advanced Materials. 30 (52), 1805308 (2018).</li><li>Sedic, 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+evaluation+of+lipid+nanoparticle-formulated+modified+mRNA+in+the+Sprague-Dawley+rat+and+cynomolgus+monkey.">Safety evaluation of lipid nanoparticle-formulated modified mRNA in the Sprague-Dawley rat and cynomolgus monkey.</a> Veterinary Pathology. 55 (2), 341-354 (2018).</li><li>Veiga, 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=Cell+specific+delivery+of+modified+mRNA+expressing+therapeutic+proteins+to+leukocytes.">Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes.</a> Nature Communications. 9 (1), 4493 (2018).</li><li>Pattipeiluhu, 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=Anionic+lipid+nanoparticles+preferentially+deliver+mRNA+to+the+hepatic+reticuloendothelial+system.">Anionic lipid nanoparticles preferentially deliver mRNA to the hepatic reticuloendothelial system.</a> Advanced Materials. 34 (16), 2201095 (2022).</li><li>Rosenblum, 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=CRISPR-Cas9+genome+editing+using+targeted+lipid+nanoparticles+for+cancer+therapy.">CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy.</a> Science Advances. 6 (47), (2020).</li><li>Fenton, O. 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=Bioinspired+alkenyl+amino+alcohol+ionizable+lipid+materials+for+highly+potent+in+vivo+mRNA+delivery.">Bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mRNA delivery.</a> Advanced Materials. 28 (15), 2939-2943 (2016).</li><li>Kauffman, 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+nanoparticle+formulations+for+mRNA+delivery+in+vivo+with+fractional+factorial+and+definitive+screening+designs.">Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs.</a> Nano Letters. 15 (11), 7300-7306 (2015).</li><li>Tomb&#225;cz, I., 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=Highly+efficient+CD4++T+cell+targeting+and+genetic+recombination+using+engineered+CD4++cell-homing+mRNA-LNPs.">Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.</a> Molecular Therapy. 29 (11), 3293-3304 (2021).</li><li>Kim, 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=Engineering+lipid+nanoparticles+for+enhanced+intracellular+delivery+of+mRNA+through+inhalation.">Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through inhalation.</a> Nano. 9 (9), 14792-14806 (2022).</li><li>Bevers, 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=mRNA-LNP+vaccines+tuned+for+systemic+immunization+induce+strong+antitumor+immunity+by+engaging+splenic+immune+cells.">mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells.</a> Molecular Therapy. 30 (9), 3078-3094 (2022).</li></ol>

There are no conflicts of interest to declare.

With this workflow, a variety of mRNA-LNPs can be formulated and tested for their in vitro and in vivo efficiency. Ionizable lipids and excipients can be swapped out and combined at different molar ratios and different ionizable lipid to mRNA weight ratios to produce mRNA-LNPs with differing physicochemical properties22. Here, we formulated C12-200 mRNA-LNPs with a molar ratio of 35/16/46.5/2.5 (ionizable lipid:helper lipid:cholesterol:lipid-PEG) at a 10:1 ionizable lipid to mRNA weight ratio. These LNPs were tested for their transfection efficiency in vitro in HepG2 cells and in vivo in C57BL/6 mice. The C12-200 mRNA-LNPs were synthesized by microfluidic mixing using a commercially available microfluidic instrument. Though other formulation methods can be used to formulate LNPs, such as pipette mixing or T-junction mixing4, microfluidic mixing formulates mRNA-LNPs that are generally smaller, less polydisperse, and have higher encapsulation efficiencies4,13,23,24.
The formulation of mRNA-LNPs with the commercially available microfluidic instrument allows for the simple and flexible formulation of LNPs. The flow rate at the beginning of a run varies from the target flow rate set as the syringe pushers accelerate to full speed. This can lead to variable particle characteristics and inconsistent results. To overcome this, the microfluidic instrument was designed to collect the LNPs formulated at the start and end of a formulation as waste separately from the LNPs formulated at steady-state conditions. This can decrease the polydispersity and lead to more consistent particle properties. The start waste can be calculated based on the combination of syringes being used for formulation as per the manufacturer&#39;s manual. However, the end waste does not experience the same degree of non-steady-state flow conditions, so the volume is often set to 0.05 mL for all syringe combinations as per the manufacturer&#39;s recommendation.
It is critical to account for dead volume in syringes and tubes when preparing the correct volumes of the organic and aqueous phases. Preparing 10% extra volume of each phase ensures that the minimum volume of LNP desired can be formulated. In this protocol, a total formulation volume of 4.7 mL was inputted with 0.7 mL of waste, resulting in 4 mL of LNP collected in steady-state conditions. However, 1.3 mL of the organic phase and 3.9 mL of the aqueous phase were prepared for formulation. This ensures that the 5 mL syringe can be filled up to the 3.6 mL mark and the 3 mL syringe can be filled up to the 1.2 mL mark. If the volume reaches the marks on the syringes, then the volume specifications used on the microfluidic instrument are safe inputs. The final screen before pressing the &#34;START&#34; button is crucial for ensuring that all the parameters are correct. It is important to do a final check on the syringe types, the volumes in the syringes, the volumes to be dispensed, the syringe placement, and the flow rates.
After the formulation of mRNA-LNPs, the dialysis step has a few important considerations to keep in mind. The dialysis cassettes used require a minimum hydration time of 2 min before the LNPs can be loaded. This increases the membrane flexibility and allows the membrane to adjust more readily as the sample is added. Additionally, care must be taken not to damage or puncture the membranes of the dialysis cassettes while loading them with mRNA-LNPs. If the membranes become punctured or damaged, the formulated mRNA-LNPs can be easily lost upon loading.
In this protocol, we tested the in vitro efficiency of mRNA-LNPs in HepG2 cells. However, other cell types can be tested for mRNA-LNP transfection efficiency. If non-adherent cells are being used, the 96-well plate requires centrifugation at 500 g for 5 min prior to any removal of the cell culture medium25. This minimizes any loss of cells during the medium removal. The cells should also be tested for the presence of mycoplasma prior to any LNP transfection studies. It will be difficult to obtain consistent results and compare different LNP formulations if the cells test positive for mycoplasma. Additionally, primary cells may require higher doses of LNP to measure transfection without toxicity compared with cell lines. A luciferase-based viability assay can be used to evaluate the mRNA-LNP toxicity in vitro at varying doses in both primary cells and cell lines18.
One of the most important steps in obtaining consistent bioluminescence measurements across different mice following treatment with the same mRNA-LNP formulation is ensuring that the time between the intraperitoneal injection of d-luciferin and the IVIS measurement is consistent26. This time interval influences the stability of the bioluminescent signal obtained. The time interval needs to be long enough so that any fluctuations in the signal are minimized. A preliminary experiment can be run in which the mice are imaged every minute following the d-luciferin injection. The time interval when the signal starts to plateau is the one most suitable for the transfection experiment. In this protocol, a 10 min interval allowed for stable luminescence signals following the 150 mg d-luciferin/kg body weight injection, which is commonly performed for imaging27,28.
Additionally, the mRNA-LNP dose used in this protocol (0.1 mg mRNA/kg body weight) is small compared to the doses used to assess toxicity and therapeutic efficacy29,30. These smaller doses help to evaluate differences in the potencies of unique mRNA-LNP formulations while not oversaturating the luminescent signal obtained. However, this dose can be increased to measure the potential mRNA-LNP toxicity. For example, liver toxicity can be evaluated by quantifying the levels of alanine transaminase, aspartate transaminase, and alkaline phosphatase in the serum at different time points following mRNA-LNP injection31,32,33. These enzymes are normally found at low levels in the serum but are increased as a result of liver damage. Commercially available kits can be used to quantify the amount of these enzymes in the serum.
In this example, we used firefly luciferase-encoding mRNA, but other reporter mRNAs can be formulated into LNPs to assess potency. mRNAs encoding for green fluorescent protein (GFP) or mCherry can be employed to investigate cell-specific delivery following mRNA-LNP treatment34,35. A single-cell suspension can be produced from tissues of interest, and GFP+ or mCherry+ cells can be assessed by flow cytometry analysis. Additionally, mRNA-LNPs can be formulated with mRNA encoding for erythropoietin in order to measure liver transfection through the secretion of erythropoietin in&#160;the serum8,36,37. In Ai9 mice, the delivery of mRNA encoding Cre recombinase induces robust tdTomato fluorescence, which can serve as another method for investigating mRNA-LNP delivery to different cell populations38,39,40. Through the demonstration of this workflow, it is our hope that mRNA-LNP formulation ceases to be a barrier for investigators as they explore new ideas and possibilities for mRNA therapeutics.

Lipid nanoparticles (LNPs) have demonstrated great promise in recent years in the field of non-viral gene therapy. In 2018, the United States Food and Drug Administration (FDA) approved the first-ever RNA interference (RNAi) therapeutic, Onpattro by Alnylam, for the treatment of hereditary transthyretin amyloidosis1,2,3,4. This was an important step forward for lipid nanoparticles and RNA-based therapies. More recently, Moderna and Pfizer/BioNTech received FDA approvals for their mRNA-LNP vaccines against SARS-CoV-24,5. In each of these LNP-based nucleic acid therapies, the LNP serves to protect its cargo from degradation by nucleases and facilitate potent intracellular delivery6,7. While LNPs have seen success in RNAi therapies and vaccine applications, mRNA-LNPs have also been explored for use in protein replacement therapies8 as well as for the co-delivery of Cas9 mRNA and guide RNA for the delivery of the CRISPR-Cas9 system for gene editing9. However, there is no one specific formulation that is well-suited for all applications, and subtle changes in the LNP formulation parameters can greatly affect the potency and biodistribution in vivo8,10,11. Thus, individual mRNA-LNPs must be developed and evaluated to determine the optimal formulation for each LNP-based therapy.
LNPs are commonly formulated with four lipid components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene-glycol (PEG) conjugate (lipid-PEG)11,12,13. The potent intracellular delivery facilitated by LNPs relies, in part, on the ionizable lipid component12. This component is neutral at physiological pH but becomes positively charged in the acidic environment of the endosome11. This change in ionic charge is thought to be a key contributor to endosomal escape12,14,15. In addition to the ionizable lipid, the phospholipid (helper lipid) component improves the encapsulation of the cargo and aids in endosomal escape, the cholesterol offers stability and enhances membrane fusion, and the lipid-PEG minimizes LNP aggregation and opsonization in circulation10,11,14,16. To formulate the LNP, these lipid components are combined in an organic phase, typically ethanol, and mixed with an aqueous phase containing the nucleic acid cargo. The LNP formulation process is very versatile in that it allows for different components to be easily substituted and combined at different molar ratios in order to formulate many LNP formulations&#160;with a multitude of physicochemical properties10,17. However, when exploring this vast variety of LNPs, it is crucial that each formulation is evaluated using a standardized procedure to accurately measure the differences in characterization and performance.
Here, the complete workflow for the formulation of mRNA-LNPs and the assessment of their performance in cells and animals is outlined.

<table><tbody><tr><td>0.1 M Hydrochloric Acid</td><td>Sigma</td><td>7647-01-0</td><td></td></tr><tr><td>0.22 &amp;mu;m Syringe Filters</td><td>Genesee</td><td>25-243</td><td></td></tr><tr><td>1 mL BD Slip Tip Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000)</td><td>Avanti Polar Lipids</td><td>880150P</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)</td><td>Avanti Polar Lipids</td><td>850725P</td><td></td></tr><tr><td>1.5 mL Eppendorf Tubes</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>200 proof Ethanol</td><td>Decon Labs</td><td>2716</td><td></td></tr><tr><td>23G Needles</td><td>Fisher Scientific</td><td>14-826-6C</td><td></td></tr><tr><td>3 mL BD Disposable Syringes with Luer-Lok tips</td><td>Fisher Scientific</td><td>14-823-435</td><td></td></tr><tr><td>3 mL Dialysis Cassettes</td><td>Thermo Scientific</td><td>A52976</td><td></td></tr><tr><td>96 Well Black Wall Black Bottom Plate</td><td>Fisher Scientific</td><td>07-000-135</td><td></td></tr><tr><td>96 Well White/Clear Bottom Plate, TC Surface</td><td>Thermo Scientific</td><td>165306</td><td></td></tr><tr><td>Ammonium Acetate, 1 Kilogram</td><td>Research Products International</td><td>&amp;nbsp;631-61-8</td><td></td></tr><tr><td>Ammonium Citrate dibasic</td><td>SIgma</td><td>3012-65-5</td><td></td></tr><tr><td>BD Luer-Lok Syringe sterile, single use, 5 mL</td><td>BD</td><td>309646</td><td></td></tr><tr><td>C12-200 Ionizable Lipid</td><td>Cayman Chemical</td><td>36699</td><td></td></tr><tr><td>C57BL/6 Mice</td><td>Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>57-88-5</td><td></td></tr><tr><td>CleanCap FLuc mRNA (5moU)</td><td>TriLink Biotechnologies</td><td>L-7202</td><td></td></tr><tr><td>Disposable cuvettes</td><td>Fisher Scientific</td><td>14955129</td><td></td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>Thermo Scientific</td><td>L2916</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Thermofisher Scientific</td><td>11965-084</td><td></td></tr><tr><td>Exel Insulin Syringes - 0.5 mL</td><td>Fisher Scientific</td><td>1484132</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Hep G2 [HEPG2]</td><td>ATCC</td><td>HB-8065</td><td></td></tr><tr><td>HyPure Molecular Biology Grade Water</td><td>Cytiva</td><td>SH30538.03</td><td></td></tr><tr><td>Infinite 200 PRO Plate Reader</td><td>Tecan</td><td>N/A</td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>Perkin Elmer</td><td>N/A</td><td></td></tr><tr><td>Large Kimwipes</td><td>Fisher Scientific</td><td>06-666-11D</td><td></td></tr><tr><td>Luciferase Assay Kit</td><td>Promega</td><td>E4550</td><td></td></tr><tr><td>NanoAssemblr Ignite Cartridges - Classic - 100 Pack</td><td>Precision Nanosystems</td><td>NIN0065</td><td></td></tr><tr><td>NanoAssemblr Ignite Instrument</td><td>Precision Nanosystems</td><td>NIN0001</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline (10x) pH 7.4, RNase-free</td><td>Thermo Scientific</td><td>AM9624</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermofisher Scientific</td><td>15140122</td><td></td></tr><tr><td>QB Citrate Buffer, (Citrate 100 mM) pH 3.0</td><td>Teknova</td><td>Q2442</td><td></td></tr><tr><td>Quant-it RiboGreen RNA Assay Kit</td><td>Thermo Scientific</td><td>R11490</td><td></td></tr><tr><td>Reporter Lysis 5x Buffer</td><td>Promega</td><td>E3971</td><td></td></tr><tr><td>RNase Away Surface Decontaminant</td><td>Thermofisher Scientific</td><td>7000TS1</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>1310-73-2</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Sigma</td><td>7601-54-9</td><td></td></tr><tr><td>TNS reagent (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt)</td><td>Sigma</td><td>T9792</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>9036-19-5</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern Panalytical</td><td>NanoZS</td><td></td></tr></tbody></table>

testing the in vitro and in vivo efficiency of mrna-lipid nanoparticles formulated by microfluidic mixing

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

mRNA-LNPs were formulated using a microfluidic instrument that possessed an average hydrodynamic diameter of 76.16 nm and a polydispersity index of 0.098. The pKa of the mRNA-LNPs was found to be 5.75 by performing a TNS assay18. The encapsulation efficiency for these mRNA-LNPs was calculated to be 92.3% by using the modified fluorescence assay and equation 4.4. The overall RNA concentration that was used for the cell treatment and animal dosing was 40.24 ng/&#181;L. This value was obtained from the modified fluorescence assay19, specifically from converting the fluorescence obtained by diluting the LNPs with 1% Triton X-100 and 1x TE buffer to certain concentrations and calculating the amount of encapsulated mRNA.
Equation 4.4
<img alt="Equation 1" src="/files/ftp_upload/64810/64810eq01.jpg" style="margin: auto;" />
CTX = Concentration of mRNA from LNPs diluted in 1% Triton X-100
CTE = Concentration of mRNA from LNPs diluted in 1x TE buffer
Increasing bioluminescence was observed with increasing doses upon the treatment of 5,000 HepG2 cells per well with different doses of mRNA-LNP. Luciferase expression was easily detected at the lowest dose (5 ng/well) used in this study. However, other cell lines may require different amounts of mRNA-LNP to readily see differences in luminescence across doses.
Mice were treated with 2 &#181;g of mRNA-LNP and assessed 6 h later for whole-body bioluminescence. As C12-200 mRNA-LNPs predominantly transfect the liver20, a strong bioluminescent signal was observed in the upper abdomen of the mice treated with our formulated LNPs. Using the imaging software, regions of interest were drawn around the liver signals to calculate the total luminescent flux. In this experiment, the total luminescent flux was approximately 5 x 109, which is consistent with other studies conducted with this LNP formulation4,21.
Table 1: Organic phase composition. Concentrations of the individual lipid stock solutions and the volumes that each component contributes to the 1.3 mL organic phase are described. Lipids are combined at a molar ratio of 35/16/46.5/2.5 (C12-200:DOPE:Cholesterol:C14-PEG2000). A 10% extra volume was prepared to account for syringe dead volumes during formulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64810/Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Microfluidic instrument settings for mRNA-LNP formulation at a 3:1 aqueous to organic flow rate ratio. The 5 mL syringe containing mRNA diluted in 10 mM citric acid buffer was inserted into the center channel, while the 3 mL syringe containing lipids was inserted into the right channel. LNPs were formulated at a 10:1 weight ratio of ionizable lipid:mRNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64810/Table2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: mRNA-LNP characterization data determined by dynamic light scattering, a modified fluorescenceassay, and a 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS) assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64810/Table3.xlsx" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64810/64810fig01.jpg" /> 
Figure 1: HepG2 cells treated with firefly luciferase mRNA-LNP. Overall, 5,000 HepG2 cells were seeded per well in a 96-well plate and allowed to adhere overnight. The cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture medium was removed before cells were treated with 5 ng, 10 ng, and 20 ng doses of a C12-200-containing LNP formulation encapsulating firefly luciferase-encoding mRNA (in 100 &#181;L of cell culture medium). At 24 h post treatment, the cell culture medium was removed, and 20 &#181;L of 1x lysis buffer was added onto the cells before the addition of 100 &#181;L of luciferase assay reagent. The bioluminescence from each well was measured using a plate reader following a 5 min incubation period. The fold increases are plotted relative to control wells that consisted of untreated HepG2 cells. The statistical significance was evaluated using a one-way ANOVA with post-hoc Tukey&#39;s test. ns, not significant; ****, p &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64810/64810fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64810/64810fig02.jpg" /> 
Figure 2: C57BL/6 mice treated with firefly luciferase mRNA-LNP. In this work, 2 &#181;g of total mRNA-LNP in 100 &#181;L or 100 &#181;L of 1x PBS was administered to C57BL/6 mice via the lateral tail vein. (A) Whole-body bioluminescence of the mice treated with either mRNA-LNP or 1X PBS was measured using the in vivo imaging system (IVIS) 6 h post administration. The IVIS Spectrum Living Image software was used to analyze and acquire the whole-body images. N = 3. (B) The total luminescent flux was quantified and plotted. The statistical significance was evaluated using a two-tailed Student&#39;s t-test. **, p &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64810/64810fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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

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

testing-vitro-vivo-efficiency-mrna-lipid-nanoparticles-formulated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.6K Views.  University of Pennsylvania. Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

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

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 ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

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

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

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

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

Biochemistry

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

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

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

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

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

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

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

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.

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 ...

Synthesizing Lipid Nanoparticles Using CIJ and &#181;MIVM Mixer Technologies

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

Author Spotlight: Enhancing Lipid Nanoparticle Formation Through Turbulent Mixing in Confined Geometries

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (&#181;MIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of ...

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

Summary

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Chapters in this video

Viral Nanoparticles for In vivo Tumor Imaging

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

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

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

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

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers