LNPs were formulated by mixing an aqueous phase containing mRNA or DNA with an ethanol phase containing ionizable lipids and excipients using a microfluidic chip device54 (link). Specifically, the ethanol phase contained a mixture of an ionizable lipid (C12–200, synthesized as previously described67 ), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipids, 850725P), cholesterol (Sigma-Aldrich, C8667), and 1,2-dimyristoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (C14-PEG 2000, Avanti Polar Lipids, 880150P) at predetermined molar ratios shown in Table 1 . High-performance liquid chromatography (HPLC) and mass spectrometry data for the ionizable lipid were shown in Fig. S4 . The aqueous phase was prepared in 10 mM citrate, pH 3.0 buffer (Teknova, Q2445) with either in-house synthesized b-mRNA, Luciferase mRNA (Trilink Biotechnologies), or b-DNA (Integrated DNA Technologies). Syringe pumps were used to perfuse the ethanol and aqueous phases at a 3:1 ratio through the microfluidic device54 (link). The resulting LNPs were dialyzed against PBS in a 20,000 MWCO cassette at room temperature for 2 hours and then extruded through a 0.22 μm sterile filter (Genesee Scientific, 25243).
Q2445
Manufactured by Teknova
The Q2445 is a laboratory equipment product. It serves a core function within the laboratory setting.
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Lab products found in correlation
5 protocols using q2445
1
Nanoparticle Formulation of Nucleic Acids
2
Robotic Formulation of Lipid Nanoparticles
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Robotic formulation of particles was achieved utilising a Bravo liquid handling robot utilising VWorks software (v12.2.0.1306, Agilent). Particles were prepared at a ratio of 50:38.5:10:1.5 (Cationic Lipid:Sterol:Phospholipid:PEG-Lipid) at a 20:1 (w/w) lipid:mRNA cargo, N:P ratio = ~6:1. 7.5 µl of each lipid component was combined per well to a final volume of 30 µl. mRNA cargo encoding for EGFP was prepared at a 4:1 ratio (Unlabelled:Cy5 labelled – TriLink: L7201/7701) in 50 mM Citrate pH 3 buffer (TekNova: Q2445) at 30 µl per well (122 µg/ml). 10 µl of the lipid-ethanol mix was added directly to mRNA mix and pipetted up and down 10 × 20 µl. An equal volume (40 µl) of PBS pH 7.4 was then added to the mRNA/lipid and mixed a further 5 × 20 µl and then incubated at 4 °C overnight prior to usage.
3
Automated Polyplex Formation for mRNA Delivery
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Polyplexes were prepared utilising a Bravo robot as for LNPs (above). 20 µl of 50 mM citrate pH 3 (TekNova #Q2445) buffered mRNA (4:1 GFP:Cy5 –TriLink CleanCap mRNA L-7201/L-7701) (100 μg/ml) was injected into wells (Greiner V-bottom #781280) that contain 20 μL of the desired polymer solution at an amine:phosphate ratio of 8:1 assuming 50% amines charged per polymer. After addition of the RNA solution, polyplex suspensions were mixed through 10 × 15 μl mix steps and incubated at RT for 30 min prior to addition of an equal volume (40 μl) 1 M Tris-HCl pH 8 (TekNova #T1080). Polyplexes were mixed 5 × 15 μl before moving to 4 °C for 16 h prior to use.
4
Formulation and Characterization of Lipid Nanoparticles
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DLin-MC3-DMA cholesterol or β-sitosterol containing LNPs were formulated using a NanoAssemblr (Precision NanoSystems) by microfluidic mixing chip. Briefly, lipids were prepared in Ethanol at a ratio of 50:38.5:10:1.5 (MC3:Sterol:DSPC:DMPE-PEG2000) at a 10:1 (w/w) Lipid:mRNA cargo, N:P ratio = ~3:1. mRNA cargo encoding for EGFP was prepared at a 4:1 ratio (Unlabelled:Cy5 labelled – TriLink: L7201/7701) in 50 mM Citrate pH 3 buffer (TekNova: Q2445). Lipid and mRNA containing solutions were mixed 3:1 (Citrate:Ethanol) at a constant flow rate of 12 ml/min to form nanoparticles. Particles were dialysed overnight into PBS pH 7.4 at 4 °C and sterile filtered using a 0.23 µm filter. Characteristics of LNP batches used in this study are shown in Supplementary Table 2 .
5
Optimized LNP Formulation for mRNA Delivery
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Commonly used and clinically relevant reference LNPs containing DLin-MC3-DMA were formulated using a NanoAssemblr microfluidic mixer (Precision NanoSystems). As described in Supplementary Fig. 2 , to achieve a 10:1 (w/w) lipid:mRNA ratio (N:P ratio = ~3:1), lipids were prepared in ethanol at a ratio of 50:38.5:9.9:1.5:0.1 (MC3:Cholesterol: DSPC:DMPE-PEG2000:DOPE-Rhod). Unlabeled and Cy5 labeled eGFP mRNA were prepared at a 4:1 ratio (TriLink:L7201/7701) in 50 mM citrate buffer (pH 3, TekNova: Q2445). Lipid and mRNA-containing solutions were mixed 1:3 (ethanol: citrate) at a constant flow rate of 12 ml/min to form LNPs. Formulated LNPs were dialyzed overnight in PBS (pH 7.4) at 4 °C. LNPs with other CILs were formulated in the same ratio of 50:38.5:9.9:1.5:0.1 (CILs:Cholesterol:DSPC:DMPE-PEG2000:DOPE-Rhod).
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