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Q2445

Manufactured by Teknova

The Q2445 is a laboratory equipment product. It serves a core function within the laboratory setting.

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5 protocols using q2445

1

Nanoparticle Formulation of Nucleic Acids

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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).
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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.
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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.
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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.
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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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