C14 peg2000

Manufactured by Avanti Polar Lipids

C14-PEG2000 is a polyethylene glycol (PEG) conjugate with a C14 fatty acid chain. It serves as a surfactant and emulsifying agent for use in various laboratory applications.

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Lab products found in correlation

1 2 dioleoyl sn glycero 3 phosphoethanolamine dope, by Avanti Polar Lipids (3 mentions) Xenolight d luciferin potassium salt, by PerkinElmer (3 mentions) Dialysis cassette, by Thermo Fisher Scientific (2 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) C12 200 1 2 distearoyl sn glycero 3 phosphocholine dspc, by Avanti Polar Lipids (2 mentions) Zetasizer, by Malvern Panalytical (2 mentions) Megascript t7 transcription kit, by Thermo Fisher Scientific (2 mentions) Q2445, by Teknova (1 mentions) Luciferase mrna, by TriLink (1 mentions) Cy5 sirna, by Merck Group (1 mentions) Dlin mc3 dma, by MedChemExpress (1 mentions) 4 methoxybenzoic acid, by Merck Group (1 mentions) Dharmafect transfection reagent, by Horizon Discovery (1 mentions) Cholesterol, by Avanti Polar Lipids (1 mentions) Dotap, by Lipoid (1 mentions) Hoeschst 33342, by Thermo Fisher Scientific (1 mentions) Igg mouse elisa kit, by Abcam (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Mouse igg and igm elisa kits, by Abcam (1 mentions)

9 protocols using c14 peg2000

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 device^{54 (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 device^{54 (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).

Guimaraes P.P., Zhang R., Spektor R., Tan M., Chung A., Billingsley M.M., El-Mayta R., Riley R.S., Wang L., Wilson J.M, & Mitchell M.J. (2019). Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening. Journal of controlled release : official journal of the Controlled Release Society, 316, 404-417.

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Polyamine-Based Lipid Nanoparticle Synthesis

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Core 200 was customized from Enamine (Monmouth Junction, NJ) and other polyamine cores were purchased from Sigma Aldrich, Tokyo Chemical Industry (TCI), and Alfa Aesar. Epoxydodecane (C12), epoxytetradecane (C14), epoxyhexadecane (C16), 4-methoxybenzoic acid, N,N’-Dicyclohexylcarbodiimide (DCC), N-Hydroxysuccinimide (NHS), HSP47 siRNA pool (NM_001111043, NM_001111044, and NM_009825) and Cy5-siRNA were purchased from Sigma Aldrich. GFP siRNA (cat. P-002048-01-50) and DharmaFECT transfection reagents were purchased from Horizon Discovery Ltd. 1% agarose gels with SYBR™ Safe (#A42100) were purchased from ThermoFisher. Recombinant mouse TGF-β1 (cat. 7666-MB) was obtained from R&D. DSPC (#850365), cholesterol (#700100), and C14-PEG2000 (#880150) were bought from Avanti Polar Lipids. DLin-MC3-DMA was purchased from MedChem Express (Monmouth Junction, NJ). Luciferase mRNA was produced through an in vitro transcription (IVT) method^{46 (link)}.

Han X., Gong N., Xue L., Billingsley M.M., El-Mayta R., Shepherd S.J., Alameh M.G., Weissman D, & Mitchell M.J. (2023). Ligand-tethered lipid nanoparticles for targeted RNA delivery to treat liver fibrosis. Nature Communications, 14, 75.

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Ionizable Lipid Nanoparticle Formulation for mRNA

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Ionizable lipid nanoparticles were prepared using a weight ratio of 10:1 of C12-200 to mRNA, following a previously described method.20 (link) Briefly, an ethanol phase consisting of ionizable lipid, cholesterol (Avanti Polar Lipids), C14-PEG2000 (Avanti Polar Lipids), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipids), was combined with an aqueous phase containing mRNA in 10 mM citrate buffer pH 5, using microfluidic mixing at a volume ratio of 3:1 to formulate LNPs. The LNPs were then dialyzed against 1x PBS for 2 h using dialysis cassettes with a molecular weight cut-off of 20 kDa (ThermoFisher), sterile filtered using a 0.22 μm filter, and stored at 4 °C.

da Silva W.N., Carvalho Costa P.A., Scalzo Júnior S.R., Ferreira H.A., Prazeres P.H., Campos C.L., Rodrigues Alves M.T., Alves da Silva N.J., de Castro Santos A.L., Guimarães L.C., Chen Ferris M.E., Thatte A., Hamilton A., Bicalho K.A., Lobo A.O., Santiago H.D., da Silva Barcelos L., Figueiredo M.M., Teixeira M.M., Vasconcelos Costa V., Mitchell M.J., Frézard F, & Pires Goulart Guimaraes P. (2024). Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression. International Journal of Nanomedicine, 19, 2655-2673.

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Lipid Nanoparticle Formulation Optimization

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Nanoparticles were prepared using a 10:1 weight ratio of ionizable lipid to nucleic acid. An ethanol phase consisting of ionizable lipid, cholesterol (Avanti Polar Lipids, no. 700100 P), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (C14-PEG 2000, Avanti Polar Lipids, no. 880150 P), and optionally 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Lipoid), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Lipoid), or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Lipoid) at varying molar ratios (Supplementary Table 1) was combined with an aqueous phase containing pDNA in 10 mM citrate buffer, pH 5, to form LNPs via microfluidic mixing at a volume ratio of 3:1. LNPs were dialyzed against 1× PBS for 2 hours using dialysis cassettes with a molecular weight cutoff of 20 kDa (Thermo Scientific, no. 66003), sterile filtered using a 0.22 μm filter, and stored at 4 °C for later use.

Guimaraes L.C., Costa P.A., Scalzo Júnior S.R., Ferreira H.A., Braga A.C., de Oliveira L.C., Figueiredo M.M., Shepherd S., Hamilton A., Queiroz-Junior C.M., da Silva W.N., da Silva N.J., Rodrigues Alves M.T., Santos A.K., de Faria K.K., Marim F.M., Fukumasu H., Birbrair A., Teixeira-Carvalho A., de Aguiar R.S., Mitchell M.J., Teixeira M.M., Vasconcelos Costa V., Frezard F, & Guimaraes P.P. (2024). Nanoparticle-based DNA vaccine protects against SARS-CoV-2 variants in female preclinical models. Nature Communications, 15, 590.

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Lipid-based mRNA Delivery Formulation

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The amine 3,3′-diamino-N-methyldipropylamine (306), the tail 1,2-epoxyhexadecane (C12), cholesterol, and 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) were purchased from Sigma-Aldrich (St. Louis. MO). The amine 2[4-2(2-aminoethyl) amino)ethylpiperazine-1-YL)ethan-1-amine (200) was purchased from Enamine (Princeton, NJ). The tail isodecyl acrylate (O_i10) was purchased from Sartomer (Colombes, France). The lipid DLin-MC3-DMA was purchased from Biorbyt (San Francisco, CA). The phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and C14-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL). All mRNAs were purchased from Trilink Biotechnologies (San Diego, CA) and included the 5-methoxyuridine (5moU) base modification. XenoLight D-Luciferin Potassium Salt was purchased from PerkinElmer (Waltham, MA). Alexa Fluor 488 Phalloidin, Hoeschst 33342, IL-6, and TNFα mouse ELISA kits were purchased from Thermo Fisher (Waltham, MA). Mouse IgG ELISA kit was purchased from Abcam (Cambridge, MA).

Hajj K.A., Melamed J.R., Chaudhary N., Lamson N.G., Ball R.L., Yerneni S.S, & Whitehead K.A. (2020). A Potent Branched-Tail Lipid Nanoparticle Enables Multiplexed mRNA Delivery and Gene Editing In Vivo. Nano letters, 20(7), 5167-5175.

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Formulation and Characterization of siRNA Lipid Nanoparticles

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12 siRNAs targeting mouse Eef2 gene sequence with the lowest off-target potential were designed as described previously^{33 (link)}. siRNAs were synthesized and chemical modifications (modified bases (2′OMe) and phosphorothioate linkages) were introduced to stabilize siRNA in-vivo reducing the off-target potential of the sense strand and minimize immune response (Supplementary Table S3). siRNAs screened in Hepa 1–6 cell line. Cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen). siRNA with the lowest IC50 (determined by western blot and qPCR) was selected for further in-vitro and in-vivo experiments. Selected siRNA was formulated into lipid nanoparticles. The water phase contained siRNA duplex and ethanol phases with lipids (C12-200, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma), C14 PEG 2000 (Avanti Polar Lipids) at a 50:10:38.5:1.5 molar ratio) were mixed together in a microfluidic chip device. LNPs were dialyzed overnight against PBS. LNP sizes were measured by dynamic light scattering (ZetaSizer, Malvern Instruments). Mean diameter of the particles prepared for injections was about 90–120 nm.

Gerashchenko M.V., Nesterchuk M.V., Smekalova E.M., Paulo J.A., Kowalski P.S., Akulich K.A., Bogorad R., Dmitriev S.E., Gygi S., Zatsepin T., Anderson D.G., Gladyshev V.N, & Koteliansky V.E. (2020). Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes. Scientific Reports, 10, 15473.

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Lipid-mRNA Nanoparticle Synthesis

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The amines 3,3′-diamino-N-methyldipropylamine (306) was purchased from Sigma-Aldrich (St. Louis, MO), and N1-{2-[4-(2-aminoethyl)piperazin-1-yl]ethyl}ethane-1,2-diamine (200) were acquired from Enamine (Princeton, NJ). The tail isodecyl acrylate (O_i10) was purchased from Sartomer (Colombes, France). Cholesterol was sourced from Sigma-Aldrich. The lipids DOPE, PS, DOTAP, and C14-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL). mRNAs were obtained from TriLink Biotechnologies (San Diego, CA) including the 5-methoxyuridine base modification or synthesized by in vitro transcription using the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific, Waltham, MA). XenoLight d-luciferin potassium salt was procured from PerkinElmer (Waltham, MA). IL-2, IL-6, and TNF-α mouse enzyme-linked immunosorbent assay (ELISA) kits were purchased from Thermo Fisher Scientific (Waltham, MA). Mouse IgG and IgM ELISA kits were obtained from Abcam (Cambridge, MA).

Melamed J.R., Yerneni S.S., Arral M.L., LoPresti S.T., Chaudhary N., Sehrawat A., Muramatsu H., Alameh M.G., Pardi N., Weissman D., Gittes G.K, & Whitehead K.A. (2023). Ionizable lipid nanoparticles deliver mRNA to pancreatic β cells via macrophage-mediated gene transfer. Science Advances, 9(4), eade1444.

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Optimized siRNA Knockdown of Mouse HAS2

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Eight siRNAs targeting mouse HAS2 gene sequence with the lowest off-target potential were designed. siRNAs were synthesised and chemically modified (methylated pyrimidine nucleotides (2′OMe) and phosphorothioate linkages) to stabilise siRNA in vivo, reducing the off-target potential of the sense strand and minimise the immune response (Supplementary Table S1). NIH 3T3 cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) to test the knockdown efficiency by qPCR for each siRNA. The siRNA with the highest potency and the lowest IC50 was selected for further experiments. The selected siRNA was formulated into lipid nanoparticles (LNPs). Briefly, the water phase contained siRNA duplex and ethanol phase with lipids (C12-200, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma), C14 PEG 2000 (Avanti Polar Lipids) at a 50:10:38.5:1.5 molar ratio) was mixed in a microfluidic PDMS chip. LNPs were dialysed overnight against PBS. LNP sizes were measured by dynamic light scattering (ZetaSizer, Malvern Instruments). The mean diameter of the particles prepared for injections was about 90–120 nm.

Halimani N., Nesterchuk M., Tsitrina A.A., Sabirov M., Andreichenko I.N., Dashenkova N.O., Petrova E., Kulikov A.M., Zatsepin T.S., Romanov R.A., Mikaelyan A.S, & Kotelevtsev Y.V. (2024). Knockdown of Hyaluronan synthase 2 suppresses liver fibrosis in mice via induction of transcriptomic changes similar to 4MU treatment. Scientific Reports, 14, 2797.

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Synthesis of Modified mRNA and Lipid Nanoparticles

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Cy5-labeled mRNA, ψ- 5’-triphosphate, m1ψ- 5’-triphosphate, m5C- 5’-triphosphate, s2U- 5’-triphosphate, and m5U- 5’-triphosphate were purchased from Trilink Biotechnologies (San Diego, CA). MEGAscript™ T7 Transcription Kit was purchased from Thermo Fisher Scientific (Waltham, MA). ScriptCap™ 2’-O-Methyltransferase Kit was purchased from CellScript (Madison, WI). Cholesterol and 1,2-epoxyhexadecane (C12) were purchased from Sigma Aldrich (St Louis, MO). The phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and C14-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL). DMG-PEG2000 was purchased from NOF America (White Plains, NY). The isodecyl acrylate (O_i10) amine and the 2[4-2(2-aminoethyl)amino)ethylpiperazine-1-YL)ethan-1-amine (200) were purchased from Sartomer (Colombes, France) and Enamine (Princeton, NJ), respectively. XenoLight D-Luciferin Potassium Salt was purchased from PerkinElmer (Waltham, MA). The lipid cKK-E12 was generously donated by the Anderson Lab at the Massachusetts Institute of Technology (Cambridge, MA). The Mouse Erythropoietin Quantikine ELISA Kit was purchased from R&D Systems (Minneapolis, MN).

Melamed J.R., Hajj K.A., Chaudhary N., Strelkova D., Arral M.L., Pardi N., Alameh M.G., Miller J.B., Farbiak L., Siegwart D.J., Weissman D, & Whitehead K.A. (2021). Lipid nanoparticle chemistry determines how nucleoside base modifications alter mRNA delivery. Journal of controlled release : official journal of the Controlled Release Society, 341, 206-214.

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