Dmg peg2000

Manufactured by Avanti Polar Lipids

Sourced in United States

DMG-PEG2000 is a lipid molecule composed of a dimyristoylglycerol (DMG) headgroup and a polyethylene glycol (PEG2000) moiety. It is used as a component in the formulation of liposomes and other lipid-based delivery systems.

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15 protocols using dmg peg2000

Artificial PD-L1 Enriched Extracellular Vesicles

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PD‐L1 ssDNA oligo (listed 5′–3′) used in this study was CTG ACA TGT CAG GCT GAG GGC TAC CCC AAG. The designed oligo was custom synthesized and purified by IDT. To fabricate artificial EVs, an aqueous solution of the PD‐L1 ssDNA oligo was mixed with a lipid formulation of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE, Avanti Polar Lipids), linoleic acid (LA, Sigma‐Aldrich), and 1,2‐dimyristoyl‐rac‐glycero‐3‐methoxypolyethylene glycol‐2000 (DMG‐PEG 2000, Avanti Polar Lipids) (50:48:2 mole ratio in 200 proof ethanol). The mixture was subsequently sonicated for 5 min, injected into PBS, and then sonicated for another 5 min. After dialysis with a 20 kDa MWCO to remove free molecules, the suspension was spiked into healthy donor EVs at different concentrations of 0.625, 1.25, 2.5, 5, and 10%.
For the calibration experiment, the gold‐coated biochip was tethered with biotinylated PD‐L1 CLN‐MBs, which was made by replacing Bis‐DSPE PEG2000 with DSPE‐PEG(2000) Biotin (Avanti Polar Lipids) in the lipid formulation of CLN‐MB fabrication. The artificial EVs at different concentrations were then captured on the biochip by fusion with the PD‐L1 CLN‐MBs for 2 h at 37°C. After rinsing with PBS, the samples were imaged using the TIRF microscope.

Nguyen L.T., Zhang J., Rima X.Y., Wang X., Kwak K.J., Okimoto T., Amann J., Yoon M.J., Shukuya T., Chiang C., Walters N., Ma Y., Belcher D., Li H., Palmer A.F., Carbone D.P., Lee L.J, & Reátegui E. (2022). An immunogold single extracellular vesicular RNA and protein (AuSERP) biochip to predict responses to immunotherapy in non‐small cell lung cancer patients. Journal of Extracellular Vesicles, 11(9), e12258.

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Lipid Nanoformulation Synthesis and Characterization

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TT3 was synthesized according to the methods reported before.^{[19 (link)]} DOPE, DMG-PEG₂₀₀₀, and other lipid materials were purchased from Avanti Polar Lipids, Inc (Alabaster, AL). Fetal bovine serum (FBS) was purchased from Life Technologies (Grand Island, NY). Lipofectamine^® 3000 Reagent (Lipo3000) was obtained from Thermo Fisher Scientific (Waltham, MA.) All the other chemicals were purchased from Sigma Aldrich or Alfa-Aesar. Lyso-Tracker® Red DND-99 and ER-Tracker™ Red E34250 were purchased from Thermo Fisher Scientific (Waltham, MA). Mitotracker™ (Green) ab176830 was ordered from Abcam (Cambridge, MA). Hoechst 33342 was purchased from Sigma Aldrich.

Zhao W., Zeng C., Yan J., Du S., Hou X., Zhang C., Li W., Deng B., McComb D.W., Xue Y., Kang D.D, & Dong Y. (2021). Construction of messenger RNA (mRNA) probes delivered by lipid nanoparticles to visualize intracellular protein expression and localization at organelles. Advanced materials (Deerfield Beach, Fla.), 33(45), e2103131.

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Lipid-based Transfection Reagent Formulation

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Opti-MEM reduced serum medium was purchased from Thermo Fisher Scientific. Cell culture plates and luminescent assay plates were obtained from Corning. MC3 was purchased from MedKoo. DOPE and DMG-PEG2000 were purchased from Avanti Polar Lipids. Cholesterol was obtained from Sigma-Aldrich. Q5 High-Fidelity PCR Kit was purchased from NEB.

Zeng C., Hou X., Yan J., Zhang C., Li W., Zhao W., Du S, & Dong Y. (2020). Leveraging mRNA Sequences and Nanoparticles to Deliver SARS-CoV-2 Antigens In Vivo. Advanced materials (Deerfield Beach, Fla.), 32(40), e2004452.

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Liposome Preparation via Extrusion

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1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoylphosphatidylcholine
(DPPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine
(DOPC), and cholesterol and 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene
glycol-2000 (DMG-PEG2000) were obtained commercially (Avanti Polar
Lipids). Liposomes were prepared through the established extrusion
method.^{104 (link)} Briefly, lipids were desiccated
for 2 h and allowed to reach RT. The lipids, cholesterol, and DMG-PEG2000
were weighed at the indicated ratios (Tables S4 and S5) and dissolved in chloroform, evaporated using a nitrogen
stream, and left under vacuum overnight to form a lipid film. The
film was rehydrated with PBS (pH 7.4) at 65 °C for 2 h with vortexing
and the vesicles were further processed with five freeze (liquid nitrogen)
and thaw (65 °C water bath) cycles. The nanoparticles were extruded
through stacked polycarbonate filters (400, 200, or 100 nm) at least
10 times using a mini-extruder (Avanti Polar lipids, cat# 610000).
The size of liposomes was measured using dynamic light scattering
(Malvern Zetasizer).

von Lersner A.K., Fernandes F., Ozawa P.M., Jackson M., Masureel M., Ho H., Lima S.M., Vagner T., Sung B.H., Wehbe M., Franze K., Pua H., Wilson J.T., Irish J.M., Weaver A.M., Di Vizio D, & Zijlstra A. (2024). Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution. ACS Nano, 18(15), 10464-10484.

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Basophil Activation Test for Vaccine Evaluation

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Whole blood preserved in heparin, as described in Mukai et al,^{6 (link)} was collected from participants. Briefly, basophil activation was assessed after stimulation for 30 minutes at 37 °C with either DMG-PEG 2000 (Avanti Polar Lipids; 1 μg/μL) or P80 (Millipore Sigma–Sigma Aldrich; 1 μg/μL). Filtered saline was used as a negative control and anti-IgE (Bethyl Laboratories; 1 μg/mL) was used as a positive control.
Vaccine-discarded remnant material was used at 0.007 μg/μL. All stimuli were prepared in Roswell Park Memorial Institute (RPMI) medium. Basophils were gated as CD123+HLA−DR− cells, and the percentage of CD63+ basophils was quantified by flow cytometry. Control participants were also consented using the same IRB-approved protocol, and SPT and BAT assays were performed (Table 1). Figure 2 illustrates an example of BAT results among control participants using anti-IgE (positive control), saline, and vaccine material as an activator.

Warren C.M., Snow T.T., Lee A.S., Shah M.M., Heider A., Blomkalns A., Betts B., Buzzanco A.S., Gonzalez J., Chinthrajah R.S., Do E., Chang I., Dunham D., Lee G., O’Hara R., Park H., Shamji M.H., Schilling L., Sindher S.B., Sisodiya D., Smith E., Tsai M., Galli S.J., Akdis C, & Nadeau K.C. (2021). Assessment of Allergic and Anaphylactic Reactions to mRNA COVID-19 Vaccines With Confirmatory Testing in a US Regional Health System. JAMA Network Open, 4(9), e2125524.

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Skin Prick Test for Histamine Sensitivity

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Single-lancet technique was performed with DMG-PEG 2000 (Avanti Polar Lipids, 1 μg/μL) or P80 (Millipore Sigma; Sigma Aldrich, 1 μg/μL). Histamine (1 mg/mL) and filtered saline (negative control) were used for internal validation. Antihistamine medication was withheld for at least 72 hours prior to the test. Wheal and erythema were measured at 15 minutes. The wheal and erythema measurements were recorded by taking the mean of the 2 perpendicular diameters in millimeters. A wheal size of 4 mm or greater was considered positive. Saline controls were used, and all were negative. Discarded, undiluted remnant vaccine was used according to the manufacturer’s concentration instructions.

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Formulation and Characterization of LNP-siRNA Complexes

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The LNP‐siRNA was formulated using previous protocols as described (Jayaraman et al, 2012 ). Briefly, LNPs were formed by mixing lipids (Selleckchem, S6683), 1, 2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) (Avanti Polar Lipids, 850365p‐200mg), cholesterol (Sigma, C3045), and DMG‐PEG2000 (Avanti Polar Lipids, 880151p‐1g) at a molar ratio of 50:10:38.5:1.5 in ethanol. siRNA solutions were diluted in 50 mM sodium citrate (pH = 4) such that the final weight ratio of lipid: siRNA was achieved from 16:1 to 4:1, accordingly, the mixture was incubated for 15 min at 37°C to encapsulate the siRNAs. LNP‐siRNAs were diluted in PBS and analyzed further. siRNAs used in this study are listed below:
control siRNA: 5′‐U.U.G.U.A.G.G.C.C.A.G.C.U.G.U.G.A.G.U.A.G‐3′ (Sense);
5′‐C.U.A.C.U.C.A.C.A.G.C.U.G.G.C.C.U.A.C.A.A‐3′ (Antisense).
PCIF1 siRNA1: 5′‐U.U.A.U.A.C.C.G.G.A.U.G.C.A.G.A.C.C.A.C.G‐3′ (Sense);
5′‐C.G.U.G.G.U.C.U.G.C.A.U.C.C.G.G.U.A.U.A.A‐3′ (Antisense).
PCIF1 siRNA2: 5′‐A.U.G.A.C.A.G.C.A.U.U.G.G.U.C.U.G.G.A.U.G‐3′ (Sense);
5′‐C.A.U.C.C.A.G.A.C.C.A.A.U.G.C.U.G.U.C.A.U‐3′ (Antisense).

Wang L., Wu L., Zhu Z., Zhang Q., Li W., Gonzalez G.M., Wang Y, & Rana T.M. (2022). Role of PCIF1‐mediated 5′‐cap N6‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD‐1 immunotherapy. The EMBO Journal, 42(2), e111673.

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Lipid-based Nanoparticle Formulation Protocol

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DOTAP (1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), cholesterol, DMG-PEG 2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DPPC (dipalmitoyl phosphatidylcholine), 18:1 PE TopFluor AF 594 (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(TopFluor^® AF594) (ammonium salt)), 18:0 PE-DTPA (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (ammonium salt)), and DSPE-PEG2000-azide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (ammonium salt)) were purchased from Avanti Polar Lipids. Ionizable lipids cKK-E12, SM-102, ALC-0315, and C12–200 were purchased from Echelon Biosciences. Anti-mouse BV421 CD41, PE-Cy7 CD62P AF700 Ly6G and APC CD42d were purchased from Biolegend. Indium-111 Chloride (In-111) was purchased from BWXT Medical. A modified Lowry assay kit (DC Protein Assay) was purchased from Bio-Rad Laboratories. tPA was purchased from Millipore.

Omo-Lamai S., Zamora M.E., Patel M.N., Wu J., Nong J., Wang Z., Peshkova A., Chase L.S., Essien E.O., Muzykantov V., Marcos-Contreras O., Myerson J.W, & Brenner J.S. (2023). Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions. bioRxiv.

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Nanoparticle-Mediated RNA Vaccine Delivery

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DOTAP, DOPE and DMG-PEG2000 were purchased from Avanti Polar Lipids (Alabama, USA); cholesterol was purchased from Sinopharm Chemical Reagent (China); FITC-DSPE-PEG2000 was obtained from AVT (Shanghai, China); Hesperetin was produced in Yuanye (Shanghai, China); eGFP mRNA was obtained from VectorBuilder (Guangzhou, China); OVA mRNA was purchased from TriLink Bio Technologies (California, USA); Quanti-iT RiboGreen RNA reagent and Kit were obtained from Invitrogen (California, USA); Mouse ovalbumin specific IgG ELISA kit was purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China); OVA323-339, OVA257-264, OVA208-216, OVA27-35 were purchased from Apeptide (Shanghai, China); Poly(I:C) (HMW) VacciGrade™ was obtained from InvivoGen (San Diego, USA); DMEM medium, RPMI 1640 medium, 1% Penicillin/Streptomycin, PBS and FBS were purchased from Procell Life Science&Technology (Wuhan, China).

Ji A., Xu M., Pan Y., Diao L., Ma L., Qian L., Cheng J, & Liu M. (2022). Lipid Microparticles Show Similar Efficacy With Lipid Nanoparticles in Delivering mRNA and Preventing Cancer. Pharmaceutical Research, 40(1), 265-279.

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Lipid Nanoparticle Synthesis Reagents

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(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA, >98%) was purchased from D&C Chemicals (China), DSPC and cholesterol were purchased from Echelon Biosciences, Inc. (USA), and DMG-PEG 2000 was purchased from Avanti Polar Lipids, Inc. (USA). Ethanol (BioUltra, ≥99.8%), citric acid monohydrate, sodium chloride, Na₂HPO₄, and KH₂PO₄ were purchased from Sigma-Aldrich (Germany).

Ray R.M., Hansen A.H., Taskova M., Jandl B., Hansen J., Soemardy C., Morris K.V, & Astakhova K. (2021). Enhanced target cell specificity and uptake of lipid nanoparticles using RNA aptamers and peptides. Beilstein Journal of Organic Chemistry, 17, 891-907.

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