Supported lipid bilayers were created using a modification of a published solvent-exchange protocol.67 Briefly, this involves mixing together 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids, 850375C), a net neutral lipid, and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP, Avanti Polar Lipids, 890890C), a net positively charged lipid, in the desired proportion (usually 2–4% DOTAP by weight). This mixture of lipids is dissolved in 50% isopropanol to a final concentration of ~1 mg/ml and 500 μl is added to a ~2 ml Teflon/Plexiglass chamber with a Hellmanex (Fisher Scientific, 14-385-944) cleaned glass coverslip bottom. Water is slowly added to the lipid mixture in seven 750 μl aliquots until the solution is >90% water. The chamber is then washed with 25 ml of sterile water to remove any residual isopropanol and non-precipitated lipid, then washed with the desired mobility buffer (10 mM Na2HPO4, pH 7.5, 5 mM Ascorbic acid and ~40–50 mM NaCl) before adding the DNA sample pre-stained with YOYO-1 (~0.5 ng of DNA stained with ~7 pmol YOYO-1 at room temperature for at least 15 minutes) and letting the DNA adsorb overnight.
Dotap
Manufactured by Avanti Polar Lipids
Sourced in United States, China
DOTAP is a cationic lipid commonly used in the formulation of liposomes and other lipid-based delivery systems. It is designed to facilitate the delivery of various payloads, including nucleic acids, proteins, and small molecules, to target cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Cholesterol, by Avanti Polar Lipids (12 mentions)
Dspe peg2000, by Avanti Polar Lipids (10 mentions)
Cholesterol, by Merck Group (9 mentions)
Opti mem, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dc chol, by Avanti Polar Lipids (5 mentions)
Chloroform, by Merck Group (5 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Avanti mini extruder, by Avanti Polar Lipids (3 mentions)
Dope peg2000, by Avanti Polar Lipids (3 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (3 mentions)
Plasmid mega prep kit, by Qiagen (3 mentions)
Glycerol monooleate, by Merck Group (3 mentions)
18 1 ethylpc, by Avanti Polar Lipids (3 mentions)
Texas red dhpe, by Thermo Fisher Scientific (3 mentions)
1 2 dioleoyl 3 trimethylammonium propane, by Avanti Polar Lipids (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Fam cysteine irgd, by LifeTein (2 mentions)
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Merck Group (2 mentions)
114 protocols using dotap
1
Solvent-Exchange Supported Lipid Bilayers
2
Lipid-based Nanoparticle Synthesis
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Cationic DOTAP, DOPC, and DOPG were purchased from Avanti Polar Lipids (Alabaster, AL). DOTAP, DOPC, and DOPG are among the most widely used lipid species for the delivery of drugs and nucleic acids. Each lipid was dissolved in chloroform, and the solvent was evaporated under vacuum for 2 h. Lipid films were hydrated with ultrapure water to a final lipid concentration of 1 mg mL−1 and stored at 4 °C. The obtained liposome suspensions of DOTAP and DOPG were extruded 20 times through a 0.1 -µm polycarbonate filter with the Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster, AL). On the other side, DOPC liposomes were sonicated for 20 min.
3
siRNA Lipoplex-Loaded Lipid Nanocarriers
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For siRNA encapsulation, the nucleic acids were firstly complexed with DOTAP/DOPE lipids. Briefly, the cationic lipid DOTAP (Avanti® Polar Lipids Inc., Alabaster, AL, USA), solubilized in chloroform, was weighted at the ratio 1/1 (mol/mol) with the neutral lipid DOPE (Avanti® Polar Lipids Inc., Alabaster, AL, USA) to obtain a final concentration of 30 mM of cationic lipid charges, based on the number of lipid charges per molecule, i.e., 1 for DOTAP. After evaporation of chloroform under vacuum, deionized water was added to rehydrate the DOTAP/DOPE lipid film overnight at 4 °C. The lipid film was then sonicated for 30 min. Lipoplexes were formulated as a simple equivolume mix of siRNA and DOTAP/DOPE lipids. In this study, the Na/K ATPase alpha 1 subunit siRNA (sense sequence: 5′-GGGCAGUGUUUCAGGCUAAdTdT-3′; antisens: 5′-UUAGCCUGAAACACUGCCCdTdT-3′; Eurogentec, Seraing, Belgium) was used.
The complexes were characterized by the charge ratio (CR), i.e., the ratio between the positive charges of the lipids and negative charges of the nucleic acids (±ratio), fixed to 5 considering our previous results.10 (link) These pre-formulated lipoplexes were added during the last phase inversion temperature (PIT) of LNC formulation leading to the formation of siRNA LNCs.
The complexes were characterized by the charge ratio (CR), i.e., the ratio between the positive charges of the lipids and negative charges of the nucleic acids (±ratio), fixed to 5 considering our previous results.10 (link) These pre-formulated lipoplexes were added during the last phase inversion temperature (PIT) of LNC formulation leading to the formation of siRNA LNCs.
4
Lipid-based Nanoparticle Synthesis
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DOTAP, cholesterol, and DSPE-PEG2000 were purchased from Avanti Polar Lipid, Inc. unless otherwise noted, all chemicals were purchased from Sigma-Aldrich, all cell culture products were purchased from Gibco BRL/Life Technologies, a division of Invitrogen, and all restriction enzymes were obtained from New England BioLabs (NEB).
5
Chemically Modified AR-ASO Lipid Nanoparticles
6
Chemically Modified Antisense Oligonucleotides for Targeted Delivery
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Chemically modified AR-ASO (sense strand, 5′TGATTTAATGGTTGCA3′) and Cy3-conjugated ASO (5′GCATTCTAATAGCAGC3′) were obtained from Ionis Pharmaceuticals (Carlsbad, CA). FAM-cysteine-iRGD was purchased from LifeTein (Somerset, NJ). DSPE-PEG2000, DPPC, DOTAP, and cholesterol were obtained from Avanti Polar Lipid (Alabaster, Alabama). DSPE-PEG2000-MAL was from Nanosoft Polymers (Winston-Salem, NC). The Lipofectamine 3000 transfection kit was from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 Medium, and trypsin were from Sigma-Aldrich (St Louis, MO). All reagents and compounds were used without further purification or modification.
7
Lipid-Based Nanoparticle Delivery of siRNA
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d -Galactosyl ceramide, PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine (ammonium salt)), cholesterol, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), rhodamine-conjugated DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and DOTAP (dioleoyl-3-trimethylammonium-propane) were purchase from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). The cationic lipid DMKE (O,O’-dimyristyl-N-lysyl glutamate) was synthesized as previously reported [32 (link)]. EX-CyTox that is cell viability and cytotoxicity test reagent is Daeil Lab Service Co., Ltd (Seoul, South Korea). Doxorubicin and CL-4B (agarose beads) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). FITC-labeled siRNA was purchased from Invitrogen™ Life Technologies (Grand Island, NY, USA). siRNA against vimentin was obtained from Santa Cruz biotechnology (Dallas, TX, USA).
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8
Liposome-Mediated siRNA Delivery for Luciferase Assay
9
DOTAP-Based Lipoplex Formulation
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DOTAP was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Chol and chondroitin sulfate C sodium salt were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DOPE was obtained from NOF Corporation (Tokyo, Japan). DXR was purchased from Wako Pure Chemical Industries Inc.
10
Liposomal Vaccine Formulation Protocol
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Female BALB/c mice (6-weeks old) were purchased from Japan SLC (Shizuoka, Japan). The mice were housed in a specific pathogen-free environment. All animal experiments followed the guidelines for laboratory animal experiments of the Tokyo University of Pharmacy and Life Sciences, and each experimental protocol was approved by the Committee for Laboratory Animal Experiments at the institution (P14–32 and P15–33). DOTAP and DC-chol were purchased from Avanti Polar Lipids (Alabama, USA). Class B CpG ODN 1826 (5′-tccatgacgtt cctgacgtt -3′; small letters indicate phosphorothioate linkage, underlining indicates the CpG motif) was synthesized by the Hokkaido System Science Co., Ltd. (Hokkaido, Japan). Ovalbumin from egg white (OVA) was obtained from Sigma-Aldrich (St. Louis, USA).
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