All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000)-maleimide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide) was obtained from Pierce (Rockford, IL, USA). O-succinyl-3′-hydroxmethyl-(±) nicotine (nicotine hapten) was purchased from Toronto Research Chemicals (Toronto, ON, Canada).
1 2 dioleoyl 3 trimethylammonium propane dotap
Manufactured by Avanti Polar Lipids
Sourced in United States
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) is a cationic lipid used in the formulation of liposomes and lipoplexes for various research applications. It is a synthetic lipid with a positively charged headgroup and two unsaturated fatty acid chains. DOTAP can be used to facilitate the delivery of nucleic acids, such as plasmid DNA, RNA, or small interfering RNA (siRNA), into cells.
Automatically generated - may contain errors
Lab products found in correlation
Cholesterol, by Merck Group (4 mentions)
1 2 dioleoyl sn glycero 3 phosphate dopa, by Avanti Polar Lipids (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine dopc, by Avanti Polar Lipids (2 mentions)
Cholesterol, by Avanti Polar Lipids (2 mentions)
Octylglucoside, by Merck Group (2 mentions)
Anti mouse cd3 apc, by BD (2 mentions)
Pe conjugated anti mouse cd73 cat no 127205, by BioLegend (2 mentions)
Isotype matched control monoclonal antibodies mabs, by BioLegend (2 mentions)
Phorbol 12 myristate 13 acetate pma ionomycin cocktail, by BD (2 mentions)
Antiil 4 pe antibodies, by BD (2 mentions)
Non targeting sirna cat no d, by Horizon Discovery (2 mentions)
Ge11 peptide cgggyhwygytpqnvi, by ChinaPeptides (2 mentions)
Anti ifn γ fitc, by BD (2 mentions)
Cd73 sirna, by Horizon Discovery (2 mentions)
Anti cd8a pe cy5, by BD (2 mentions)
Pe cy5 anti cd4, by BD (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
41 protocols using 1 2 dioleoyl 3 trimethylammonium propane dotap
1
Synthesis of Nicotine Hapten Conjugates
2
Lipid-based Vaccine Formulation Protocol
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1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Additionally, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] (DC-chol) and egg white OVA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Class B CpG ODN 1668 (5′-tccatgacgttcctgatgct-3′; small letters indicate phosphorothioate linkage) was synthesized by Sigma-Aldrich (Woodlands, TX, USA). Endotoxin-free phosphate-buffered saline (PBS) was acquired from FUJIFILM Wako Pure Chemical Industries (Osaka, Japan). Rat anti-mouse IL-6 receptor (IL-6R) antibody (clone MR16-1) was provided by Chugai Pharmaceutical Co., Ltd. The corresponding rat IgG1 k isotype control monoclonal antibody (clone RTK2071) for MR16-1 was purchased from BioLegend (San Diego, CA, USA).
3
Lipid-Coated Gold Substrates
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Different thiols were used in combination with the different lipids to be coated onto them by electrostatically-driven physisorption. We used two thiols, namely 4-mercaptobenzoic acid (MbA) and 11-amino-1-undecanethiol hydrochloride (AT), from Sigma (Milan, Italy), and three lipids, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), from Avanti Polar Lipids (Alabaster, Alabama, US). All solvents were purchased from Sigma-Aldrich.
First, the substrates were incubated at rt for 2 h with a 1 mM aqueous solution of the thiol molecule, either MbA or AT, to let the sulfur of the –SH group bind covalently to the Au surface (chemisorption). The substrates were then gently washed with their aqueous solutions and dried under nitrogen flow.
All the lipids were dissolved in chloroform/methanol 2:1 vol/vol, dried under a gentle nitrogen flux in a test tube, and put under a mild vacuum overnight to remove all solvent traces. POPC/POPS in a 9:1 mol/mol ratio and DOTAP were then re-suspended in PBS at a 5 g/L concentration, let to swell for 30 min, and extruded 11 times through a polycarbonate filter (Whatman, USA) with 100 nm pore diameter to form unilamellar vesicles.
First, the substrates were incubated at rt for 2 h with a 1 mM aqueous solution of the thiol molecule, either MbA or AT, to let the sulfur of the –SH group bind covalently to the Au surface (chemisorption). The substrates were then gently washed with their aqueous solutions and dried under nitrogen flow.
All the lipids were dissolved in chloroform/methanol 2:1 vol/vol, dried under a gentle nitrogen flux in a test tube, and put under a mild vacuum overnight to remove all solvent traces. POPC/POPS in a 9:1 mol/mol ratio and DOTAP were then re-suspended in PBS at a 5 g/L concentration, let to swell for 30 min, and extruded 11 times through a polycarbonate filter (Whatman, USA) with 100 nm pore diameter to form unilamellar vesicles.
4
Lipid-based Nanoparticle Delivery of Antisense Oligonucleotides
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Cholesterol (Chol), didecyldimethylammonium bromide (DDAB), polyethylenimine 1200 (PEI1200, MW 1200), d-α-tocopherol polyethylene glycol succinate (TPGS), human holo-transferrin (Tf), 2-iminothiolane (Traut's reagent), and other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and distearoyl phosphatidylethanolamine-N-[maleimide-polyethylene glycol, MW 2000] (Mal-PEG2000-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL). All tissue culture media and supplies were purchased from Invitrogen (Carlsbad, CA). Antisense oligonucleotides used in this study were fully phosphorothioated. These include G3139 (5′-TCT CCC AGC GTG CGC CAT-3′) and fluorescein-labeled G3139, FAM-G3139, which were custom-synthesized by TsingKe (Chengdu, China).
5
Radiolabeled Liposomes for Targeted Therapy
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177Lutetium Chloride (177LuCl3) was purchased from PerkinElmer (Waltham, MA). N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG2000) was purchased from NOF America Corporation (White Plains, NY). DSPE-PEG2000-Anisamide (DSPE-PEG-AA) was synthesized in our lab as described previously [19 (link)]. 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL). Primary antibody against p-H2AX was purchased from Cell Signaling (Rabbit mAb #9718). Anti-rabbit secondary antibody was purchased from Cellsignaling (#4414). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or ThermoFisher (Waltham, MA).
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177Lutetium Chloride (177LuCl3) was purchased from PerkinElmer (Waltham, MA). N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG2000) was purchased from NOF America Corporation (White Plains, NY). DSPE-PEG2000-Anisamide (DSPE-PEG-AA) was synthesized in our lab as described previously [19 (link)]. 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL). Primary antibody against p-H2AX was purchased from Cell Signaling (Rabbit mAb #9718). Anti-rabbit secondary antibody was purchased from Cellsignaling (#4414). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or ThermoFisher (Waltham, MA).
6
Lipid-based Nanocarrier Formulation
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Materials. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Avanti Polar Lipids, Inc.); 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-(glutaryl) (Glutaryl PE) (Avanti Polar Lipids, Inc); Tween® 80 (Croda chocques, France); egg-Phosphatidylcholine (egg-PC) and sodium tetraborate decahydrate (Sigma-Aldrich®, Italy); Sodium enoxaparin (Clexane® 10000 IU/mL, Sanofi Aventis, Italy), calcium nadroparin (Fraxiparina 19500 IU/mL, Glaxo-SmithKline, Italy), sodium unfractionated heparin (UFH) (kindly provided by LDO S.p.A., Milan, Italy). HPLC-grade and analytical-grade organic solvents were also purchased from Sigma-Aldrich (Milan, Italy). HPLC-grade water was prepared with a Milli-Q water purification system. Carbazole was purchased from Merck, Italy. Osmium tetroxide (OsO4) (Sigma-Aldrich, Italy). All other reagents were of analytical grade unless specified.
7
Lipid-Peptide Nanoparticle Formulations
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The lipids (Supplementary Fig. 1 ) 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE) and Rhodamine-DOPE were purchased from Avanti Polar Lipids (Birmingham, Alabama, USA). The peptide sequences K16GACSERSMNFCG (K16E) and K16GACYGLPHKFCG (K16Y) were purchased from China Peptides (Shanghai, China) and dissolved to 10 mg/mL in DNase/RNase free water (Invitrogen, Paisley, UK). The plasmid pCI-Luc comprised the luciferase gene of pGL3 (Invitrogen, Paisley, UK) subcloned into the eukaryotic expression vector pCI (Promega, Southampton, UK).
8
Synthesis and Characterization of Lipid-PEG Conjugates
9
Fabrication of Lipid-Polymer Hybrid Nanoparticles
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SU-8 epoxy photoresist (SU-8 100) was purchased from MicroChem Inc. (Newton, MA, USA) and polysiloxane acrylate (PSA) precursor (ST1010s) was obtained from MCNet Inc. (Gwangju, Republic of Korea). 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)2000] (PEG2000-DSPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Polydimethylsiloxane (PDMS, Sylgard 184), cholesterol (CHOL), N,N-diisopropylethylamine (DIPEA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), chloroform, anhydrous N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and Flamma® Fluor 648-NHS ester dye (Flamma 648) was available from BioActs (Incheon, Republic of Korea). Doxorubicin hydrochloride (DOX) and acetyl-Ala-Val-Pro-Ile-Ala-Gln-Phe-Arg-Arg-Gly (Ac-AVPIAQ-FRRG; SMAC-P-FRRG) were obtained from FutureChem (Seoul, Republic of Korea), and recombinant human cathepsin B protein was purchased from R&D Systems (Minneapolis, MN, USA).
10
Lipid-based Nanoparticle Synthesis and Characterization
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1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol (Chol) was obtained from Chengdu Kelong Chemical Co. Ltd & ChengDu Kelong Chemical Reagent Company (Chengdu, China). Cholesterol succinic anhydride ester (Chol-suc) was synthesized as described previously23 (link). Poly (ethylene glycol) diamine (H2N-PEG-NH2) was provided by BioMatrik Inc. (Jiaxing, Zhejiang, China). MPEG-succinyl-Cholesterolconjugate (mPEG-Chol) was synthesized and purified by our research group24 (link)25 (link). The plasmid green fluorescent protein (pGFP), PEDF plasmid PAAV2-PEDF (PEDF gene) and null plasmid PAAV2 were constructed according to our previous report16 (link). Plasmid DNA (pDNA) was extracted according to the EndoFree Plasmid Purification Handbook (QIAGEN, Germany). DNA ladder and pre-stained protein ladder were provided by Fermentas (Thermo Fisher Scientific Inc., Waltham, MA, USA). All reagents were of analytical grade and were used without further purification except chloroform used to prepare nano-liposomes.
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