In this work, pH-sensitive liposomes were prepared. Different lipid formulations were studied using the lipids dioleoylphosphatidylethanolamine (DOPE, from Avanti Polar Lipids, Birmingham, AL, USA), dipalmitoylphosphatidylethanolamine (DPPE, from Sigma-Aldrich, St. Louis, MO, USA), cholesterol (Ch, from Sigma-Aldrich, St. Louis, MO, USA) and cholesteryl hemisuccinate (CHEMS, from Sigma-Aldrich, St. Louis, MO, USA) at the molar ratios DOPE:CHEMS (7:3), DOPE:Ch:CHEMS (45:45:10) and DPPE:Ch:CHEMS (45:45:10). Liposomes composed of these formulations were synthetized following the ethanolic injection method [40 (link)], where an ethanolic lipid solution (1 × 10−3 M) was initially prepared according to the desired proportion. The volume of the lipid solution equivalent to this concentration was evaporated under an ultrapure nitrogen flow and subsequently redissolved in absolute ethanol in the same volume. Finally, the ethanolic solution was injected, drop by drop and under vortexing, to 3 mL of ultrapure water, inducing the formation of liposomes. The injection was performed while ensuring that the aqueous medium was at a higher temperature than the main phospholipid phase transition temperature (−16 °C for DOPE and 63 °C for DPPE) [41 (link)].
Chems
Manufactured by Merck Group
Sourced in United States
CHEMS is a line of laboratory equipment produced by Merck Group. It is designed for general use in scientific research and analysis environments. The core function of CHEMS products is to provide reliable and precise tools for various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Dspe peg2000, by Lipoid (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Dipalmitoylphosphatidylethanolamine, by Merck Group (1 mentions)
Dioleoylphosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
Cholesteryl hemisuccinate, by Merck Group (1 mentions)
Sonifier 450, by Emerson (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Formalin, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt salt, by Merck Group (1 mentions)
Sephadex g 75, by Merck Group (1 mentions)
Zymosan a, by Merck Group (1 mentions)
Hepes salt, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
5 protocols using chems
1
Formulation and Synthesis of pH-Sensitive Liposomes
2
Liposomal Delivery of Peptide and CpG-DNA
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The liposomes DOPE and CHEMS were obtained from Sigma-Aldrich Co. Liposome complexes consisting of peptide (50 µg) and CpG-DNA (50 µg) co-encapsulated with DOPE:CHEMS (at a 1:1 ratio) were formulated as reported previously.31 (link) Briefly, DOPE and CHEMS were mixed in 10% ethanol at a molar ratio of 1:1, evaporated with nitrogen gas to produce a solvent-free lipid film, and resuspended in a mixture containing equal volumes of water-soluble MB-ODN 4531(O) (50 µg) and peptide (50 µg), followed by vigorous stirring at room temperature for 30 minutes. After adjusting the pH to 7.0, the peptide and CpG-DNA co-encapsulated with the DOPE:CHEMS [Lipoplex(O)] complex were sonicated lightly for 30 seconds with a sonicator (Sonifier 450; Branson Ultrasonics, Danbury, CT, USA). The complex was filtered with a 0.22 µm filter and freeze-thawed three times with liquid nitrogen.
3
Lipid Composition Analysis Protocol
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Tween 20, cholesterol, CHEMS, Sephadex G75, HEPES salt (N-(2-idroxyethyl) piperazine-N-(2-ethanesulfonic acid)), zymosan A, formalin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) salt, and Trypan blue were purchased from Sigma-Aldrich SRL (Milan, Italy). Fetal bovine serum, 0.1% trypsin/0.02% ethylenediamine tetraacetic acid, phosphate-buffered saline, and Dulbecco’s Modified Eagle’s Medium were purchased from Gibco® Cell Culture Basics (Life Technology Italia, Monza, Italy). Diphenylhexatriene was obtained from Acros Organics BVBA (Geel, Belgium). The AG was a kind gift from Bernett (Pavia, Italy). The cellulose membrane dialysis tubing was purchased from Spectrum Laboratories (Prodotti Gianni SpA, Milan, Italy). Cellulose filters (0.45 μm) were obtained from Millipore (Billerica, MA, USA). Tissue culture test plates were purchased from TPP Tissue Cell Culture Products (Delchimica Scientific Glassware, Naples, Italy). Male CD-1 mice were purchased from Harlan Laboratories (Correzzana, Italy). All other chemicals used during the experiments were of analytical grade and no additional purification was carried out before analysis. Double-distilled water was used throughout the study.
4
Preparation and Characterization of Doxorubicin-Loaded Liposomes
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DOPE and DSPE-PEG2,000 were supplied by Lipoid GmbH (Ludwigshafen, Germany). DSPE-PEG3,400-N-(N-hydroxysuccinimide) (DSPE-PEG3,400-NHS) was purchased from NanoCS (New York, NY, USA). CHEMS, sodium AL, DOX, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) sodium salt, magnesium chloride, sodium chloride, sodium hydroxide, Triton X-100, sodium dodecyl sulfate, 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum, penicillin, and streptomycin were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dimethyl sulfoxide (DMSO) and ethanol were obtained from Thermo Fisher Scientific. All other chemicals used in this study were of analytical grade. The human breast adenocarcinoma cell line (MDA-MB-231) was purchased from the American Type Culture Collection (Manassas, VA, USA).
5
Breast Cancer and Glioblastoma Cell Lines
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DOPE and DSPE-PEG2000 were supplied by Lipoid GmbH (Ludwigshafen, Germany). UA, CHEMS, phosphate saline buffer, and sodium hydroxide were obtained from Sigma Chemical Company (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), trypsin, and penicillin/streptomycin solution were purchased from Gibco Life Technologies (Carlsbad, USA). BD Matrigel was purchased from BD Biosciences (Mississauga, ON, Canada). Pellets of 17-β-estradiol (0.72 mg/pellet, 60-day release) were obtained from Innovative Research of America (Sarasota, FL, USA). Gadoterate dimeglumine (Dotarem) was purchased from Guerbet (Aulnay, France). All other chemicals and reagents used were of analytical grade. Human breast adenocarcinoma MCF-7 cell line transfected with luciferase gene and puromycin resistant was supplied by Dr Bertrand Tavitian (INSERM-U970, Paris, France). Rat gliosarcoma 9L cell line expressing the luciferase gene was supplied by Dr Alexandra Winkeler (CEA, Orsay, France). Female nude (NMRI-Foxn1nu/Foxn1nu) mice were purchased from Elevage Janvier (Le Genest-St-Isle, France).
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