The composition of the lipid mix was 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA[Ni2+]), and 1.1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiD; 79.5:15:5:0.5 mol%). Appropriate volumes of lipid stocks (Avanti Polar Lipids, Alabaster, AL) were aliquoted into a glass vial, diluted to a final concentration of 1 mM in chloroform, and stored at −80°C. Before use, the vial was brought to room temperature. A small aliquot (1–5 nmol of total lipid) was spread on a PEGylated glass coverslip (Dar et al., 2015 (link)) and assembled in a flow cell that was filled with filtered and degassed assay buffer. The flow cell was left undisturbed for 10 min at room temperature before flowing assay buffer to remove excess membrane reservoir.
Lipid stocks
Manufactured by Avanti Polar Lipids
Sourced in United States
Lipid stocks are a collection of different lipid compounds commonly used in research laboratories. These stocks provide a convenient way to obtain a variety of lipids for various experimental purposes. The core function of lipid stocks is to serve as a source of specific lipid molecules for use in research, analysis, and development activities.
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4 protocols using lipid stocks
1
Reconstitution of Membrane Proteins
2
Supported Membrane Tube Formation
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Appropriate volumes of lipid stocks (Avanti Polar Lipids) were aliquoted into glass vials, diluted to a final concentration of 1 mm in chloroform, and stored at −80 °C. lipid stocks contained a trace amount (0.5 mol%) of the fluorescent DiD (Invitrogen) lipid probe. Stocks were brought to room temperature before use. A small aliquot (∼1–5 nmol of total lipid) was spread on a PEGylated glass coverslip and kept under high vacuum for 5 min to remove all traces of chloroform. A ∼35-μl flow cell (Bioptechs) was assembled by placing a 0.1-mm silicone spacer between the PEGylated coverslip and an indium tin oxide-coated slide. The flow cell was filled with filtered and degassed HKS containing 1% w/v BSA (Sigma) and left undisturbed for 10 min at room temperature. Hydration of the dry lipid causes the formation of large vesicles inside the chamber. Supported membrane tubes are created by extrusion of the large vesicles to narrow membrane tubes by flowing excess HKS buffer containing 1% BSA at high (∼30 mm/s particle velocity inside the chamber) flow rates. SMrT templates were judged ready for experiments when the entire membrane reservoir was extruded into tubes that remained lightly tethered to the surface, even in the absence of external buffer flow.
3
Lipid Bilayer Preparation and Functionalization
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BPLE (141101; Avanti Polar Lipids, Alabaster, AL) or Folch lipids (B1502; Sigma Aldrich, St. Louis, MO) were used as is. Lipid stocks (Avanti Polar Lipids) were aliquoted in the following proportions to generate PIP2-, NTA-, or PIP2 plus NTA–containing mixtures: DOPC:DOPS:DOPIP2 (84:15:1 mol%), DOPC:DOPS:NTA (80:15:5 mol%), and DOPC:DOPS:DOPIP2:NTA (79:15:1:5 mol%). When necessary, trace amounts of the fluorescent lipid probe p-Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (DHPE) was incorporated to a final concentration of 1 mol%. For liposomes, dried lipid mixtures were hydrated in assay buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 150 mM KCl) to a final concentration of 1 mM and extruded through 100-nm polycarbonate membranes (Avanti Polar Lipids). SUPER templates were prepared with PIP2- or NTA-containing liposomes as previously reported (Pucadyil and Schmid, 2010 (link)). SMrT templates were prepared as described previously (Dar et al., 2015 (link), 2017 ).
4
Targeted Microbubble-Liposome Complexes for Cancer
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Microbubble-liposome complexes were prepared as previously described (7 (link)). Lipid stocks (Avanti Polar Lipids, Albaster, AL, USA) comprising 15.4 mg of 1,2-dipalmitory-sn-glycero-3-phosphatidylcholine, 3.5 mg of cholesterol, 1 mg of dicetyl phosphate, 1.2 mg of 1,2-dipalmitory-sn-glycero-3-phosphoethanolamine, and 5 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(PDP[polyethylene glycol]-2000) were dissolved in 5 mL of 99.9% chloroform (Sigma-Aldrich). The mixture was lyophilized for 24 hours to remove chloroform, and mixed with 2 mL of solvent comprising glycerin, propylene glycol, and H2O (1:2:7). Cores of synthesized microbubbles were filled with sulfur hexafluoride gas (SF6). Liposomes were formed by freezing and thawing the mixture 5 times using liquid nitrogen, followed by agitation in a sonicator at 60℃ for 5 minutes with 2 mL of H2O. The resultant multilamellar vesicles were extruded using polycarbonate filters (filter size, 200 nm) to obtain liposomes smaller than 200 nm. Microbubbles and liposomes were shaken together at 25℃ for 2 hours to form MLC. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (5 mg; Sigma-Aldrich) was added to MLCs, followed by conjugation with anti-Her2 antibodies (Herceptin®; Roche, Berlin, Germany) by shaking at 4℃ for 24 hours to allow MLCs to target Her2-expressing prostate cancer cells (12 (link)).
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