Liposomes containing M40401 were prepared using a mixture of distearoyl phosphatidylcholine (DSPC) and PEGylated distearoyl phosphatidyl-ethanolamine (DSPE)-PEG(2000) (90:10 mol/mol). Lipids were dissolved in chloroform and a thin lipid film was created by rotary evaporation. Water solutions of M40401 were added to the film and the lipids were hydrated for 60 min with the formation of multilamellar vesicles at 65°C. Unilamellar liposomes were formed by extruding the liposomes using Liposo-Fast (Avestin) assembly through stacked Nuclepore membranes with a 200 nm pore size at above the phase transition temperature 20 times. Unencapsulated M40401 was removed using a PD-10 Sephadex G-25 size exclusion spin-column (GE Healthcare) equilibrated with DPBS buffer, pH 6.8.
Liposofast
Manufactured by Avestin
Sourced in Canada
The LiposoFast is a compact and versatile instrument designed for the extrusion of lipid vesicles (liposomes). It utilizes an extruder and a mini-extruder to produce unilamellar liposomes with a defined and reproducible size distribution. The device is suitable for a wide range of applications, including drug delivery, membrane research, and the preparation of liposomal formulations.
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Lab products found in correlation
Tl100.1 rotor, by Beckman Coulter (4 mentions)
Bio beads sm 2, by Bio-Rad (4 mentions)
1 2 dioleoyl sn glycero 3 phospho 1 myo inositol 4 5 bisphosphate pi 4 5 p2, by Avanti Polar Lipids (1 mentions)
Zetasizer ultra red, by Malvern Panalytical (1 mentions)
Malvern zetasizer, by Malvern Panalytical (1 mentions)
Polycarbonate membrane, by Merck Group (1 mentions)
Phosphatidylserine, by Avanti Polar Lipids (1 mentions)
Phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Chloroform, by Büchi (1 mentions)
Rotary evaporator, by Büchi (1 mentions)
Zetasizer 3000hs, by Malvern Panalytical (1 mentions)
Membrane protein extraction kit, by Beyotime (1 mentions)
Octet htx, by Molecular Devices (1 mentions)
38 protocols using liposofast
1
Liposomal Encapsulation of M40401
2
FAAH-ΔTM Activity Assay Using Radiolabeled AEA
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All reagents used in this work were of the highest analytical grade. [3 (link)H]-AEA used for activity assay was purchased from Larodan Fine Chemicals AB (Malmo, Sweden). The LiposoFast apparatus for liposome formation was purchased from Avestin (Ottawa, Canada). pTrcHisA-FAAH-ΔTM was kindly provided by Prof. Benjamin F. Cravatt (Cellular Biology Department, University of San Diego, La Jolla, California). All other reagents were from Merck KGaA (Darmstad, Germany).
3
Preparation of Lipid Vesicles for Experiments
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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-4′,5′-bisphosphate) (PI(4,5)P2), and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid) succinyl] (DGS-NTA(Ni)) lipids were purchased from Avanti Polar Lipids (Alabaster, Al, USA). Dipalmitoyl phosphatidylinositol 3-phosphate (PI3P) lipids were from Echelon Biosciences Incorporated (EBI). Lyophilized lipids were dissolved in 1:1 (v/v) chloroform: methanol mixed in glass flasks in the desired amount. The organic solvent was first evaporated under a gentle stream of nitrogen while gently turning the flask to form a thin lipid film onto the wall of the flask. The lipid film was further dried with nitrogen for at least 30 min. Lipid films were hydrated in a buffer containing 10 mM Tris–HCl (pH 7.5) (Merck), and 150 mM NaCl at a final concentration of 2 mg/ml. After vortexing for 30 min, the resulting multilamellar vesicle solutions were extruded 25 times through a polycarbonate membrane with 50 or 200 nm nominal pore diameter (LiposoFast, Avestin) leading to a stock solution of unilamellar vesicles, which were stored at 4°C and used for 5 d at most.
4
Evaluating Liposome Membrane Elasticity
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The membrane elasticity of the different surface-charged liposomes was evaluated by previously reported method [25 (link)]. Briefly, the liposomal dispersions were continuously extruded (LiposoFast, Avestin, Ottawa, ON, Canada) at room temperature through a 100 nm pore-size membrane (rp) for 5 min by applying an external pressure of 2.5 bar, and the average diameter of the liposomes following extrusion (rv) as along with the mass of the extruded liposomes (J) were determined. For the assessment of AZT-liposomes, the non-encapsulated drug was pre-separated from the liposomally-encapsulated AZT by ultracentrifugation (2.7). A calculation of the degree of bilayer elasticity (E) was performed using the following equation:
rv of the different liposomes was determined by dynamic light scattering on Zetasizer Ultra Red (Malvern Panalytical Ltd., Malvern, UK) at scattering angle of 90°. The liposomal dispersions were properly diluted by PB, pH 7.5 and the measurements were performed at 25 °C using disposable DTS0012 cuvettes. The results are expressed as the mean ± S.D (n = 3).
rv of the different liposomes was determined by dynamic light scattering on Zetasizer Ultra Red (Malvern Panalytical Ltd., Malvern, UK) at scattering angle of 90°. The liposomal dispersions were properly diluted by PB, pH 7.5 and the measurements were performed at 25 °C using disposable DTS0012 cuvettes. The results are expressed as the mean ± S.D (n = 3).
5
Liposome-Mediated Antisense Delivery
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DOTAP, DOPE, and PEG were dissolved in chloroform. Dried lipid film was formulated by exposing the lipid mixture to a slow N2 stream for evaporation of chloroform and then to a vacuum system overnight to ensure complete removal of chloroform traces. The lipid film was hydrated using sterile PBS with pH 7.4 at room temperature. The suspension was left at room temperature for 1 hour to allow assembly and formulation of liposomes. Liposomes were exposed to freeze/thaw cycles (7–9 cycles) to facilitate size management using liquid nitrogen and warm tap water. The size was homogenized using manual graded extrusion with 800‐, 400‐, and 100‐nm polycarbonate membranes (LiposoFast; Avestin, Inc). The final size of the liposomes was 100 to 200 nm with a polydispersity index of 0.1 measured using a dynamic light scattering technique (Malvern Zetasizer; Malvern Instruments). Antisense or sense of Giα‐2 or Giα‐3 was added to the final concentration of 200 μg/mL in sterile PBS with pH 7.4 and an N:P ratio of 2 calculated according to the Felgner equation.44 Lipoplexes were left for 1 hour at room temperature to allow encapsulation of antisense or sense. To ensure that antisense was encapsulated inside the liposome, fluorescently labeled antisense was used.
6
Rat GLUT5 Proteoliposome D-Fructose Uptake Assay
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The proteoliposome D-fructose uptake assay was modified from that previously described for human GLUT146 (link). In brief, purified rat GLUT5 was reconstituted by the freeze–thaw/extrusion method. Crude lipids were extracted from bovine liver and sonicated to make unilamellar liposomes. 500 μL of a mixture containing ~10 μg of purified GLUT5 and 20 mg of liposomes in 10 mM TrisSO4 (pH 7.5) was flash frozen and thawed at room temperature. Large, unilamellar proteoliposomes were prepared by extrusion (LiposoFast, Avestin; membrane pore size, 400 nm). For each time point, 10 μl proteoliposomes (0.4 mg lipid; 0.2 μg GLUT5) was added to 10 μl transport buffer containing 10 mM TrisSO4 and 2 mM MgSO4 (pH 7.5) with or without the addition of 0.5 mM HgCl2. Time course of 0.1 mM [14C]-D-fructose transport was measured at 25 °C at indicated time intervals and stopped by the addition of cold buffer containing 10 mM TrisSO4, 2 mM MgSO4 (pH 7.5) and 0.5 mM HgCl2 and immediately filtered. Non-specific uptake was estimated with 0.1 mM [14C]-L-glucose. The radioactivity corresponding to the internalized substrate was measured by scintillation counting. Each experiment was performed in triplicate and data-points shown indicate average values of two technical replicates.
7
Rat GLUT5 Proteoliposome D-Fructose Uptake Assay
8
Unilamellar Vesicle Preparation by Lipid Film Hydration
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Unilamellar vesicles (roughly 100 nm diameter) were obtained by the following procedure: lipid powders (d 75 -DPPC or d 62 -DPPC:GM1 10:1 mol) were weighted in glass ball-shaped containers, dissolved the appropriate organic solvent, then evaporated under continuous rotation so that lipid films were deposited over the balloons surface. Chloroform evaporation was completed under vacuum for 30 minutes. Then the films were submitted to a gentle stream of humidified nitrogen for 30 minutes, to disentangle multilayer compact stacks. Finally, 150 mM NaCl water solution was added, to the final concentration of 0.5 mg/ml. The d 62 -DPPC:GM1 10:1 mol system spontaneously forms unilamellar vesicles, whereas the d 75 -DPPC multilamellar system was extruded through twinned polycarbonate filters (800 Å porosity) with a manual extrusor (LiposoFast, Avestin Inc.). Samples were then stored at 45°C, above the chain gel-to-fluid transition, to ensure vesicle stability. GM1 micelles were prepared by dissolving 1 mg of GM1 powder in 1ml of pure water (MilliQ).
9
Ethosomal Delivery of SPACE-Peptide and GAPDH-siRNA
10
Liposome Preparation and Characterization
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Lipid stock solutions were prepared in chloroform and quantified by measuring phosphate concentration [35 (link)]. Lipid films were prepared by mixing an adequate volume of each lipid stock solution, followed by solvent evaporation under a stream of argon, and complete drying under vacuum for at least two hours.
Lipid films were suspended in aqueous Tris-HCl 10 mM, pH 7.4, buffer solution yielding multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) with a hydrodynamic diameter of 100 nm were obtained by extrusion of MLV through polycarbonate membranes (Millipore, MA, USA) in a LiposoFast syringe-driven extruder (AVESTIN, Ottawa, Canada).
For the CF leakage assay, LUV were prepared in 10 mM Tris-HCl buffer, pH 7.4 containing CF 50 mM. Bulk solution-free CF was separated from the LUV by size-exclusion chromatography using a prepacked Sephadex G-25 filter column (GE Healthcare, Buckinghamshire, UK) equilibrated with 10 mM Tris-HCl buffer, pH 7.4, with 300 mM NaCl. The collected lipid suspension was quantified by measuring phosphate [35 (link)].
Lipid films were suspended in aqueous Tris-HCl 10 mM, pH 7.4, buffer solution yielding multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) with a hydrodynamic diameter of 100 nm were obtained by extrusion of MLV through polycarbonate membranes (Millipore, MA, USA) in a LiposoFast syringe-driven extruder (AVESTIN, Ottawa, Canada).
For the CF leakage assay, LUV were prepared in 10 mM Tris-HCl buffer, pH 7.4 containing CF 50 mM. Bulk solution-free CF was separated from the LUV by size-exclusion chromatography using a prepacked Sephadex G-25 filter column (GE Healthcare, Buckinghamshire, UK) equilibrated with 10 mM Tris-HCl buffer, pH 7.4, with 300 mM NaCl. The collected lipid suspension was quantified by measuring phosphate [35 (link)].
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