Chromatographically pure EYPC was isolated and purified from hen eggs according to 36 (link) as modified in. 37 ULL were prepared by extrusion using LiposoFast Basic Extruder and 100 nm polycarbonate filter purchased from Avestin Europe (Germany) as described in. 38 (link) CHOL and C 12 NO were purchased from Sigma Aldrich (Germany). NaCl, NaOH and KH 2 PO 4 were obtained from Centralchem (Slovakia) and K 2 HPO 4 from Lachema (Czech Republic). Calcein, also known as fluorexon, was purchased from Acros Organics (USA). Redistilled water was prepared before use. All chemicals used, except of EYPC, were of the analytical grade. Sephadex™ G-50 (fine) (Pharmacia, Fine Chemicals AB, Sweden), Whatman GF/B glass microfiber filter (GE Healthcare, UK), 5 ml disposable syringes and 15 ml disposable polypropylene centrifuge tubes were used for column preparation. Quartz cells were purchased from Hellma Müllheim (Germany).
Liposofast basic extruder
Manufactured by Avestin
Sourced in Canada, Germany
The LiposoFast-Basic extruder is a lab equipment product designed for the extrusion of liposomes and other lipid-based vesicles. It allows for the controlled and uniform size reduction of these vesicles through a polycarbonate membrane.
Automatically generated - may contain errors
Lab products found in correlation
Lipid mixture, by Merck Group (2 mentions)
Whatman gf b glass microfiber filter, by GE Healthcare (1 mentions)
Calcein, by Thermo Fisher Scientific (1 mentions)
Hi 190m, by Hanna Instruments (1 mentions)
Soybean phosphatidylcholine, by Lipoid (1 mentions)
Tween 85, by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Membrane and cytosol protein extraction kit, by Beyotime (1 mentions)
17 protocols using liposofast basic extruder
1
Liposome Preparation and Purification
2
Preparation of Large Unilamellar Vesicles
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Large unilamellar vesicles (LUVs) of approximately 100 nm of diameter were prepared by extrusion [72 (link),73 (link)]. Lipids were dissolved in chloroform; the solvent was removed under a nitrogen steam and the lipid film was placed on a vacuum pump overnight. In the case of the composition containing lipopolysaccharide (LPS), film formation was obtained dissolving LPS in chloroform:methanol (2:1), and the solution was vortexed and bath-sonicated at 40 °C for 15 min [73 (link)]. The resulting lipid films were then rehydrated with buffer, frozen and thawed, and extruded through a membrane with a pore size of 100 nm (Whatman, Florham Park, NJ, USA), using a LiposoFast-Basic extruder (Avestin Europe, Mannheim, Germany)56. Information on the compositions used are shown in Table 2 .
3
Optimization of Liposomal Composition
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To identify the optimal composition of liposomes, the concentration of the components was varied over a wide range: 15 mM, 20 mM, and 30 mM (toward PC/Chol or PC/Chol/TOC) (Table 4 ). The PC/TPPB-14 ratio in all cases was 50/1. The lipid film hydration method was chosen as a method for the preparation of liposomes. The corresponding weights of lipids were dissolved in 100 µL of chloroform. To include TOC and TPPB-14 in the lipid bilayer, their stock solutions (in ethanol and chloroform, respectively) were prepared and dosed to the lipid mixture to obtain the appropriate concentrations (Table 4 ). The organic solvents were then evaporated on a rotary evaporator RE-52AA (Shanghai Jingke Scientific Instrument Co., Ltd., Shanghai, China) under vacuum until a lipid film formed. The final film was hydrated with Milli-Q water (in the case of empty liposomes) or an aqueous solution of DNP (in the case of drug-loaded liposomes). In both cases, the liposome dispersions were frozen in liquid nitrogen and thawed in a water bath (5 cycles). The size of the resulting liposomes was controlled by passing the dispersions through a polycarbonate membrane with a pore size of 100 nm using an LiposoFast Basic extruder (Avestin, Ottawa, ON, Canada). Liposomes were stored at 4 °C.
4
Encapsulation of Anti-miR-155 Oligonucleotides in Lipid Particles
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Following published procedures [23 (link)], 13 μmol of a lipid mixture (Sigma Aldrich) in 200 μL of 100% ethanol was slowly added under strong vortex to 0.086 μmol of anti-miR-155 oligonucleotides (Beijing Yijie Biotech; Beijing, China) in 300 μL of 20 mM citrate buffer (pH 4) (the solutions were heated to 60°C). The resulting particles were extruded 13 times through a polycarbonate membrane (100-nm-diameter) using a LiposoFast basic extruder (Avestin, Toronto, Canada). A Sepharose CL-4B column was prepared and equilibrated with HBS pH 7.4. The samples were passed through the column to remove ethanol and nonencapsulated anti-miR-155. The total lipid concentration was assessed by cholesterol quantification using the Liebermann–Burchard test [24 (link)].
5
Liposome Preparation for S. aureus
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Liposomes were prepared as described previously, with minor modifications (41 (link)). The phospholipids (Avanti Polar Lipids) were resuspended in chloroform and mixed in a molar ratio that mimicked the composition of the cell membrane of S. aureus (41 (link)): phosphatidyl-ethanolamine, phosphatidyl-dl -glycerol, phosphatidyl-choline, and cholesterol in a molar ratio of 5:6:2:9. The chloroform-liposome mixture was dried for 4 hours in a 200-ml evaporation flask in a rotatory vacuum evaporator at 37°C and 20 rpm to create a homogenous lipid film. The lipid film was resuspended in phage buffer [50 mM tris (pH 8.0), 10 mM CaCl2, and 10 mM NaCl], flash-frozen and thawed five times, and sonicated for 120 s using an ultrasonic cleaner (Sonica). Subsequently, the solution was passed 20 times through a membrane with 400-nm pores in the LiposoFast Basic Extruder (Avestin).
6
Multilamellar Vesicle Preparation and Photobleaching Analysis
7
Preparation of Biotinylated and Fluorescent Liposomes
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Vesicles were prepared by the lipid film hydration and extrusion method. Appropriate volumes of chloroform solution of DOPG or DOPS were transferred to a round-bottomed flask. Biotinylated vesicles were prepared by mixing 1% of the total lipid mass of DSPE-PEG(2000) Biotin (dissolved in methanol) with DOPG or DOPS in the organic solution. In the case of fluorescently labeled vesicles, rhodamine DHPE was also added (1 wt %) to the vesicles. The organic solvents were removed by rotary evaporation, and the resultant film was dried under vacuum for at least 3 h, hydrated with 20 mM phosphate buffer, 1 mM EDTA, pH 6.5, and vortexed for 10 min. The size of the vesicles was reduced by extrusion using Avestin LiposoFast-Basic extruder and 100-nm-pore-size polycarbonate membranes.
8
Liposome Preparation with Surface-Active Agents
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MLV, LUV and SUV liposomes with incorporated SA were prepared according to the thin-film hydration method, as previously described (Milenkovic et al., 2013 (link), Petrović et al., 2014 ) with slight modifications. The PC lipid was dissolved in chloroform at the molar concentration of 5 × 10−4 M. The SA dissolved in PBS was added to the lipid at the concentration of 1 × 10–5 M, considering the final buffer volume of liposome suspension. The obtained liposome dispersions after preparation procedure were MLVs. In order to obtain LUVs and SUVs, the liposome dispersion was vortexed (HI 190M, Hanna instruments, Italy) at a speed of 500 rpm, and then extruded (LiposoFast-Basic Extruder, Avestin, Inc. Canada) through poly-carbon filters of 400 and 100 nm pore size respectively. UV–Vis absorption spectroscopy was used to rapidly estimate the dimension and lamellarity of liposomes with incorporated SA. All the operations have been performed above the critical temperature (Tc) of lipid in order to avoid defects. After preparation, all dispersions were stored at 4 °C and after 24 h their characterization was performed. The spectral data have been processed by using the software Origin 8.0.
9
Liposomal Delivery of Lipophilic Compounds
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Liposomes were obtained by lipid film hydration method according to the algorithm published in [88 (link)] at a PC/Chol molar ratio of 9/1 (total concentration was 15 mM). Liposomes were modified by incorporating TPPB-n and IA-n(OH) with 10, 12, 14, and 16 carbon atoms in hydrocarbon tail into the lipid bilayer at surfactant/lipid ratio of 50/1, 35/1, and 25/1. ROT was also incorporated into the lipid bilayer at the stage of lipid film formation. The lipid film was then hydrated with Milli-Q water, incubated in a water bath at 60 °C within 1 h, followed by 5 cycles of freezing and thawing using liquid nitrogen. The resulting dispersions were then extruded through a polycarbonate membrane using an LiposoFast Basic extruder (Avestin, Ottawa, ON, Canada) to obtain aggregates with diameter of 100 nm. Liposomes were stored at 4 °C.
10
Preparation of Modified Liposomes
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The preparation of modified liposomes was carried out by the method of hydration of a thin lipid film according to a previously developed technique [51 (link),52 (link),53 (link)]. Briefly: phospholipid and surfactant were taken in certain amounts and dissolved in ethanol (300 μL) to obtain a surfactant/lipid molar ratio of 0.02/1, 0.029/1, or 0.04/1. The alcohol was then evaporated on a rotary evaporator for 30 min at 40–45 °C under reduced pressure until the solvent was completely removed. The resulting fine lipid film was dispersed with water under stirring at a temperature of 60 °C for 1 h. The spontaneously formed hydrated lipid phase underwent five freeze/thaw cycles. The solution was then extruded 20 times through a 100 nm polycarbonate membrane (Whatman Nuclepore Track-Etched Membranes) using a LiposoFast Basic extruder (Avestin, Germany) to obtain unilamellar liposomes.
The preparation of liposomes loaded with substrates was carried out according to the same procedure, but the hydration of a thin lipid film was carried out with aqueous solutions of the substrates. The concentration of rhodamine B for loading into liposomes was 0.5 mg/mL, while for 2-PAM, it was 10 mg/mL or 20 mg/mL.
The preparation of liposomes loaded with substrates was carried out according to the same procedure, but the hydration of a thin lipid film was carried out with aqueous solutions of the substrates. The concentration of rhodamine B for loading into liposomes was 0.5 mg/mL, while for 2-PAM, it was 10 mg/mL or 20 mg/mL.
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