The lipids used in the present study are listed in Table 4 . The lipids were dissolved in chloroform, flushed with liquid nitrogen, and kept in glass flasks at –20°C. For each experiment, 400 nmol of the respective lipid mixture or, in case of complex lipid mixtures such as total brain lipids, 500 μg of the lipid mixture was dried under nitrogen gas in a 2-mL round-bottom plastic tube and rehydrated in 25 μL of hydration buffer (as indicated below) for 1 h in a 60°C water bath. An Avanti Mini Extruder (Sigma-Aldrich, catalog no. 610020-1EA) was assembled. The turbid lipid mixture was ultrasonicated for 90 s (Branson Ultrasonic CL-40549) and filled into an extruder-compatible 1-mL Hamilton syringe (Sigma-Aldrich, catalog no. 610017-1EA). Whatman Nuclepore Track-Etched polycarbonate membranes (pore size, 0.2 μm) were inserted. The lipid mixture was equilibrated on the Mini Extruder heating bloc (Sigma-Aldrich, catalog no. 610024) for 10 min at 60°C and then pressed 21 times through the extruder. The resulting liposomes were stored at 4°C until use, with a maximum of 5 days.
Avanti mini extruder
Manufactured by Merck Group
Sourced in United States, Germany
The Avanti mini-extruder is a compact laboratory instrument used for the extrusion of various materials, including polymers, lipids, and other substances. It is designed to produce small and uniform sample sizes for research and development purposes. The core function of the Avanti mini-extruder is to apply controlled mechanical force to materials, enabling their transformation into thin, continuous sheets or films.
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5 protocols using avanti mini extruder
1
Preparation of Lipid Vesicles by Extrusion
2
Extracellular Vesicle Isolation Protocol
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To prepare ENVs, MSCs were resuspended in phosphate buffered saline (PBS, Servicebo, Wuhan, China) at 5×106 cells/mL and sequentially extruded 10 times each through 1um, 400 nm, and 200 nm polycarbonate porous membranes (Sigma‒Aldrich, St. Louis, USA) in the Avanti mini extruder (Sigma‒Aldrich, St. Louis, USA). The goal is to isolate ENVs from a mixture of cellular debris and free proteins. An iodixanol gradient solution (Axis-Shield, Dundee, UK) was performed, 50% iodixanol was placed at the bottom of an ultracentrifuge tube, overlaid with 10% iodixanol and the extruded samples, and then ultracentrifuged at 100,000 × g for 2 h at 4 °C. ENVs were obtained from the interface of the 50% and 10% iodixanol layers and further pelleted at 100,000 × g for 2 h at 4 °C.
3
Preparation of Liposomal Drug Delivery System
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The MT-LIPs were prepared by film dispersion followed by the extrusion method.42 (link) Specifically, HSPL (10 mg/mL) and cholesterol (1 mg/mL) were dissolved in chloroform separately and sonicated till dissolved. The MT was dissolved in ethanol at 1 mg/mL. The HSPL-, cholesterol-, and MT-solution were mixed in a glass tube at ratios upon the experimental design. A thin film was formed at the bottom of the tube with nitrogen treatment to remove the solvents. The liposomes were obtained by dispersing this thin film in PBS buffer and hydrated for 2 h under sonication. To enhance their uniformity, we repeatedly manual-extruded the liposome suspensions to pass through the polycarbonate membranes (pore size 0.2 μm) for 10 cycles using an Avanti mini-extruder (Merck). The TAT-modified liposome was prepared using the same method, except that TAT-PEG2000-DSPE partly replaced the HSPL at the designed proportion.
4
Preparation of Large Unilamellar Vesicles and Lipid Nanotubes
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Large unilamellar vesicles (LUVs) were prepared as previously described (46 (link)). For LUVs, 70% PS (840032C, Avanti), 10% biotinPE (870285X, Avanti), and 20% cholesterol (700000, Avanti) (w/v), and for lipid nanotubes, 40% NFA Galactocerebrosides (Sigma C1516), 40% PC (840051C, Avanti), 10% PI(4,5)P2 (524644, Calbiochem), and 10% cholesterol (700000, Avanti) (w/v) were mixed and dissolved in 250 μl of chloroform and 75 μl methanol in Mighty Vial No.01 4 ml (5-115-03, Maruemu). Then the solvent was evaporated using slow-flow nitrogen gas to produce a lipid film on the glass and then completely dried in a vacuum desiccator for 1 h. The dried lipid was rehydrated by water-saturated nitrogen gas followed by addition of 250 μl of filtered 0.3 M sucrose for 2 h at 37 °C. The resultant LUVs and lipid nanotubes were passed through 0.4 μm- and 0.2 μm-polycarbonate filters, respectively, 11 times using Avanti Mini extruder (Merck). The LUVs and lipid nanotubes (1 mg/ml of final concentration) were stored at 4 °C in dark to avoid photooxidation.
5
Preparation and Characterization of LUVs
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Large unilamellar vesicles (LUVs), with 100 nm of diameter, were prepared by extrusion of multilamellar vesicles [54 (link)]. The liposomes were prepared according to methods previously described [55 (link)]. Briefly, lipid mixtures composed of adequate amounts of lipids (POPC and POPG) were prepared in chloroform to a final lipid concentration of 2 mM. The solvent was slowly vaporized under a nitrogen flux and the resulting lipid film was left in vacuum for 3 h to ensure the complete removal of chloroform. Afterwards, the lipid was resuspended in 2 mL of DPBS (Thermo Fisher Scientific) and freeze–thaw cycles (liquid nitrogen/water bath at 60 °C) were performed to re-equilibrate and homogenize the samples. LUVs were finally obtained by extrusion of the solutions at 50 °C with an Avanti Mini-Extruder (Merck, Darmstadt, Germany) using 100 nm pore size polycarbonate membranes. All lipid stock solutions were prepared in chloroform and the respective concentrations were determined by the colorimetric quantification of inorganic phosphate.
Liposome size was determined by dynamic light scattering (DLS) using a Nanosizer ZS (Malvern Instruments). The POPC/POPG vesicles were incubated with L-OEI-h for 5 min. Data was collected at 25 °C and a backscattering angle of 173°. Two independent experiments were performed.
Liposome size was determined by dynamic light scattering (DLS) using a Nanosizer ZS (Malvern Instruments). The POPC/POPG vesicles were incubated with L-OEI-h for 5 min. Data was collected at 25 °C and a backscattering angle of 173°. Two independent experiments were performed.
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