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Mini-extruder

Manufactured by Avestin
Sourced in Canada

The Mini-extruder is a compact laboratory instrument designed for small-scale extrusion processes. It features a simple and intuitive operation for controlled material processing.

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17 protocols using Mini-extruder

Lipid solutions in chloroform were mixed in glass test tubes, evaporated to dryness under N2 gas, then dried 30 min under vacuum. For liposome sedimentation assays, lipid films were rehydrated under 20 mM Tris pH 7.5, 100 mM NaCl, 0.5 mM TCEP at 85°C for 1 hour, then vortexed to generate crude liposomes. Crude liposomes were frozen in liquid nitrogen and thawed at the rehydration temperature 7 times. Liposomes were used immediately or stored at −80°C. For lipid transfer assays, lipid films were rehydrated under 20 mM Tris pH 7.5, 100 mM NaCl (for light liposomes) or 20 mM Tris pH 7.5, 100 mM NaCl, 0.75 M sucrose (for heavy liposomes) at 37°C for 1 hour. Rehydrated lipids were vortexed to generate crude liposomes. Crude liposomes were frozen in liquid nitrogen and thawed at 37°C 5 times, then extruded through 100 nm (light lip.) or 400 nm (heavy lip.) pore size cellulose acetate filters in a mini-extruder (Avestin). Liposomes were clarified by centrifugation at 16,100×g for 15 min. Light liposome supernatants were transferred to a clean tube, while heavy liposome pellets were washed twice, then resuspended, all in sucrose-free buffer. Liposomes were stored at 4°C and used within 48 hours.
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To prepare nanoliposomes, cholesterol (Chol), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) (Avanti Polar Lipid; Alabaster, USA) lipids were dissolved in chloroform at the molar ratios of 15:10:75, respectively. Lipid solution was dried to a thin lipid film under reduced pressure using rotary evaporation (Heidolph, Germany). The prepared lipid film was then freeze-dried (VD-800F, Taitech, Japan) overnight to completely remove the solvent. Subsequently, the dried lipids were hydrated with 10 mM HEPES buffer (pH 7.2) containing 5% dextrose, and vortexed and bath-sonicated to disperse completely in the buffer. To obtain small unilamellar vesicles (SUVs) with a uniform size of 100–200 nm, the multilamellar vesicles (MLVs) were sequentially extruded using a mini extruder (Avestin, Canada) with polycarbonate membranes of 600, 400, 200, and 100 nm pore size, respectively. Physical properties of the prepared nanoliposomes, including particle size (diameter, nm), polydispersity index (PDI), and surface charge of the nanoliposomal formulation, were determined using dynamic light scattering (DLS) technique on a Zetasizer (Nano-ZS, Malvern, UK) at the room temperature (RT). The morphology and structure of the manufactured nanoliposomes were also visualized using a Philips CM10 transmission electron microscope (TEM).
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P5 peptide was encapsulated in liposomes using a method we optimized to gain higher encapsulation efficiency in our earlier study (16 (link)). Briefly, Phospholipid mixtures containing DMPC:DMPG: Chol:DOPE (30:4:6:10, molar ratio) were added to a glass tube from their stock chloroform solutions. Control liposomes (Lip) were also prepared in the same molar ratio as above without using DOPE. The lipid mixture was dried by rotary evaporator at 30 °C and freeze drier. Then, the lipid film was dissolved in 300 µl ethanol and 700 µl HEPES-dextrose buffer containing 10% (v/v) of DMSO. The resulting dispersion was sonicated for about 15 sec and extruded 5 times through 400 nm and 11 times through 100 nm polycarbonate membranes at 25 °C using a mini extruder (Avestin, Canada) to form 100 nm small unilamellar vesicles (SUVs) with a uniform size. 20 µl of P5 solution (10 µg/µl) in DMSO was slowly added to preformed liposomes while vortexing. Subsequently, the ethanolic mixture of liposome and P5 was incubated at 25 °C for 1 hr and then dialyzed to remove unencapsulated peptide, ethanol and DMSO. Liposomes were stored at 4° C under argon.
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Co-extrusion method was used to prepare M/PCOD@PLGA. Briefly, the harvested macrophage membrane vesicles (1 mg protein) were mixed with PCOD@PLGA (100 μg). Then, the mixture was ultrasonicated (100w, 2 min) on ice. Subsequently, the mixture solution was extruded for 10 times (400, 200 nm polycarbonate porous membrane successively) using an Avestin mini-extruder (Avestin, 610000, Canada).
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We used large unilamellar vesicles
(LUVs) composed of a mixture of zwitterionic and negatively charged
lipids (DMPC and DMPS in 3:1 ratio) or the brain total lipid extract
(bTLE). Model membranes were prepared as described elsewhere.85 (link) Briefly, appropriate aliquots of lipid stock
solutions in chloroform were dried by using a stream of dry nitrogen
and evaporated overnight under high vacuum to dryness in a round-bottomed
flask. Initially, multilamellar vesicles (MLVs) were obtained by hydrating
the lipid film with an appropriate amount of buffer (aCSF buffer 10
mM (pH 7.4) or MOPS buffer 10 mM and 100 mM NaCl (pH 7.4)) and dispersing
by vigorous stirring. MLVs were then extruded through polycarbonate
filters (pore size = 100 nm, Nuclepore, Pleasanton, CA) mounted in
a mini-extruder (Avestin, Ottawa, Canada) fitted with two 0.5 mL Hamilton
gastight syringes (Hamilton, Reno, NV) to obtain LUVs. Samples were
typically subjected to 23 passes through two filters in tandem and
as recommended elsewhere.86 (link)
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We used large unilamellar vesicles (LUVs) composed of zwitterionic lipids with different hydrocarbon tail length as model membranes, which were prepared as described elsewhere.18 (link),62 Briefly, appropriate aliquots of lipid stock solutions in chloroform were dried by using a stream of dry nitrogen and evaporated overnight under high vacuum to dryness in a round-bottomed flask. Multilamellar vesicles (MLVs) were obtained by hydrating the lipid film with an appropriate amount of phosphate buffer (10 mM buffer, 100 mM NaCl, pH 7.4) and dispersing it by vigorous stirring. LUVs were obtained by extruding MLVs through polycarbonate filters (pore size = 100 nm, Nuclepore, Pleasanton, CA) mounted in a mini-extruder (Avestin, Ottawa, Canada) fitted with two 0.5 mL of Hamilton gastight syringes (Hamilton, Reno, NV). Samples were typically subjected to 23 passes through two filters in tandem and as recommended elsewhere.63
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MM/RAPNPs were fabricated by coating RAPNPs with MMs by a direct extrusion method. Briefly, MM vesicles and RAPNPs were mixed at a membrane protein-to-polymer ratio of 1:1 (w/w) and sonicated for 3 min in a sonicator bath (FS30D, 42 kHz, 100 W). The mixture was then extruded 10 times through a 200 nm polycarbonate porous membrane using an Avestin mini extruder (Avestin, LF-1, Canada) to harvest the MM/RAPNPs.
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OEMs were prepared as previously described.23 (link) OEMs and ETBNPs were co-cultured for 30 min at 37 °C to harvest OEM-ETBNPs. For OEM-ETBPD synthesis, OEMs and ETBPD were co-extruded using an Avestin mini-extruder (Avestin, LF-1, Canada, 100 nm polycarbonate porous membrane) for 10 times to harvest OEM-ETBPD.
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We used large unilamellar vesicles (LUVs) composed of TLBE or DMPC/DMPS (7/3 molar ratio). Model membranes were prepared as described elsewhere [47 (link)]. Briefly, aliquots of lipid stock solutions in chloroform were dried by using a stream of dry nitrogen gas and evaporated under high vacuum to dryness in a round-bottomed flask. To obtain multilamellar vesicles (MLVs), the resulting lipid film was hydrated with an appropriate amount of phosphate buffer (10 mM buffer, 100 mM NaCl, pH = 7.4) and dispersed by vigorous stirring in a water bath. LUVs were obtained by extruding MLVs through polycarbonate filters (pore size = 100 nm, Nuclepore, Pleasanton, CA) mounted in a mini-extruder (Avestin, Ottawa, ON, Canada) fitted with two 0.5 ml Hamilton gastight syringes (Hamilton, Reno, NV). Samples were typically subjected to 23 passes through two filters in tandem and as recommended elsewhere. An odd number of passages were performed to avoid contamination of the sample by vesicles that might not have passed through the filter.
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RBC membrane vesicles were prepared as previously described.[7, 30] RBC vesicles and LVTNPs were cocultured for 30 min at 37 °C to harvest RBC‐LVTNPs. For eRBC‐LVTNPs synthesis, RBC vesicles and LVTNPs were coextruded using an Avestin mini‐extruder (Avestin, LF‐1, Canada, 100 nm polycarbonate porous membrane) for 10 times to harvest eRBC‐LVTNPs.
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