The procedure of making nanoTCEs has been previously described [16 (link)]. Briefly, nanoTCEs were prepared with three components: cholesterol, DPPC, and DSPE-PEG2000 with a molar ratio equivalent to 30: 65: 5. Lipids were mixed and solubilized in chloroform, and evaporated through a rotary evaporator (Heidolph, Schwabach, Germany) to form a thin lipid film. The film was then hydrated, and the resulting suspension was extruded using the Avestin LiposoFast LF-50 (Ottawa, ON, Canada) with 100 nm polycarbonate membranes [31 (link), 32 (link)]. The biotinylated antibodies (Isotype, CD3, and/or CD33) were conjugated to the liposomes using streptavidin and biotin reaction [33 (link)]. Malvern Zetasizer Nano ZS90 (Malvern, Worcestershire, United Kingdom) was used to determine zeta potential, diameter, and polydispersity index. Fluorescent liposomes were prepared by dissolving DiO in the lipid/chloroform mixture before film formation.
Liposofast lf 50
Manufactured by Avestin
Sourced in Canada, Germany, United States
The LiposoFast LF-50 is a laboratory instrument designed for the extrusion of liposomes and other lipid-based vesicles. It operates using a hydraulic hand-operated extruder system to produce uniform and reproducible vesicle sizes.
Automatically generated - may contain errors
Lab products found in correlation
Polycarbonate membrane filter, by Merck Group (2 mentions)
100 nm pore size membrane, by Avanti Polar Lipids (2 mentions)
Ultrafiltration centrifuge tube, by Merck Group (2 mentions)
Crl 1573, by Avestin (2 mentions)
Polycarbonate membrane filters, by Avanti Polar Lipids (2 mentions)
Nano zs90, by Malvern Panalytical (2 mentions)
Rotary evaporator, by Heidolph (1 mentions)
Nuclepore track etch membrane, by Cytiva (1 mentions)
Whatman anodisc inorganic filter membrane, by Cytiva (1 mentions)
S400 high prep column, by GE Healthcare (1 mentions)
Akta start, by GE Healthcare (1 mentions)
Ultra 15 centrifugal filter, by Merck Group (1 mentions)
Ultracel 100 membrane, by Merck Group (1 mentions)
Laborota 4000 efficient, by Heidolph (1 mentions)
Zetasizer nano zs90, by Malvern Panalytical (1 mentions)
14 protocols using liposofast lf 50
1
Fabrication of Targeted Nanoparticle Therapeutic Carriers
2
Isolation and Characterization of Membrane Particles
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MSC were collected, counted, washed twice with phosphate-buffered saline (PBS), and centrifuged at 2,000 × g for 5 min. The MSC pellet was incubated in Milli-Q water at 4°C for ~20 min to induce osmotic lysis and release of cell nuclei. This step was carefully monitored by an optical microscope and stopped when nuclei were released from the cells. Cell extracts were isolated from unbroken cells and nuclei by centrifugation at 2,000 × g for 20 min. Then, the supernatant was transferred to Amicon Ultra-15 filter tubes (100 kDa pore size) and concentrated by centrifugation at 4,000 × g for 45 min. The concentrated pellet consisted of crude membranes and was diluted in filtered PBS. To prepare a small and uniform size of MP, the membranes were extruded three times through polycarbonate membrane filters (Merck, KGaA, Darmstadt, Germany) using LiposoFast LF-50 (AVESTIN Europe, Mannheim, Germany) at 20 psi, first with a pore size filter of 800 nm, then 400 nm, and finally 200 nm. All procedures were performed on ice. To obtain fluorescent MP, MSC were labeled with the red fluorescent PKH-26 dye (PKH-MP), which intercalates into lipid bilayers, according to the manufacturer's instructions (Sigma-Aldrich), before generation of MP.
3
Azide Liposome Functionalization with HA-Alkyne
4
Carvedilol-Loaded Transfersomal Delivery
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Carvedilol loaded transfersomes were prepared using a thin-film hydration method, as described in previous reports [12 (link),13 (link)]. Briefly, lipids, surfactants, and carvedilol were solubilized in chloroform:methanol (2:1, v/v) in a round bottom flask and mixed well. The organic solvent was gradually removed under reduced pressure in a rotary evaporator at 45 °C for 30 min until a thin film was formed. The thin film was kept in a desiccator overnight to completely remove the residual solvent before it was hydrated with phosphate-buffered saline (PBS) (pH 7.4) at the transition temperature (51 °C) of the lipid components using a rotary evaporator for 30 min. For selected formulation F18 and the control transfersome without drug loading (plain transfersome), after the suspension was sonicated in a bath for 5 min, it was extruded through a 100 nm pore size membrane (Avanti Polar Lipids, Alabaster, AL, USA) through an extruder (Liposofast LF-50, Avestin, Ottawa, ON, Canada) for five cycles to obtain the final formulations.
5
Nanodecoy Derivation from Cells
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Nanodecoys were derived from LSCs or HEK293 cells (ATCC® CRL-1573™) by an extruder (AVESTIN LIPOSOFAST LF-50, AVESTIN, Inc). Cells were collected and suspended in PBS at a concentration of 5 × 106 cells/mL. A large volume of cells could be extruded immediately or stored at −80°C until ready. The cells were passed through the extruder twice through 5 μm, 1 μm, and 400 nm pore-sized polycarbonate membrane filters (Avanti Polar Lipids, Inc.) sequentially. The resulting nanodecoys were purified and concentrated using an ultrafiltration centrifuge tube (100 kDa MWCO; Millipore) and centrifuged at 4,500 g for 10 min and washed with PBS. The size and concentration of nanodecoys were measured using Nanoparticle Tracking Analysis system (Nanosight, Malvern). Nanodecoys were stored at 4°C for one week or placed in long-term storage at −80°C. The ACE2 receptors on the nanodecoys were detected using immunoblot, immunostaining, flow cytometry, and transmission electron microscopy (TEM) with immunogold labeling.
6
Nanodecoy Derivation from Cells
7
Liposome Preparation Using Pad-PC-Pad and DPPC-DSPE-PEG2000
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Two phospholipids, the commercially available natural DPPC with 5 M DSPE-PEG2000 (Lipoid, Zug, Switzerland) and the 1,3-palmitoyl-amido-1,3-deoxy-sn-glycero-2-phosphatidylcholine (Pad-PC-Pad) synthesized according to the recently reported protocol [7] , were used to prepare liposomes. Briefly, the liposomes were formulated via the standard thin-film method [34] , [35] (link) and hydrated with ultrapure water. Each suspension obtained has a lipid content of 20 mg/mL, which corresponds to the highest concentration achievable using Pad-PC-Pad. The suspensions were freeze-thawed in a twelve-step series of liquid nitrogen cooling and water bath heating (60 °C). To obtain liposomes with a diameter of about 100 nm with a narrow size distribution, multiple barrel extrusions using Liposofast LF-50 (Avestin Inc., Canada) through track-edged polycarbonate filter membranes (Whatman Nuclepore, Sigma-Aldrich, Buchs, Switzerland) were applied. The pore sizes were reduced from 400 nm (five times) via 200 nm (five times) to 100 nm (15 times).
8
Nanoparticle Isolation and Purification
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Firstly, 1–5 × 107 cells were harvested with 0.25% trypsin–EDTA (SLB57926) and washed with Phosphate Buffered Saline (PBS) (10010023, Gibco, Paisley, Scotland, UK). To disrupt the cells, they were resuspended in 5 mL PBS (10010023, Gibco, Paisley, Scotland, UK) and sonicated. Subsequently, the sonicated sample was recovered, transferred to the LiposoFast LF-50 (Avestin, York, UK) and extruded under compressed nitrogen through Nuclepore Track-Etch Membrane Whatman polycarbonate filters in a serial manner, i.e., starting with a 1.0 µm (110610, Whatman, Maidstone, UK) followed by 0.4 µm (110607, Whatman, Maidstone, UK), and finally four times through a 0.1 µm Whatman® Anodisc inorganic filter membrane (6809-6012,, Whatman, Maidstone, UK),) to yield a homogenous population size. The crude NV sample was loaded on the S400 high prep column (GE Healthcare, Uppsala, Sweden) using AKTAStart (GE Healthcare) with a 280 nm UV flow cell at 4 °C. The NVs-containing fractions were combined, filtered (0.45 µm), and concentrated with an Amicon Ultra-15 Centrifugal Filter with an Ultracel-100 membrane (UFC910024, Merck, Darmstadt, Germany). See Figure 1 a for a schematic illustration of the process.
9
Isolation and Purification of Extracellular Membrane Vesicles
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AT-MSC were trypsinized and washed twice with PBS. Then, the MSC were incubated in milliQ water at 4°C to induce osmotic lysis and liberation of the cell nuclei (after about 20 min, monitored by microscope). Cell extracts were cleared of unbroken cells and nuclei by centrifugation at 2,000 x g for 20 min. The obtained supernatant was transferred to Amicon Ultra-15 filter tubes (100 kDa pore size) and concentrated by centrifugation at 4,000 x g at 4°C. The concentrated pellet consisted of crude membrane and was diluted in 0.2 µm filtered PBS. A population of MP, homogeneous in size was obtained by extruding the plasma membranes 3 times through polycarbonate membrane filters (Merck, KGaA, Darmstadt, Germany), first with a pore diameter of 800 nm, secondly with a 400 nm and last with a 200 nm pore size filter. The extrusion process was performed using LiposoFast LF-50 (AVESTIN Europe, Mannheim, Germany) at 20 psi (Figure 1
10
Transfersomes for Topical Delivery of R-carvedilol
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Transfersomes were prepared by a thin film hydration method as described previously [18 (link)]. In brief, the lipids, surfactants and R-carvedilol (5 mg) were dissolved in chloroform:methanol (2:1, v/v). To form a thin film, the organic solvent was gradually evaporated under reduced pressure in a rotary evaporator at 45 °C for 30 min. Next, the thin film was hydrated in 10 mL PBS (pH 7.4) at 51 °C. Then, the formulation was sonicated in a water bath for 5 or 30 min before passing through a 100 nm pore size membrane (Avanti Polar Lipids, Alabaster, AL, USA) through an extruder (Liposofast LF-50, Avestin, Ottawa, ON, Canada) to reduce the particle size and obtain stable transfersomes. The plain transfersome (PT), used as no drug control, was prepared in the same way, except that no drug was added. Carbopol gel was prepared because it was previously reported in mouse studies that it increases skin retention [17 (link)]. The transfersomal formulations were mixed with 0.5% Carbopol® 934 and triethanolamine (TEA) (1:1.5, w/w) and then vortexed until a clear gel was formed.
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