Track etch membrane

LUVs that contain DOPC or DOPC plus 0.1% Lipid II were prepared for a carboxyfluorescein (CF) leakage assay. The lipids were dried by a nitrogen stream and followed under vacuum for 2 h. After that, the lipids were hydrated by adding 25 mm Tris-HCl, 150 mm NaCl, and pH 7.5 containing 50 mm CF. The suspensions were frozen and thawed 10 times, followed by extrusion through 200nm membrane filters (Whatman Nuclepore, Track-Etch Membranes) 10 times. The excess dye was removed by loading vesicles on a spin column (Sephadex G50) for 2 min at 500 × g. Subsequently, the vesicles were diluted to 5 μm, and the brevibacillins-induced release of CF from the vesicles was monitored by measuring the increase in fluorescence intensity. The maximum fluorescence was reached by adding 10 μl of 20% Triton X-100. A Cary Eclipse Fluorescence Spectrophotometer (Agilent, United States) was used to determine the changes of fluorescence signals, and the excitation wavelength and emission wavelength were adjusted to 492 nm and 515 nm, respectively.

LUVs containing Lipid II (2%, mol/mol) were prepared by mixing the appropriate volumes of Lipid II and DOPC stock solutions in CHCl₃/MeOH (2:1, v/v). The lipid solutions were dried by a nitrogen stream and hydrated with 10 mm Tris-HCl, 100 mm NaCl, and pH 8 buffer to a lipid-phosphate concentration of ~ 20 mm. LUVs were obtained after 10 times freeze-thaw cycles followed by 10 rounds of extrusion through 200 nm membrane filters (Whatman Nuclepore, Track-Etch Membranes). The concentration of lipid-phosphate was determined as described (Rouser et al., 1970 (link)).
Isothermal titration calorimetry was performed with the Low Volume Nano ITC (Waters LLC, New Castle, DE, United States) to determine the interaction between LUVs and brevibacillins. Brevibacillins were diluted in a buffer (10 mm Tris-HCl, 100 mm NaCl, and pH 8) to a final concentration of 50 μm. Samples were degassed before use. The chamber was filled with 177 μl of the brevibacillins solutions, and the LUVs were titrated into the chamber at a rate of 2 μl/300 s with a stirring rate of 300 rpm. Experiments were performed at 25°C. Control experiments were performed with Lipid II-free LUVs. The Kd values of brevibacillins to Lipid II were calculated using the Nano Analyze Software (Waters LLC).

Cell plasma membrane mixed with 300 μg of DSPE-PEG2000 (Nanocs, catalog no. 121109) were physically extruded through a 200-nm Track-Etch membrane (Whatman, catalog no. 800281) for ten passes. PPDCNPs were coated in the membrane vesicles by an extruder set with holder block (Avanti, catalog no. 610000) through a 200-nm Track-Etch membrane to form m-PPDCNPs.