Experimental data
Raw data for scanning electron microscopy (SEM), confocal microscopy, flow cytometry experiments and code for the theoretical modelling simulation from the paper entitled "Engineering a nanoantibiotic system displaying dual mechanism of action" published in Proceedings of the National Academy of Sciences (PNAS). All other data are included in the manuscript and/or SI appendix.
Steps to reproduce
Computational modelling: theoretical modelling was performed using a molecular theory (MOLT) for peptide amphiphile self-assembly. MOLT explicitly incorporates the chemical structure of the different molecules found in the system (at a coarse grain level), as well as the different interactions and the presence of acid-based chemical equilibrium. As an output, the theory provides structural and thermodynamic information about the system. The code is provided here, and the references and theoretical approaches are detailed in the SI Appendix of the paper "Engineering a nanoantibiotic system displaying dual mechanism of action" from PNAS.
Scanning Electron Microscopy (SEM) procedure: 20 mL of A. baumannii (A.b) or MRSA JE2 at 108 cells/mL were treated with C16uK2, LJC 1-26, the Combination or the Encapsulation solutions for 2 h at 37 ˚C while shaking at 75 rpm. The suspension was washed with saline and was fixed by immersion in a solution of 2% glutaraldehyde, 2% paraformaldehyde in a 0.1M Sorenson’s phosphate buffer (pH 6.2) for a minimum of 24 h at 4 °C. Samples were then washed three times with phosphate buffer to clear excess fixative. They were post-fixed in a 1% aqueous solution of osmium tetroxide for 30 minutes to aid in conductivity. Subsequently, samples were dehydrated in a graded ethanol series (50, 70, 90, 95, 100% x3). Following dehydration, samples were dried at critical point and attached to aluminum SEM stubs with double-sided carbon tape. Silver paste was applied to increase conductivity. The following day, samples were coated with ~50nm Gold-Palladium alloy in a Hummer VI Sputter Coater (Anatech USA) and imaged at 30kV in a FEI Quanta 200 SEM operating in high vacuum mode.
Confocal Microscopy (membrane analysis): 2 mL of A. baumannii (A.b) or MRSA JE2 at 108 CFU/mL was treated with C16uK2, LJC 1-26, combination, and encapsulation strategies for 2 h at 37 ˚C with shaking (75 rpm). The bacteria suspension was centrifuged at 4500 rpm for 15 min and stained with the membrane staining FM4-64 (final concentration 1.5 µM) and Syto 9 (final concentration 1.0 µM) for a final volume of 0.2 mL in PBS. 10 µL stained solution was dropped in the center of a 1.5% agarose pad mounted on glass slides, covered with coverslips, and then sealed with nail painting. Images were acquired on a Zeiss AxioImager.Z2 equipped with an Apotome2 using a 100X lens objective.
Flow cytometry: A. baumannii (A.b) at 106 CFU/mL was treated with C16uK2, LJC 1-26, Combination, Encapsulation, and polymyxin B at 37 ˚C, 75 rpm. At 6 h of treatment, 8 mL of bacteria was taken from the culturing solution. The bacteria suspension was washed with PBS and then stained with DAPI and PI (total volume 1 mL, final concentration at 10 µg/mL and 1 µg/mL respectively) at room temperature for 30 mins before scanning. For machine calibration, an unstained bacteria suspension, a DAPI single-stained suspension, and a PI single-stained suspension were used (106 CFU/mL). Further, a dead bacteria control was used (death induced by heat-shock, 70 ˚C for 30 mins). DAPI permeates into the cells regardless of their viability and intercalates to the DNA, resulting in a blue-fluorescent complex. PI is a fluorescent dye that is not permeable into live cells. If the plasma membrane is damaged, PI enters the cell, binds to the DNA, and forms a bright red fluorescent complex. Dead cells (PI positive) were distinguished from live cells with intact membranes (PI negative) by PI uptake versus exclusion. Samples were analyzed using a LSR II flow cytometer (BD Biosciences).
