NP Characterization: NP Morphology, Size, BAR Conjugation

Unhydrated NP morphology, diameter, and size distribution were determined using scanning electron microscopy (SEM) (XL-30 ESEM-FEG SEM, FEI Company, USA). Lyophilized NPs were mounted on carbon tape and sputter coated with a thin layer of gold/palladium. Average diameters of 500 particles were determined from SEM images (n=3) using image analysis software (ImageJ, National Institutes of Health, version 1.5a, ImageJ.nih.gov). Dynamic light scattering and zeta potential analyses were performed to determine the hydrodynamic diameter and surface charge of hydrated NPs. The unhydrated and hydrated diameters of NPs are typically assessed to establish the size characteristics within different conditions of dry storage and more physiologically relevant aqueous environments. Briefly, a 1 mg/mL sample of BAR-modified PLGA NPs in diH2O was prepared. After vortexing and sonication, samples were diluted at a 1:50 ratio in diH2O. One mL was aliquoted to the cuvette for analysis [Malvern, Malvern, UK (Zetasizer Nano ZS90), courtesy of Dr. Martin O’Toole, Univ. of Louisville] to measure dynamic light scattering and zeta potential with Zetasizer Nano software. Samples were run in triplicate, using a refractive index of 1.57 for PLGA, absorption coefficient of 1, and water refractive index of 1.33. The equations used by the Zetasizer to calculate nanoparticle size are shown in Supplementary Data.

To measure the amount of BAR peptide that was conjugated to the NP surface, a fluorescence binding assay was conducted with F-BAR NPs. After conjugation, NPs were centrifuged and washed twice with diH2O to remove unbound BAR from the formulated NPs. NPs were then suspended in 1X PBS to create a 1 mg/mL NP solution and the resulting samples were transferred to a microtiter plate in triplicate. Total NP-associated fluorescence was determined using Victor3 Multilabel spectrophotometer (488/518 nm excitation/emission), and peptide quantity was determined from a standard curve of known F-BAR concentrations [35]. The stability of the avidin palmitate interaction with the NP surface has been previously tested by assessing the release of avidin and biotinylated ligand from the NP surface with respect to time [37, 41]. In addition, the functional stability of BNPs was tested through in vitro inhibition assays against biofilms prior to these in vivo experiments [35].