Sa monomer

Chemicals used for monomer and polymer synthesis included sodium hydroxide, hydrobenzoic acid, dibromohexane, 1-methyl-2-pyrrolidinone, SA monomer, and triethylene glycol purchased from Sigma Aldrich (St. Louis, MO, USA). Acetic anhydride, ethyl ether, petroleum ether, chloroform, methylene chloride, hexane, acetone, sulfuric acid, potassium carbonate, dimethyl formamide, toluene, acetonitrile, N,N-dimethylacetamide, and acetic acid were purchased from Fisher Scientific (Fairlawn, NJ, USA); 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK). For ¹H NMR analysis of the copolymers, deuterated chloroform and dimethyl sulfoxide were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). The N-terminal region of a recombinant PspA (UAB055, PspA/Rx1 AA1 to 303, clade 2 PspA of the PspA family 1) was produced by Dr. David McPherson (University of Alabama at Birmingham) as described previously (55 (link)). Prior to immunization, endotoxin was removed from the protein using endotoxin removal beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions followed by dialysis and lyophilization. The final endotoxin content of the protein was determined to be less than 1.9 EU/mg as determined by a limulus amebocyte lysate chromogenic endotoxin quantification kit (Thermo Fisher Scientific).

SA monomer was purchased from Sigma Aldrich (St. Louis, MO). CPH and CPTEG monomers were synthesized as previously described^{52 (link),53 (link)}. The 20:80 CPTEG:CPH, 20:80 CPH:SA, and 10:90 CPTEG:SA copolymers were synthesized by melt condensation to number average molecular weights of approximately 7.5, 17.5, and 17.5 kDa, respectively^{49 (link),52 (link)–54 (link)}, and composition and molecular weight were confirmed by ¹H NMR analysis using a Varian MR-400 (Varian, Inc., Palo Alto, CA). Nanoparticles were synthesized by flash nanoprecipitation^{29 (link)}. Nanoparticles were collected by vacuum filtration, scanning electron microscopy (SEM, FEI Quanta 250, Hillsboro, OR) was used to image all formulations, and size distributions were calculated using Fiji image analysis software^{55 (link)}. Nanoparticle Zeta potential was measured (see below) using a Zetasizer Nano (Malvern Instruments Ltd., Worcester, UK).