Dxr 500

Monomers based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy)hexane (CPH) were synthesized as described previously (30 (link), 31 (link)) Using these monomers, 20:80 CPTEG:CPH copolymer was synthesized using melt polycondensation for ~6 h, as described (31 (link)). The final copolymer composition, purity, and molecular weight of the copolymer were characterized using ¹H HNMR (DXR 500, Bruker, Billerica, MA). Next, 20:80 CPTEG:CPH nanoparticles containing 1% H1 HA, 1% NP, and 2% CpG1668 were synthesized via solid-oil-oil double emulsion (32 (link)). Briefly, HA and NP protein antigens (Sino Biological, Beijing, China) were dialyzed to nanopure water and lyophilized overnight. The 20:80 CPTEG:CPH copolymer, along with HA, NP, and CpG (ODN 1668, Invivogen, San Diego, CA), was dissolved at a polymer concentration of 20 mg/mL in methylene chloride. The solution was sonicated for 30 s and then precipitated into chilled pentane (at a methylene chloride:pentane ration of 1:250). The resulting nanoparticles were collected via vacuum filtration and scanning electron microscopy (FEI Quanta 250, FEI, Hillsboro, OR) was used to characterized morphology and size.

1D and 2D NMR spectra were recorded on Bruker DXR-600 instrument (600 and 150 MHz) and Bruker DXR-500 instrument (500 and 126 MHz). The UV data were detected by Hitachi UH5300 spectrophotometer (Hitachi, Kyoto, Japan). IR spectra were conducted on IRT racer-100 (SHIMADZU, Kyoto, Japan) with KBr pellets. HR-ESI–MS data were obtained on a UPLC-Q Exactive MS system (Thermo Fisher, Santa Clara, CA, USA). The packing for column chromatography (CC) is silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) or Sephadex LH-20 (Amersham Biosciences, Upssala, Sweden). The semi-prepared HPLC was carried out on an Agilent Technologies 1260 Infinity II system with a diode array detector. And the chromatographic column was C₁₈ reversed phase column (5 μm, 10 × 250 mm) (Agela, Tianjin, China).