Styragel columns hr3 and hr4

Poly(lactic-co-glycolic acid) polymers (PLGA; LA:GA molar ratio 50:50; acid end group) with inherent viscosities of 0.05 – 0.15 dL/g (PLGA1A, Mw ~ 5.6 kDa and PDI ~ 2.5), 0.15 – 0.25 dL/g (PLGA2A, Mw ~ 18 kDa and PDI ~ 3.1) and 0.35 – 0.45 dL/g (PLGA4A, Mw ~ 55 kDa and PDI ~ 1.7), respectively, were purchased from Lakeshore Biomaterials (Evonik Corporation, Birmingham, AL). The molecular weights of the three polymers were provided by the supplier. The block copolymers of PLGA(LA:GA 50:50) and monomethoxy poly(ethylene glycol), PLGA_45k-PEG_5k and PLGA_15k-PEG_5k, were custom-synthesized by the Daigang Biomaterial Co., Ltd, (Jinan, China) and characterized by ¹H NMR and GPC. A Waters GPC system equipped with a refractive index detector and two Waters Styragel® columns (HR3 and HR4) were used. The analysis was performed at 30°C using chloroform as the mobile phase and a flow rate of 0.3 mL/min. The GPC was calibrated with polystyrene standards (Walters) of known MW. Cholic acid sodium salt (CHA), polyethylene glycol (Mw 5 kDa) and mucin from bovine submaxillary glands (type I-S) were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 555 cadaverine (AF555) was purchased from Invitrogen (Eugene, OR).

All reagents were purchased from Sigma-Aldrich and used as received unless stated otherwise. CH₂Cl₂ was distilled over calcium hydride. Styrene was washed with 1 N NaOH, followed by water to remove inhibitors, dried over MgSO₄ and then purified by vacuum distillation over calcium hydride. AIBN was recrystallized from MeOH twice prior to use. Lactose-oxime 8 was synthesized according to previously reported procedures.^{[25 (link)]} The NMR spectra were recorded on a Varian Mercury 300 MHz or Varian Inova 500 MHz spectrometers using CDCl₃ as a solvent unless stated otherwise. ¹H NMR-based M_n of dodecyl trithiocarbonate-terminated polymers was calculated by comparing the integral areas under CH_ar peaks of repeating units with integrals of CH₂–S signal of ω-chain end. The weight of CTA (566 g mol⁻¹) was then added to the sum of weights of repeating units. GPC analyses were performed on Shimadzu LC-20AD liquid chromatography instrument, equipped with RI detector. Two Waters Styragel columns (HR3 and HR4) were placed in series. THF was used as eluent at 1 mL min⁻¹ flow rate; the column oven was set to 40 °C. Molecular weights were calculated against polystyrene standards. TEM images were obtained on Philips/FEI Technai 20 instrument using copper TEM grids. A 2% (wt/vol) uranyl acetate solution was used to stain the polymeric nanoparticles before recording.