Dialyzer

Transmission electron microscopy (TEM) imaging was carried out by first glow discharging carbon-coated 400 square mesh copper grids (Electron Microscopy Sciences). Particle suspensions (25 μL) were left on the grid for 1 min before being washed off with 10 drops of water. Grids were then negatively stained with 3 drops of 1% uranyl acetate (Sigma Aldrich). For filtration study, the polymerization precursor solution was added with calcein (excitation/emission = 495/515 nm) (5 mg/mL), followed by the same synthesis procedure. Unencapsulated calcein was removed by dialysis using a dialyzer (Harvard Apparatus, Holliston, USA) equipped with a dialysis membrane (0.05 μm Isopore Membrane filters, Merck Millipore Ltd. Billerica, USA). The RBC membrane-derived vesicles and the RBC membrane-coated nanogel samples were then treated with Trition X-100 and filtered through an Amicon ultra filter with a molecular weight cutoff of 100 kDa. The fluorescence intensity of the filtrates was measured with a fluorescent spectrophotometer (Infinite M200, TECAN, Switzerland). The dynamic light scattering (DLS) measurements were carried on a Zetasizer Nano ZS (model ZEN3600 from Malvern Instruments). To dissolve RBC membranes, the solutions were added with 0.25 mL 5 wt% Triton X-100 together with 20 μg Proteinase K (New England Biolabs, Inc., Beverly, USA).

Vancomycin was encapsulated into the RBC-nanogels by mixing 5 mg/mL drug solution with the reactant mixture during the nanogel synthesis process described above. Nanogels were lyophilized and weighted for total mass. To quantify encapsulated vancomycin, nanogels were resuspended with DI water and the suspension was stirred for 24 h to allow for equilibrium. Vancomycin concentration was quantified with high-performance liquid chromatography (HPLC, Perkin Elmer Flexar series 200, Waltham, MA, USA) equipped with a Brownlee SPP C18 column (100 mm × 4.6 mm and 2.7 µm beads). The mobile phase contained 25 mM KH₂PO₄ solution and ACN (9:1, volume ratio) with a flow rate of 1.0 mL/min. The detection wavelength was set at 230 nm. The loading yield was calculated as the weight percentage of vancomycin to the total nanogels. Vancomycin release was measured by loading 1 mL RBC-nanogel suspension into a dialyzer (Harvard Apparatus, Holliston, USA). Dialysis membranes with pores of 50 nm in diameter (Isopore membrane filters, Merck Millipore Ltd. Billerica, USA) were used. The dialyzer was immersed into 1000 mL PBS, PBS with Triton X-100 (5% w/v), or PBS with Triton X-100 (5% w/v) and TCEP (10 mM) at 37°C under agitation. The released vancomycin at pre-determined time intervals was quantified with the same HPLC method.