2000fx tem

¹H NMR spectra were recorded in 400 MHz Bruker NMR spectrometer with the residual proton of the solvent as internal standards, chemical shifts are reported in parts per million (ppm). Transmission electron microscopy (TEM) was performed on JEOL 2000FX TEM. Dynamic light scattering (DLS) was performed using Malvern nanozetasizer with a 637 nm laser source with noninvasive backscattering detected at 173°. Fluorescence measurements were performed using Molecular Devices SpectraMax M5 fluorescence plate reader.

Polymeric vesicles (PVs) were prepared by the solvent evaporation method. Briefly, 40 mg of mPEG-b-P(Ser-PBE) was dissolved in 5 mL of THF, followed by injection of 10 mL of DI water with or without 7.5 mg of human insulin and 0.75 mg of GOx dissolved in it. The mixture was stirred at room temperature for 30 min, and then THF was removed by bubbling with N₂. The unloaded insulin was removed by centrifugation at 4000g for 10 min with a centrifugal filter (25 000 Da molecular mass cutoff, Millipore) and washed with PBS buffer for several times. The obtained PVs suspension was stored at 4 °C for further studies. The loading content and loading efficiency of insulin was tested using a Coomassie Plus protein assay. The absorbance was detected at 595 nm on the Infinite 200 PRO multimode plate reader (Tecan Group Ltd., Switzerland) and the concentration was interpolated from an insulin standard curve. The size distribution and zeta-potential of the PVs were measured using the Zeta sizer (Nano ZS; Malvern). The TEM images were acquired using a JEOL 2000FX TEM.

10 μL of the superstructure solution was drop-cast on to a TEM grid (carbon film- 400 mesh copper, electron microscopy sciences) and the sample allowed to dry at room temperature overnight. Superstructures were inspected by using JEOL 2000FX TEM.

Freshly prepared sample solution was drop-cast onto a TEM grid (carbon film, 400 mesh copper, Electron Microscopy Sciences), and the sample was allowed to dry at room temperature overnight. Negative staining was employed to enhance the imaging contrast.⁴⁴ 2% uranyl acetate solution was dropped on the sample for 30 seconds, following by drying using a filter paper. The structural of samples were inspected using a JEOL 2000FX TEM with an accelerating voltage of 200 kV.