The native and engineered RBCs were fixed with 4% glutaraldehyde and washed with distilled water, and then SEM images were obtained under an S-4800 microscope (Hitachi, Japan) at an acceleration voltage of 5 kV. Biological TEM was conducted with a JEM-1230 microscope (JEOL, Japan). The samples were fixed with glutaraldehyde, OsO4, and K2Cr2O7, and after dehydration in ethanol/acetone, they were embedded in Epon 812/Araldite M resin. Thin sections were cut using a Reichert ultratome (Zeiss, Germany) and then stained with uranyl acetate and lead citrate. RhD-positive RBCs were encapsulated via the surface engineering method described above, and only the alternative frameworks were covalently coupled with fluoresceinamine isomer I (Sigma-Aldrich). Then, the native and engineered RBCs were rinsed with PBS three times, cultured in PBS buffer for 1, 5, 10, 15, 20, and 25 days, and then subjected to CLSM (Nikon, Japan). All images were captured and analyzed using image analysis software (Zen Light Edition 2009). FTIR spectra were collected on a Nicolet iS10 spectrometer (Thermo Fisher Scientific) using a GS10800-B Quest sampling accessory (Specac, England) with a diamond attenuated total reflectance sampling plate to study the composition of the PSA and PSA-tyramine.

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