All handling and care of animals were performed according to the guidelines on the care and use of animals for scientific purposes issued by Zhejiang University. The distribution of native and engineered mouse RBCs was examined in vivo in mice obtained from Institute of Cancer Research (ICR). Whole blood was acquired from different donor mice and washed with 10 mM PBS, with erythrocytes collected by centrifugation. Half of the RBCs were engineered with frameworks as described above, and the same volume of native RBCs was labeled with DIR dye (GeneCopoeia, MD). Labeled RBCs (200 μl, 1 × 109 cells ml−1) were intravenously injected into each recipient mouse (n = 3, in each group). The DIR dye signals were subjected to Vevo LAZR imaging (Visual Sonics, Canada) to measure the RBCs’ distribution in the major organs (heart, liver, spleen, and kidneys). The in vivo survival assay of the RBCs was carried out during the same process, although the dye was substituted with PKH-26 (Sigma), which is a fluorescence molecule that can label the cell membrane. The survival profiles of RBCs were tested and recorded until no signals were determined by detecting the PKH-26 fluorescence using a FC500 MPL flow cytometer (Beckman Coulter, CA). Approximately 0.5 ml of whole blood from each mouse was harvested for routine blood tests by eyeball extirpation. The major organs (heart, liver, spleen, lung, and kidneys) were harvested and fixed in a 10% formalin solution. For histopathological examinations, the tissue samples were embedded in paraffin blocks, sectioned into slices, and mounted onto glass slides. After H&E staining, images were captured via Eclipse TE2000-S microscopy (Nikon, Japan). The serum levels of complement 3 (MU30594, Bioswamp), complement 4 (MU30595, Bioswamp), TNF-α (ab208348, Abcam), and IL-6 (ab100712, Abcam) were determined using an ELISA kit.

The ICR mice removed 200 μl of blood from the retro-orbital sinus, and then an equal volume of native (type-matched) or engineered RBCs was intravenously injected. HR, MBP, and SBP were tested using a BP-98A mice noninvasive sphygmomanometer (Softron, Japan).

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