Preparation of nano-Ag@erythrosomes

The whole blood were first collected from the orbital sinus of C57 mice and stored in PBS containing EDTA. Then RBCs were separated from whole blood by centrifugation (2000 rpm, 5 min). The RBC membrane was obtained by a previously reported hypotonic treatment.(1) Briefly, deionized water containing EDTA was added into the obtained RBCs and the mixture was shaken gently for 5 min. For this process, large amount of deionized water is recommended. The mixture was centrifuged at 4000 rpm for 10 min and the supernatant was collected and further centrifuged at 14800 rpm for 20 min at 4 ℃. Deionized water containing EDTA was added to the precipitation at first step and the solution was sonicated for 10 s, followed by centrifugation at 14800 rpm for 20 min at 4 ℃. The procedure was repeated twice. The obtained RBC membrane was washed twice with deionized water containing EDTA to remove the hemoglobin. The obtained RBC membrane would be pink to white in color.

In order to obtain B16 membrane, the cells were suspended with homogenization medium (0.25 M sucrose, 1mM EDTA, 20 mM Hepes-NaOH, protease inhibitor cocktail, pH 7.4). The cells were sonicated using Selecta Sonopuls for 30 rounds on ice (T_on = 3s, T_off = 7s). Note that the cell suspension should not be sonicated for a long time; otherwise the obtained cell debris would be too small to collect by centrifugation. The solution was centrifuged at 4000 rpm for 10 min and the supernatant was collected and further centrifuged at 14800 rpm for 20 min. The obtained B16 cell membrane was washed twice with PBS. Large amount of cells were required in order to get enough cell membrane for experiment. The obtained B16 membrane would be white in color.

In order to obtain cell membrane from tumor tissue, B16-luc or 4T1-luc tumor tissues were collected and cut into pieces, followed by sonication using Selecta Sonopuls for 30 rounds on ice (T_on = 3s, T_off = 7s). The solution was centrifuged at 4000 rpm for 10 min and the supernatant was collected. For B16-luc tumor, the mixture was centrifugated at 6000 rpm for 10 min in order to remove melanin (for 4T1-luc tumor, this step was skipped) and supernatant was collected and further centrifuged at 14800 rpm for 20 min. The obtained 4T1 tumor cell membrane would be white in color and the obtained B16 tumor cell membrane would be slightly grey.

The protein amount in both RBC membrane and B16 membrane was determined using a BCA kit. The mixture of both membranes was sonicated for 15 mins until the mixture solution became transparent. During this step, ice was added into the sonicator to avoid protein denaturation caused by heat. Then the mixture was gently shaken using a dry bath incubator at 37 ℃ for 30 min before extrusion through a 400 nm membrane. The temperature is required to facilitate membrane fusion.

1. M. Gao et al., Erythrocyte‐Membrane‐Enveloped Perfluorocarbon as Nanoscale Artificial Red Blood Cells to Relieve Tumor Hypoxia and Enhance Cancer Radiotherapy. Advanced Materials 29, 1701429 (2017).