Characterization of nano-Ag@erythrosomes

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.(34) 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.

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.

Characterization of nano-Ag@erythrosomes

RBCs and B16 cells were labeled with DiD and DiL respectively and washed twice with PBS before obtaining membrane. The labeled nano-Ag@erythrosomes were prepared using the protocol listed in the previous section. The labeled nano-Ag@erythrosomes as well as the simple mix of labelled RBC membrane vesicles and B16 cell membrane vesicles were cocultured with DC2.4 cells for 12 hours. Then, the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) and analyzed using a confocal microscope.

In vivo distribution study

The B16 cells were labeled with DiD before obtaining cell membrane and fusing with the erythrocyte membrane (unlabeled) at different membrane protein ratios (1:0, 1:5, and 1:20). The fused membrane vehicles were intravenously injected into healthy C57 mice. For each group, 10-µg proteins from the B16 cell membrane were used. After 1 hour, the mice were sacrificed, and the major organs were collected and imaged using an IVIS spectrum imaging system.