Facscanto flow cytometry machine

Flow cytometry was used to validate the successful encapsulation of RBCDVL and VAN-L [32] . RBCs were washed twice with ice-cold PBS. The percentage of the volume of RBC membrane accessible by liposomes in preparation before incubation was determined by mixing the same volume of RBC membrane preparation and liposome preparation. Liposomes loaded with fluorescent probes were prepared using a procedure similar to that used to produce liposomes containing VAN and DAPT, with VAN replaced by FAM (Ex/Em, 493/517 nm) and DAPT by DIL (Ex/Em, 549/565 nm). To determine the EE, 5 µl FAM/DIL was dissolved in chloroform with a mixture of EPC, cholesterol, mPEG2000-DSPE, and DAPT-PEG-DSPE before being subjected to evaporation and drying under vacuum. Rehydration was subsequently performed with the FAM solution before incubation with the RBC membrane to allow the transition of liposomes loaded with VAN into the RBC membrane's pores across the concentration gradient. The RBC membrane was suspended in 1 × PBS to close the pores and then centrifuged at 400 × g. The RBC membrane was washed three times with cold PBS and centrifuged at 400 × g for removing unloaded drug molecules. The supernatant was removed, and the pellets were collected and diluted with 200 µl of isotonic solution. The final formulation results were validated using the BD FACSCanto flow cytometry machine and TEM imaging.