4T1 cells seeded on coverslip A were pretreated with free CPT, CSSP NPs, or CP NPs (10 μM, CPT equivalent) for 12 hours. Coverslip A was washed and then coincubated with fresh cells on coverslip B for 24 hours. After coincubation, coverslip B was withdrawn and coincubated with fresh cells on another coverslip C for 24 hours. The cells (A, B, and C) were washed with PBS, stained with Hoechst 33342, and observed by CLSM.

To explore the mechanism of intercellular delivery of CSSP NPs, after incubating 4T1 cells with CSSP NPs (10 μM, CPT equivalent) for 12 hours, the cells were washed and cultured with fresh medium for another 24 hours. Then, the supernatant medium was sucked and centrifuged twice at 50g (5 min) to remove apoptotic cells and debris. The ApoBDs were collected by centrifugation at 1000g (10 min). The size and morphology of ApoBDs were observed using a particle size analyzer and transmission electron microscopy (TEM), respectively. Western blot was also performed to characterize the protein constitution of ApoBDs. In addition, after incubating 4T1 cells with ApoBDs (approximately 10 μM, CPT equivalent) for 24 hours, the cells were washed, stained with propidium iodide (PI), and observed by CLSM. To further verify the cytotoxicity of ApoBDs, the collected ApoBDs of different protein concentrations were incubated with 4T1 cells under normoxia for 48 hours, and then the cell survival rate was measured (n = 6).

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