*2.8. Hemolysis Test*

To determine the endosomolytic activity of the EV samples, a hemolysis test was conducted using red blood cells (RBCs) collected from BALB/c mice (7-week-old female) [31,41,42]. The RBC solutions (10<sup>6</sup> cells/mL) at pH 7.4 or 6.5 were incubated with the EV samples (equivalent to EVs of 30 µg/mL, without DOX) at 37 ◦C for 1 h. The RBC solutions were centrifuged at 1500 *g* for 10 min at 4 ◦C and the supernatant was collected. The light absorbance (LA) value of the supernatant was measured using a spectrophotometer at a wavelength of 541 nm. A 0% (as a negative control) LA was acquired from a PBS-treated intact RBC solution and the 100% (as a positive control) of LA value was obtained from completely lysed RBC solution using 2 wt.% Triton X-100. The hemolysis (%) of each EV sample was determined as the LA of the RBC solution treated with each sample relative to the control LA value [31,41,42].

#### *2.9. Cell Culture*

Human breast carcinoma BT-474 cells, human neuroblastoma SK-N-MC cells, and human liver carcinoma Huh7 cells were purchased from the Korean Cell Line Bank. When the cells were grown as a monolayer (1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL), they were harvested by trypsinization using a 0.25% (wt./vol.) trypsin/0.03% (wt./vol.) EDTA solution. Subsequently, the cells suspended in the RPMI-1640 or DMEM medium were seeded in well plates before cell test [37–40].

#### *2.10. In Vitro Cellular UPTAKE test*

BT-474, SK-N-MC, or Huh7 tumor cells were incubated with EV samples (equivalent to DOX of 5 µg/mL) or free DOX (5 µg/mL) for 4 h. After washing the cells using fresh PBS (150 mM, pH 7.4), the fluorescence intensity of the cells was determined using a FACSCaliburTM flow cytometer (FACS Canto II, Becton Dickinson, Franklin lakes, NJ, USA) [3,31,37–39]. In addition, to visualize the cellular uptake of the EV samples, BT-474 or SK-N-MC tumor cells were incubated with the EV samples for 4 h and then fixed using 3.7% formaldehyde in PBS. The fixed cells were monitored using a Nikon microscope equipped with a VNIR hyperspectral camera system (Cytoviva high-resolution adapter, Cytoviva, Auburn, AL, USA) [43–45]. For confocal microscopy analysis, the treated cells were stained with WGA-Alexa Fluor® 488 and DAPI. The stained cells were then fixed using 3.7% formaldehyde in PBS and analyzed using a confocal laser scanning microscope (LSM710, Carl Zeiss, Oberkochen, Germany) [3,30,31].

#### *2.11. In Vitro Cytotoxicity Test*

To determine the cytotoxicity, BT-474, SK-N-MC, or Huh7 tumor cells were incubated with the EV samples (equivalent to DOX of 5 µg/mL) or free DOX (5 µg/mL) at pH 7.4 for 4 h. After washing the cells using fresh PBS (150 mM, pH 7.4), the treated cells were further incubated with fresh RPMI-1640 or DMEM medium at 37 ◦C for 24 h. CCK-8 assay was used to determine the cell viability [3,31,38–40]. In addition, the viability of the cells incubated with drug-free EVs for 24 h was measured using the CCK-8 assay to determine the original toxicity of the EVs and polymers [3,31,38–40].

#### *2.12. In Vitro Cellular Uptake Test of EV Blends*

BTEVs (with fluorescent Ce6 dye incorporation) and SKEVs (with fluorescent FITC dye incorporation) were used to visualize the cellular uptake of the EV blends. Here, Ce6 dye or FITC dye was incorporated into the EVs through sonication [3,30,36]. Briefly, Ce6 dye (200 µg) or FITC dye (200 µg) dissolved in DMSO (0.1 mL) were mixed with EVs (200 µg) suspended in PBS (10 mL, 150 mM, pH 7.4) at 25 ◦C; subsequently, the solution was sonicated using a tip sonicator, vcx-130 with cv-18 (Sonics, Newtown, CT, USA) with 30% amplitude for 30 s. The obtained solution was incubated at 37 ◦C for 60 min to recover the EVs. The filtration method using 0.22 µm membrane was performed to remove Ce6 or FITC aggregates. Ultracentrifugation at 100,000 *g* at 4 ◦C for 70 min was

performed to remove free Ce6 or FITC. The measured weight of the Ce6 or FITC dye incorporated in the EVs was 1–2 wt.%, as evaluated using a fluorescence spectrofluorometer. The obtained EV blends [HDEA@BTEVs/HDEA@SKEVs (50/50 wt.%)] (equivalent to EVs of 30 µg/mL, without DOX) were added to the tumor cells seeded on coverslips in a culture plate at 37 ◦C for 4 h. After washing the cells using fresh PBS (150 mM, pH 7.4), the treated cells were fixed using 3.7% formaldehyde in PBS. The fixed cells were analyzed using a confocal laser scanning microscope (LSM710, Carl Zeiss, Oberkochen, Germany) [3,30,31].

### *2.13. In Vitro Cytotoxicity Test of EV Blends*

The mixed tumor cells (BT-474/SK-N-MC = 50:50 ratio of the number of cells) were prepared before the cell test and incubated with EV blends [HDEA@BTEVs/HDEA@SKEVs (50/50 wt.%)] (equivalent to DOX of 5 µg/mL), HDEA@BTEVs (equivalent to DOX of 5 µg/mL), or HDEA@SKEVs (equivalent to DOX of 5 µg/mL) for 4 h at 37 ◦C. After washing the cells using fresh PBS (150 mM, pH 7.4), the treated cells were additionally cultured with fresh RPMI-1640/DMEM mixed medium (50/50 vol.%) at 37 ◦C for 24 h. The CCK-8 assay was used to determine the cell viability [3,31,38–40].

### *2.14. Statistics*

All the experimental results were analyzed using Student's t-test or ANOVA at a significance level of *p* < 0.01 (\*\*) [37].
