*3.12. Anti-Tumor Effect In Vitro*

BV2 cells were seeded into 96-well plates (n = 5) at a density of 4000 cells per well and cultured for 24 h. The cell culture medium was replaced with 100 µL of fresh medium containing an increased concentration of DOX and GQDs/DOX@CCM complexes, respectively. The treatment concentration of GQDs was 100 µg/mL in the GQDs/DOX@CCM group. After co-cultivation for 24 h, the cells were washed twice with PBS, and then they were incubated with 10 µL of CCK-8 solution for another 2 h. The absorbance at 450 nm was detected using a Multifunctional plate reader ((PerkinElmer Inc., Waltham, MA, USA). The PTT groups were exposed to an 808 nm laser with a power density of 1.44 W/cm<sup>2</sup> for 3 min after co-cultivation for 4 h, with other conditions kept the same. The formula for calculating the cell viability (%) was listed as follows:

$$\text{Cell viability} = \frac{\text{A}}{\text{A}\_0} \times 100\% \tag{8}$$

Note: A is the OD<sup>450</sup> of the treated BV2 cells; A<sup>0</sup> is the OD<sup>450</sup> of the untreated BV2 cells.

For the Calcein-AM/PI double-staining assay, BV2 cells were seeded in 35 mm confocal dishes (n = 3) at a density of 40,000 cells per well and co-cultivated with free DOX, GQDs, GQDs@CCM, and GQDs/DOX@CCM for 4 h. The concentration of GQDs was kept consistent at 100 µg/mL. Then, cells were washed three times with PBS and subjected to the light irradiation (808 nm, 1.44 W/cm<sup>2</sup> ) for 3 min. After being washed by PBS, cells were stained with Calcein-AM and PI for 15 min at 37 ◦C. Finally, cells were visualized by CLSM.

#### *3.13. Statistical Analysis*

Data was reported as mean ± SD. The differences among groups were determined by a one-way ANOVA analysis followed by the Tukey's post-test: (\*) *p* < 0.05, (\*\*) *p* < 0.01, (\*\*\*) *p* < 0.001.

#### **4. Conclusions**

In summary, a cancer cell membrane biomimetic drug delivery system for chemophotothermal combination therapy of homogeneous cancer cells was developed. The synthesized GQDs with favorable properties of excellent dispersibility, controllable size, stable fluorescence, and superior photothermal performance was utilized as a therapeutic agent and co-encapsulated with DOX in a BV2 cell membrane. The GQDs were not only capable of converting near-infrared light to heat for photothermal therapy, but they also improved the release of DOX for chemotherapy. Besides this, benefiting

from the homologous targeting of the cancer cell membrane, the GQDs/DOX@CCM can actively target BV2 cells in vitro, resulting in a higher cellular uptake. The antitumor results demonstrated the superior killing efficiency of GQDs/DOX@CCM to cancer cells through chemo-photothermal treatment. Based on this study, the fabricated GQDs/DOX@CCM are capable of providing an effective combination strategy for precision oncology therapy.

**Supplementary Materials:** The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/ph15020157/s1: Figure S1: characterization of GQDs. (a) UV-Vis-NIR spectrum of GQDs. (b) Photothermal effect of the GQDs solution (300 µg/mL) exposed to the NIR laser (808 nm, 1.44 W/cm<sup>2</sup> ). The lasers were shut off after 300 s irradiation. (c) Plot of cooling time versus negative natural logarithm of the temperature driving force obtained from the cooling period after the NIR irradiation (808 nm, 1.44 W/cm<sup>2</sup> ). Figure S2: Flow cytometry analysis. (a–c) Mean fluorescence intensity of BV2 cells after 10, 30, and 60 min incubation with GQDs or GQDs@CCM; the final concentration of GQDs was 200 µg/mL. (d–f) Mean fluorescence intensity of MCF-7 and BV2 cells after 10, 30, and 60 min incubation with GQDs@CCM; the final concentration of GQDs was 200 µg/mL. Figure S3. Homologous targeting of GQDs@CCM. (a) CLSM images of GMI-R1 and Rat astrocytes cells incubated with GQDs@CCM at GQDs concentration of 200 µg/mL for different time period. Scale bar = 10 µm. (b,c) Quantitative analysis of cell uptake by FCM in different cell lines.

**Author Contributions:** Conceptualization, F.C., S.F., Y.R. and C.M.; writing—review and editing, Y.R. and C.M.; methodology, Y.R., C.M. and L.T.; project administration and funding acquisition, F.C. and S.F.; formal analysis, Y.L. and P.N.; software, H.L. and Y.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (52002239).

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Shanghai University (protocol code ECSHU-2020-030).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are contained within the article and Supplementary Materials.

**Conflicts of Interest:** The authors report no conflict of interest.

#### **References**


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