3.4. Cytotoxicity Assay
oxygen to kill tumor cells under the laser irradiation. Fur-
thermore, the combination of PLG-g-mPEG-CA4 and PLG-
g-mPEG-TPP nanoparticles showed the obvious inhibition
of cell proliferation in vitro, which would obtain the out-
standing antitumor effect in the solid tumor due to the blood
vessel blocking of tumor tissue. The combination of PLG-
g-mPEG-CA4 and PLG-g-mPEG-TPP nanoparticles will be
promising for cancer treatment in the field of tumor recur-
rence and metastasis.
The cytotoxicities of PGC, PGT, PGC, and PGT with or without
laser were measured using the MTT assay in 4T1 cells, HeLa
cells, and MCF-7 cells at 24 and 48 h, respectively (Figure 6). As
shown in Figure 6, the cell viabilities of PGC, PGT and PGC
and PGT group without laser irradiation were above 80% at
24 h (Figure 6A,C,E). However, the cytotoxicities of PGC and
PGT group without laser irradiation on three types of cells were
different (MCF-7<HeLa<4T1) at 48 h (Figure 6B,D,F). The cyto-
toxicities of PGT, PGC, and PGT with laser irradiation were
much lower than those without laser irradiation, which was due
to that PGT with porphyrin could produce singlet oxygen to kill
cancer cells. In the 4T1 cells (Figure 6A,D), the cell viability at 24
and 48 h in the PGT group were 90.2% and 88.0%, respectively,
and in the PGC and PGT combination group were 73.0% and
50.0%, respectively. However, under the light condition, the cell
viability of PGT group at 24 and 48 h decreased to 33.5% and
15.9%, respectively, and cell viability of the PGC and PGT com-
bination group was reduced obviously, which were 31.0% and
11.8%, respectively. In HeLa cells (Figure 6B,D) and MCF-7 cells
(Figure 6E,F), the cell viabilities were same as that in 4T1 cells.
The results showed that both the PGT group and the PGC/PGT
combined group showed significant dose dependence, and
the results also proved that the combination of photodynamic
therapy and chemotherapy had a better effect on inhibition of
cancer cell proliferation than the single chemotherapy, which
suggested combined PGC and PGT had good antitumor effect
and had great potential for cancer treatment in vivo.
Acknowledgements
This work was financially supported by National Natural Science
Foundation of China (Project Nos. 51973025, 51503202, 51673189,
51873206, 51829302, 51833010, and 51520105004), Jilin Science
and Technology Bureau (20200801054GH), and Jilin Province
(20190103033JH), Ministry of Science and Technology of China (Project
No. 2018ZX09711003-012),
Conflict of Interest
The authors declare no conflict of interest.
Keywords
chemotherapy, combination therapy, combretastatin A4, photodynamic
therapy
Received: May 30, 2020
Revised: August 26, 2020
Published online:
3.5. Intracellular ROS Generation Detection
Intracellular ROS could be produced under the laser irradia-
tion, thus exhibiting the cytotoxicity to cancer cells. The ROS
generated in the cells were determined by DCFDA. DCFDA
could be oxidized green fluorescent- DCF by intracellular ROS
due to the deacetylation of DCFDA. Under the laser irradiation,
the obvious green fluorescence signals were observed in 4T1
cells treated with PGT group and the combined PGC and PGT
group (Figure 7A). The quantitative analyses for the fluores-
cence intensities/pixel of DCF were showed in Figure 7B, the
results showed that the amount of singlet oxygen produced by
PGT group and PGC combined with PGT group was signifi-
cantly higher than that of the control group, while the amount
of singlet oxygen produced by PGC combined with PGT group
was slightly higher than that of the PGT group, which revealed
that PGT nanoparticles could generate singlet oxygen signifi-
cantly in 4T1 cells.
[1] Z.-H. Tang, X.-S. Chen, Acta Polym. Sin. 2019, 50, 543.
[2] W. Song, Z. Tang, D. Zhang, M. Li, J. Gu, X. Chen, Chem. Sci. 2016, 7, 728.
[3] A. Hasani, N. Leighl, Clin. Lung Cancer 2011, 12, 18.
[4] G. M. Tozer, C. Kanthou, B. C. Baguley, Nat. Rev. Cancer 2005, 5, 423.
[5] G. M. Tozer, V. E. Prise, J. Wilson, M. Cemazar, S. Q. Shan, M. W. Dewhirst,
P. R. Barber, B. Vojnovic, D. J. Chaplin, Cancer Res. 2001, 61, 6413.
[6] D. W. Siemann, Cancer Treat. Rev. 2011, 37, 63.
[7] R. Nallamothu, G. C. Wood, C. B. Pattillo, R. C. Scott, M. F. Kiani,
B. M. Moore, L. A. Thoma, Aaps PharmSciTech 2006, 7, E7.
[8] A. Close, Future Med. Chem. 2016, 8, 443.
[9] P. G. Tardi, N. Dos Santos, T. O. Harasym, S. A. Johnstone,
N. Zisman, A. W. Tsang, D. G. Bermudes, L. D. Mayer, Mol. Cancer
Ther. 2009, 8, 2266.
[10] D. Toshihiko, N. Fuse, A. Ohtsu, Y. Matsumura, T. Hamaguchi,
K. Kato, Y. Shimada, T. Nakajima, Y. Yamada, M. Takanashi, Ejc
Suppl. 2008, 6, 133.
[11] W. Song, Z. Tang, D. Zhang, H. Yu, X. Chen, Small 2015, 11, 3755.
[12] W. Song, Z. Tang, D. Zhang, X. Wen, S. Lv, Z. Liu, M. Deng,
X. Chen, Theranostics 2016, 6, 1023.
4. Conclusions
[13] Y. Wang, H. Yu, D. Zhang, G. Wang, W. Song, Y. Liu, S. Ma, Z. Tang,
Z. Liu, K. Sakurai, X. Chen, Acta Biomater. 2019, 92, 229.
[14] T. Liu, D. Zhang, W. Song, Z. Tang, J. Zhu, Z. Ma, X. Wang, X. Chen,
T. Tong, Acta Biomater. 2017, 53, 179.
[15] J. Jiang, N. Shen, W. Song, H. Yu, K. Sakurai, Z. Tang, G. Li, Bio-
mater. Sci. 2019, 7, 5283.
In summary, we successfully prepared PLG-g-mPEG-CA4
and PLG-g-mPEG-TPP nanoparticles using the Yamaguchi
esterification reaction and condensation reaction, respec-
tively. The PLG-g-mPEG-CA4 nanoparticles showed that
CA4 released faster at acidic pH than at slight alkaline pH.
And PLG-g-mPEG-TPP nanoparticles could produce singlet
[16] N. Shen, J. Wu, C. Yang, H. Yu, S. Yang, T. Li, J. Chen, Z. Tang,
X. Chen, Nano Lett. 2019, 19, 8021.
2000192 (8 of 9)
Macromol. Biosci. 2020, 2000192
© 2020 Wiley-VCH GmbH