Supporting information
Influence of Surface Coatings of PLGA Particles on HepG2 Cell
Behavior and Particle Fate
Dahai Yu 1, Yuying Zhang 1, Guangyang Zou, Xiaojing Cui, Zhengwei Mao
1*
,
Changyou Gao 1,2*
1 MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou
310027, China
2 State Key Laboratory of Diagnosis and Treatment for Infectious Diseases, First
Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003,
China
*Corresponding author.
Email: zwmao@zju.edu.cn, cygao@mail.hz.zj.cn
Fax: +86-571-87951108
100
PLGA-PEI
PLGA-BSA
Mass(g)
80
60
40
20
0
0
6
Time (h)
10
20
Figure S1. Protein mass of the cells after treated with particles as a function of
incubation time.
After the cells were incubated with 50 g/mL PLGA particles for 24 h (which is
designated as the 0 time point in Figure S1), the total protein content of the cells was
measured via a BCA kit as a function of culture time (the exocytosis time).
Figure S1 shows that the cells did not duplicate during the exocytosis time until 20h
since their protein content kept unchanged. Therefore, exocytosis rather than cell
proliferation was the major reason for the decrease of fluorescent intensity of each
cell during this period.
*
PLGA-PEI
PLGA-BSA
600
*
400
* *
200
*
0
(A)
0
50
100
Concentration (μg/ml)
Average fluorescence intensity per cell
Average fluorescence intensity per cell
800
PLGA-PEI
PLGA-BSA
600
*
*
*
400
*
*
200
150
(B)
0
0
5
10
15
20
25
Time (h)
Figure S2. Uptake of the PLGA-PEI and PLGA-BSA particles by HepG2 cells as a
function of (A) particle concentration with a culture time of 3 h, and (B) culture time
with a particle concentration of 50 µg/mL. Data were measured by flow cytometry
and averaged to each cell. Asterisk indicates significant difference at p < 0.05 level vs
respective inhibitors free control.
Figure 2
As shown in Figure S2A, the PLGA particles that internalized into or adsorbed onto
the cells increased almost linearly along with the increase of particle concentration
regardless of their surface chemistry at a fixed co-culture time of 3 h, but the
PLGA-PEI particles were more significantly uptaken than the PLGA-BSA particles at
each fixed particles concentration (p<0.05). When the particles concentration was
fixed at 50 µg/mL, the cellular endocytosis amount of both types of PLGA particles
increased significantly along with the co-culture time, especially during the first 6 h
(Figure S2B), suggesting the fast cellular endocytosis rate. Again, the PLGA-PEI
particles were uptaken with a larger amount at each detection time interval (p<0.05).
This result is apparently caused by the difference in surface chemistry.
Cell mortality rate (%)
30
Control
PLGA-PEI
PLGA-BSA
20
10
0
Control
10
25
50
100
Concentration (g/mL)
150
Figure S3. Percentage of dead cells as a function of particle concentration with a
culture time of 24 h. The particle free cells were used as a control (about 6% dead
cells).
The ratios of dead cells after exposure of the PLGA particles of various
concentrations were determined by PI staining. As shown in Figure S3, about 90% of
the cells survived even after 24 h co-incubation with the PLGA particles of the highest
concentration (150 µg/mL). The results suggested that both types of PLGA particles
did not cause significant impact on cell mortality.
Table S1. Influence of PLGA particles on HepG2 cell cycle.
G1 (%)
S (%)
G2/M (%)
Particle free control
54.4±1.3
18.4±1.3
27.1±2.6
PLGA-PEI (50μg mL−1）
54.4±1.7
18.1±1.1
27.6±0.8
PLGA-PEI (150μg mL−1）
51.4±0.2
17.8±0.6
30.8±0.7
PLGA-BSA (50μg mL−1）
57.7±2.0
15.9±0.8
26.5±1.2
PLGA-BSA (150μg mL−1） 57.4±0.8
16.8±1.1
25.8±1.4
Cell cycle is a series of events which can induce cell division and duplication,
comprising interphase gap1 (G1), synthesis (S), gap2 (G2) and mitosis (M) phases.
Therefore, the cell cycle analysis is powerful to reflect the influence of cellular
endocytosis on cell proliferation. Several studies have been focused on the influence
of the particles, genes and inhibitors on the cell cycle.[1-4] Since G2 and M phases
could not be distinguished only by DNA content, they were put together (G2/M) to
represent cell division period. As shown in Table S1, no significant difference of the
cell cycle for all the samples was found, revealing that exposure to both types of
PLGA particles did not cause obvious influence on cell cycle.
Relative cell adhesion (% )
150
120
90
60
30
(A)
0
PLGA-PEI
PLGA-BSA
A)
25μm
(B)
B)
(C)
(D)
C)
Figure S4. (A) Cell adhesion percentage after the cells were pre-cultured without or
with 50 μg/mL PLGA-PEI and PLGA-BSA particles for 24 h, respectively.
Cytoskeleton organization of HepG2 cells treated without (B) or with (C) 50 μg/mL
PLGA-PEI and (D) PLGA-BSA particles, respectively. The F-actin was stained by
rhodamine-conjugated phalloidin.
Figure S4A shows that the adhesion ability of the cells did not change significantly
after exposure to both types of the PLGA particles. F-actin staining was performed to
examine the effects of the PLGA particles on the cytoskeleton of HepG2 cells. As
shown in Figure S4B, the microfilaments in the control cells were well ordered. After
the cells were exposed to 50 µg/mL PLGA particles for 12 h, the organization of
microfilaments changed to some extent (Figure S4C, D), especially of the cells treated
with PLGA-PEI particles (Figure S4C).
(A)
20
-20
-15
-10
-5
15
15
10
10
5
5
0
0
-5
0
5
10
15
20
25 -25
-20
-15
-10
-5
-5
(C)
20
(B)
20
B
-25
25
25
25
15
10
5
0
0
5
10
15
20
25
-25
-20
-15
-10
-5
-5
0
5
10
15
20
25
A
A
-10
-10
-10
-15
-15
-15
-20
-20
-20
-25
-25
-25
Migration Rate (μm/h)
10
8
*
6
*
4
2
(D)
0
Control
PLGA-PEI PLGA-BSA
Figure S5. Migration trajectories of HepG2 cells pre-cultured without (A) or with 50
µg/mL (B) PLGA-PEI and (C) PLGA-BSA particles. Quantitative analysis of the cell
migration rate (D). Asterisk indicates significant difference at p < 0.05 level.
The cell migration plays a central role in a wide variety of biological phenomena. It is
a dynamic adhesion process requiring coordinate activity of cytoskeleton, membrane
and signaling system.[5] The cell mobility was reduced to some extent after the cells
were exposed to 50 µg/mL PLGA-PEI particles for 24h (p<0.05). By contrast, the cell
mobility was not significantly changed after the cells were exposed to 50 µg/mL
PLGA-BSA particles for 24 h (Figure S5D).
[1] L. N. Wei, J. L. Tang, Z. X. Zhang, Y. M. Chen, G. Zhou, T. F. Xi, Biomed Mater
2010, 5.
[2] V. Robert, P. Michel, J. M. Flaman, A. Chiron, C. Martin, F. Charbonnier, B.
Paillot, T. Frebourg, Carcinogenesis 2000, 21, 563.
[3] M.-j. Gao, Z.-w. Xu, F.-m. Wang, X.-y. Chen, W.-l. Hu, R.-c. Xu, Zhongguo
Yaolixue Tongbao 2010, 26, 452.
[4] Z.-W. Xu, F.-M. Wang, M.-J. Gao, X.-Y. Chen, W.-L. Hu, R.-C. Xu, Biological &
Pharmaceutical Bulletin 2010, 33, 743.
[5] D. A. Lauffenburger, A. F. Horwitz, Cell 1996, 84, 359.