Figure Captions
Fig.1 Physicochchemical characterization of LDH nanoparticles and 5-FU/LDH nanocomplex.
A) XRD, B) Particle size distribution and zeta potential distribution of LDHs and 5FU(10)/LDH nanohybrids, C) TEM image of 5-FU(10)/LDH nanohybrids, D) FT-IR of
Na-5FU, LDH-Cl and 5-FU(50)/LDH nanohybrids.
Fig.2 DNA-binding ability of 5-FU/LDH nanocomplex. Lane 1: DNA ladder, Lane 2: dsDNA
only, Lane 3: 5-FU/LDH nanocomplex only, Lane 4-7: dsDNA-5-FU/LDH with the
dsNDA:LDH mass ratio from 1:5 to 1:40.
Fig.3 Confocal microscopy images of MCF-7 cells taking up red fluorescence siRNA
AF456/LDH nanohybrids (A and C: cells cultured at 37oC and 4oC, respectively) and
siRNA AF456-5-FU/LDH nanocomplex (B and D: cells cultured at 37oC and 4oC,
respectively). The scale bars: 50 μm. The mass ratio of LDH to siRNA: 40:1.
Fig.4 Cytotoxicity of LDHs at different concentrations to MCF-7 cell lines
Fig.5 MTT assay analysis of effects of treatments with 5-FU, 5-FU(10)/LDH, DC-siRNA/LDH,
and CD-siRNA-5-FU/LDH on the viability of MCF-7 cells at the 5-FU concentration
from 0-9.6 µg/mL and the CD-siRNA concentration at 40 nM in all relevant treatments
for 72 h at 37oC. Data represent mean ±SD (n=5). ** p<0.05 versus 5-FU treatment, *
p<0.01 versus 5-FU treatment. **** p<0.0001 versus 5-FU treatment.
Fig.6 FACS analysis of cell distribution for apoptosis and necrosis on MCF-7 cells after
treatment with LDH, 5-FU/LDH, CD-siRNA/LDH and CD-siRNA-5-FU/LDH for 24 h.
The concentration of 5-FU: 2.4 µg/mL and CD-siRNA: 40 nM.
Fig.7 Suppression of Bcl-2 protein expression in MCF-7 cells after single or combined
treatment with 5-FU and CD-siRNA delivered by LDHs. (A) Western blot analysis after
1
transfection with 5-FU and CD-siRNA delivered by LDH for 24h. (B) Quantitative
evaluation of the percent change in expression levels of Bcl-2 protein against α-tubulin.
The 5-FU, CD-siRNA concentration in all samples was 1.2 μg/mL and 40 nM,
respectively. LDH concentration is 50 μg/mL.
2
A
B
(006) 5-FU(50)/LDH
5-FU(10)/LDH
(003)
(((006)0
C
1650
1540
LDH
D
767
667
549
1220
1365
5-FU(50)/LDH
1615
1545
Na-5FU
LDH
100
Fig. 1 Physicochchemical characterization of LDH nanoparticles and 5-FU/LDH nanocomplex.
A) XRD, B) Particle size distribution and zeta potential distribution of LDHs and 5-FU(10)/LDH
nanohybrids, C) TEM image of 5-FU(10)/LDH nanohybrids, D) FT-IR of Na-5FU, LDH-Cl and
5-FU(50)/LDH nanohybrids.
3
Lane 1
2
3
4
Ladder
dsDNA
5-FU/LDH
1:5
5
1:10
6
1:20
7
1:40
Fig. 2 DNA-binding ability of 5-FU/LDH nanocomplex. Lane 1: DNA ladder, Lane 2: dsDNA
only, Lane 3: 5-FU/LDH nanocomplex only, Lane 4-7: dsDNA-5-FU/LDH with the dsDNA:5FU/LDH mass ratio from 1:5 to 1:40.
4
Nuclei
Red siRNA 456
Merged
A
B
C
D
Fig. 3 Confocal microscopy images of MCF-7 cells taking up Red siRNA 456/LDH nanohybrids
(A and C: cells cultured at 37oC and 4oC, respectively) and Red siRNA 456-5-FU/LDH
nanocomplex (B and D: cells cultured at 37oC and 4oC, respectively). The scale bars: 50 μm.
The mass ratio of LDH to siRNA: 40:1.
5
120
Cell viability (%)
100
80
60
40
20
0
0
50
100
200
Concentration of LDH (µg/mL)
Fig. 4 Cytotoxicity of LDHs at different concentrations to MCF-7 cell lines.
6
C e ll v ia b ilit y ( % )
****
*
****
**
Fig. 5 MTT assay analysis of effects of treatments with 5-FU, 5-FU(10)/LDH, DC-siRNA/LDH,
and CD-siRNA-5-FU/LDH on the viability of MCF-7 cells at the 5-FU concentration from 0-9.6
µg/ml and the CD-siRNA concentration at 40 nM in all relevant treatments for 72 h at 37oC.
Data represent mean ±SD (n=5). ** indicated p<0.05 versus 5-FU treatment, * p<0.01 versus 5FU treatment. **** p<0.0001 versus 5-FU treatment.
7
5-FU/LDH
5-FU
CD-siRNA-5-FU/LDH
CD-siRNA/LDH
Fig 6. FACS analysis of cell distribution for apoptosis and necrosis on MCF-7 cells after
treatment with 5-FU, 5-FU/LDH, CD-siRNA/LDH and CD-siRNA-5-FU/LDH for 24 h. The
concentration of 5-FU: 2.4 µg/ml and CD-siRNA: 40 nM.
8
LDH
A
5-FU
5-FU/LDH CD-siRNA/ CD-siRNA-5LDH
FU/LDH
Bcl-2
α-tub
B
Fig 7. Suppression of Bcl-2 protein expression in MCF-7 cells after single or combined
treatment with 5-FU and CD-siRNA delivered by LDHs.
(A) Western blot analysis after
transfection with 5-FU and CD-siRNA delivered by LDH for 24h. (B) Quantitative evaluation of
the percent change in expression levels of Bcl-2 protein against α-tubulin. The 5-FU, CD-siRNA
concentration in all samples was 2.4 μg/mL and 40 nM, respectively. LDH concentration is 50
μg/mL.
9
Supplement
Co-delivery of siRNA and an Anticancer Drug Using Layered Double
Hydroxide Nanoparticles in Effective Cancer Treatment
Li Lia,, Wenyi Gua,*, Jiezhong Chenb,c, Weiyu Chena, Zhi Ping Xua,*
a
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland,
Brisbane, QLD 4072, Australia
b
School of Biomedical Sciences, University of Queensland, St Lucia, QLD 4072
c
Faculty of Science, Medicine and Health, University of Wollongong, Northfields Avenue, NSW
2522, Australia.
Fax: 61 7 33463973; Tel: 617 33463809; E-mail: [email protected]; [email protected]
10
Lane 1
2
3
4
5
6
7
Ladder
dsDNA
LDH
1:5
1:10
1:20
1:40
Fig. S1 DNA-binding ability of LDH nanoparticles. Lane 1: DNA ladder, Lane 2: dsDNA only,
Lane 3: LDHs only, Lane 4-7: dsDNA/LDH with the dsDNA: LDH mass ratio from 1:5 to 1:40.
11
Nuclei
Red siRNA 456
Merged
A
B
Fig. S2 Confocal microscopy images of MCF-7 cells with Red siRNA 456/LDH nanonybris (A)
and Red siRNA 456-5-FU/LDH nanocomplex (B) cultured at 37oC for 4h. The scale bars are 50
μm. The mass ratio of the particles to Red siRNA 456 is 20:1.
12
C e ll V ia b ilit y ( % )
A
*
C e ll V ia b ilit y ( % )
B
*
Fig. S3 The effect of combined treatment with CD-siRNA and 5-FU co-delivered by LDHs on
viability of HCT-116 and U2OS after 72-hour treatment at 37oC. (A) HCT-116 cells and (B)
U2OS cells. Data represent mean ±SD (n=4 or 5).* indicated P<0.05.
13