Supporting Information for
Recycling Gene Carrier with High Efficiency and Low Toxicity
Mediated by L-Cystine-Bridged Bis(β-cyclodextrin)s
Yu-Hui Zhang,a Yong Chen,a Ying-Ming Zhang,a Yang Yang,a Jia-Tong Chen,b and Yu Liu a*
a
Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai
University, Collaborative Innovation Center of Chemical Science and Engineering, and
b
Department of Biochemistry and Molecular Biology, College of Life Sciences, Tianjin
300071, P. R. China
*
Address correspondence to yuliu@nankai.edu.cn
S1
Experimental Section
Instruments. NMR spectra were recorded on Bruker AV400 instruments. Gel
electrophoresis was run on a 1% (w/v) agarose gel at 60 V for 1h and photographed by means
of a UV transilluminator and WD-9415B gel documentation system (Beijing Liuyi Instrument
Factory, P. R. China). For the AFM measurements, 10 μL of sample solution was dropped
onto newly clipped mica and air-dried, then the sample was examined using an atomic force
microscope (Veeco Company, Multimode, Nano IIIa) in tapping mode in the air under
ambient conditions. High-resolution transmission electron microscope (HR-TEM) images
were obtained on a Tecnai G2 F20 microscope (FEI) with an acceleratig voltage of 200 kV.
The samples were prepared by placing a drop of solution onto a carbon-coated copper grid
and air-dried. The sample solutions for DLS measurements were prepared by filtering the
solution through a 0.45 μm filter (JET BIOFIL) into a clean cuvette. The samples were
examined with scattering spectrometer (Brookhaven company) at λ648 nm and a scattering
angle of 90 °C. The zeta potential were recorded on NanoBrook 173Plus (Brookhaven
company) at 25 °C. The fluorescence microscope images were recorded on Nikon ECLIPSE
TE2000-U with a CCD camera. The gene transfection efficiencies were determined with a
flow cytofluorometer (BD FACS Calibur) equipped with an argon laser at λ488 nm, and
10000 cells were counted. The fluorescent confocal images were performed on a Leica TCS
SP8 fluorescence microscope at λex = 405 nm for DAPI, λex = 561 nm for rhodamine.
Synthesis of adamantyl polyethyleneimine (PEI-Ada). EDC (144.54mg, 0.754 mmol)
and NHS (86.78 mg, 0.754 mmol) were added to solutions containing various amounts of
1-adamantaneacetic acid (56.34-112.68 mg, 0.29-0.58 mmol) in ethanol (20 mL), and the
S2
mixture was stirred at 25°C under an atmosphere of N2 for 30 min. Then a solution of
polyethyleneimine (Mw = 10000) (100 mg, 2.32 mmol) in ethanol (10 mL) was added, and the
mixture was stirred for 24 h at room temperature. The resulting solution was dialyzed against
an excess amount of ethanol for 5 days. After being freeze-dried, PEI-Ada with different
degrees of substitution was obtained as a white power. 1H NMR ( 400 MHz, D2O, ppm): (1)
PEI-Ada-8.2: δ 1.60-1.71 ( m, 12H, H of methylene of Ada), 2.00 (d, 5H, H of methyne of
Ada and H of methylene of amido bond), 2.48-3.39 (m, 113H, H of methylene of
polyethyleneimine and H of amino of amido bond); (2) PEI-Ada-10: δ 1.60-1.71 (m, 12H, H
of methylene of Ada), 2.00 (d, 5H, H of methyne of Ada and H of methylene of amido bond),
2.48-3.39 (m, 93H, H of methylene of polyethyleneimine and H of amino of amido bond); (3)
PEI-Ada-12.2: δ 1.60-1.71 ( m, 12H, H of methylene of Ada), 2.00 (d, 5H, H of methyne of
Ada and H of methylene of amido bond), 2.48-3.39 (m, 76H, H of methylene of
polyethyleneimine and H of amino of amido bond); (4) PEI-Ada-13.7: δ 1.60-1.71 ( m, 12H,
H of methylene of Ada), 2.00 (d, 5H, H of methyne of Ada and H of methylene of amido
bond), 2.48-3.39 (m, 68H, H of methylene of polyethyleneimine and H of amino of amido
bond).
Cell culture. 293T human embryonic kidney cells and HeLa human cervical carcinoma cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) at 37 °C under a humidified atmosphere with 5% CO2.
Cytotoxicity experiments. 293T cells and HeLa cells were seeded in 96-well plates (1 × 105
cells mL-1, 100 μL per well) for 24h at 37 °C in 5% CO2. The cells were incubated with
PEI-Ada-13.7, PEI-Ada-LCD and 25 kDa bPEI at different concentrations for another 24 h.
S3
Then 50 μL of MTT solution (5 mg/mL) was added into each well. The cells were further
cultured for 4 h, then the medium was removed, and 100 μL of DMSO was added into each
well. After 15 min, the absorbance of the dissolved formazan was measured with a Bio-Rad
microplate reader at 490 nm. All the experiments were carried out in triplicate, and the data
were presented as the mean results ± standard deviation.
Confocal fluorescence images. HeLa cells were seeded on 14 mm2 coverslips that were
placed in 6-well plates (2 × 105 cells mL-1, 1 mL per well) for 24 h at 37 °C in 5% CO2. The
cells
were
incubated
with
PEI-Ada-13.7@Rhodamine-labled
DNA,
PEI-Ada-LCD@Rhodamine-labled DNA at an N/P ratio of 20 for 4 h (3.2 μg DNA per
well). Then the medium in each well was replaced with 1 mL of fresh DMEM medium with
10% FBS. The cells were further incubated for 20 h. Then the culture medium was removed,
and the cells were washed with PBS for three times and fixed with 4% paraformaldehyde for
15 min. Then the cell nuclei were stained with DAPI (1 μg/mL) for 15 min. The cells were
subjected to observation by a confocal laser scanning microscope (λex = 405 nm, 561 nm).
S4
Figure S1. 1H NMR spectra of PEI-Ada-(a) 8.2, (b) 10, (c) 12.2 and (d) 13.7 in D2O at 25 °C.
Figure S2. 1H NMR spectra of PEI-Ada-(a) 33, (b) 15.5 in CDCl3 at 25 °C.
S5
Figure S3．1H NMR spectral titration of AdAA with β-CD in D2O containing 0.9% sodium
chloride at 25 °C (a) 1H NMR spectra of 1 mM AdAA upon addition of 0, 0.2, 0.4, 0.6, 0.8,
1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 mM β-CD from 1 to 12, (b) Nonlinear least-squares analysis of
the chemical shift changes of the adamantyl proton peak at 9 as a function of the
concentrations of β-CD.
S6
Figure S4. (a) 1H NMR spectra of AdAA (1 mM) upon addition of 0, 0.2, 0.4, 0.6, 0.8, 1.0,
1.2, 1.4, 1.6, 1.8, 2.0, 2.2 mM β-CD in D2O containing 5% glucose at 25 °C. (b) Nonlinear
least-squares analysis of the chemical shift changes of the adamantyl proton peak at 9 as a
function of the concentrations of β-CD.
S7
Figure S5. TEM images of (a) PEI-Ada, (b) PEI-Ada-LCD, (c) PEI-Ada-LCD treated by 10
mM DTT, (d) PEI-Ada-LCD + 10 mM DTT after treated by 50 U/ml HRP for 4 h at 25 °C.
S8
Figure S6. (a) Particle sizes and (b) zeta potentials of PEI-Ada-13.7@pDNA,
PEI-Ada-13.7-LCD@pDNA, PEI-Ada-137-LCD@pDNA + 10 mM DTT complexes at
different N/P ratios.
S9
Figure S7. Zeta potential of (a) PEI-Ada-13.7: 39.38 mV, (b) PEI-Ada-13.7-LCD: 40.65 mV.

Figure S8. TEM images showing the condensation effect of the polycations (a), (c)
PEI-Ada-13.7; (b), (d) PEI-Ada-13.7-LCD at the N/P ratios of 10 and 20, respectively.
S10
Figure S9. Agarose gel electrophoresis of PEI-Ada-LCD@pDNA complex (a) in the absence
and (b) in the presence of DTT.
Figure
S10.
Gel
electrophoresis
of
(a)
PEI-Ada-13.7@pDNA,
(b)
PEI-Ada-13.7-LCD@pDNA, (c) PEI-Ada-13.7-LCD@pDNA + 10 mM DTT (N/P ratio = 20)
S11
in the presence of heparin.
Figure S11. Gene transfection efficiency of PEI-Ada-13.7 and PEI-Ada-13.7-LCD in (a)
293T and (b) HeLa cells at the N/P ratios of 10, 20, 30 in the presence of serum. 25 kDa bPEI
at N/P ratio of 10 was used as positive control.
S12
S13
Figure S12. Flow cytometry analysis of EGFP expression in 293T cells in the presence of
serum by (a)-(c) PEI-Ada-13.7 at N/P ratios of 10, 20, 30; (d)-(f) PEI-Ada-LCD at N/P ratios
of 10, 20, 30; (g) 25 kDa bPEI at a N/P ratio of 10.
Figure S13. Fluorescence microscopy images of HeLa cells transfected by (a)-(c)
PEI-Ada-13.7; (d)-(f) PEI-Ada-LCD; (g)-(i) 25 kDa bPEI after 48h at N/P ratios 10, 20, 30 in
the presence of serum.
S14
S15
Figure S14. Flow cytometry analysis of EGFP expression in HeLa cells in the presence of
serum by (a)-(c) PEI-Ada-13.7 at N/P ratios of 10, 20, 30; (d)-(f) PEI-Ada-LCD at N/P ratios
of 10, 20, 30; (g) 25 kDa bPEI at a N/P ratio of 10.
Figure S15. Fluorescence microscopy images of 293T cells transfected by (a)-(c)
PEI-Ada-13.7; (d)-(f) PEI-Ada-LCD after 48h at N/P ratios 10, 20, 30 in the absence of
serum.
S16
Figure S16. Flow cytometry analysis of EGFP expression in 293T cells in the absence of
serum by (a)-(c) PEI-Ada-13.7 at N/P ratios of 10, 20, 30; (d)-(f) PEI-Ada-LCD at N/P ratios
of 10, 20, 30; (g) 25 kDa bPEI at a N/P ratio of 10.
S17
Figure S17. Fluorescence microscopy images of Hela cells transfected by (a)-(c)
PEI-Ada-13.7; (d)-(f) PEI-Ada-LCD after 48h at N/P ratios 10, 20, 30 in the absence of
serum.
S18
Figure S18. Flow cytometry analysis of EGFP expression in HeLa cells in the absence of
serum by (a)-(c) PEI-Ada-13.7 at N/P ratios of 10, 20, 30; (d)-(f) PEI-Ada-LCD at N/P ratios
of 10, 20, 30; (g) 25 kDa bPEI at a N/P ratio of 10.
S19
Figure S19. Fluorescent microscopy images of 293T cells transfected with PEI-Ada-LCD at
N/P ratio of 20 for 24 h. PEI-Ada-LCD was incubated (a) without DTT; (b) with 5 equiv of
DTT for 1 h at 37 °C before complexation with EGFP gene.
Table S1. Characterization of PEI-Ada with Different Degrees of Substitution.
Sample
Feed ratioa
DS
Mwb
PEI-Ada-33
2
33
15816.9
PEI-Ada-15.5
0.5
15.5
12732.2
PEI-Ada-13.7
0.25
13.68
12411.4
PEI-Ada-12.2
0.2
12.2
12150.5
PEI-Ada-10
0.15
10
11762.7
PEI-Ada-8.2
0.125
8.23
11454.2
a
Molar feed ratio of 1-adamantaneacetic acid to PEI. bMolecular weights were estimated by
1
H NMR spectrum.
S20