Low cytotoxicity of a fluorescent PAMAM dendrimer
used as a gene carrier for monitoring the delivery of
siRNA
Lingmei Guan · Saipeng Huang ·Zhao Chen ·Libo Du · Yanchao Li ·Ke Liu
·Yang Liu
L. Guan ·K. Liu
State Key Laboratory of Bio-resources and Eco-environment, The Ministry of
Education, College of Life Sciences, Sichuan University, Chengdu, 610064, P.R.
China.
S.Huang ··Y. Li ·Y. Liu · L. Du
State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Center
for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences,
100190 (P.R.China)
Z. Chen ·
School of Science, Xi’an Jiaotong University, Xi’an 71004 9(P.R.China)
Corresponding author: Yang Liu, Tel: +861062571074; Fax: +861062559373;
E-mail: yliu@iccas.ac.cn; dulibo@iccas.ac.cn
Experimental Section
Materials
Fluorescein was obtained from Sigma-Aldrich and used as
received
unless
otherwise
noted.
Methyl
alcohol
(GR),
dichloromethane, thionyl chloride were obtained from Beijing
Chemical work. Acrylic acid was obtained from Sinopharm
chemical Reagent co.Ltd. All reactions were conducted in
flame-dried glassware with magnetic stirring under an atmosphere
of dry nitrogen. Hydroxy-terminated Boc-PAMAM G3 was got from
previous work in our lab.
Methods
Ultraviolet-visible
(UV/Vis)
spectra
were
recorded
in
a
conventional quartz cell (light path 10 mm) by using a Hitach
UV-3310 spectrophotometer. 1H NMR spectra were recorded at 400
MHz on a Bruker Advance II 400 spectrometer (Bruker, S. A.
Wissembourg, Germany). Fourier transform infrared spectroscopy
(FTIR) was obtained by using a Bruker Tensor-27 Fourier-transform
infrared spectrometer. The samples were prepared as tablets by
using spectroscopic-grade KBr. 1H-NMR spectra were obtained
with a Bruker AVANCE 400 NMR spectrometer in D2O solution at
room temperature. Elemental analyses were performed on a Flash
EA 1112 instrument.
Synthesis of acryloyl chloride
Acrylic acid (5g, 69 mmol) dissolved in 30 mL dichloromethane in a
100 mL round bottom flask which was put into an ice bath. Under
violent stirring, thionyl chloride (10g, 84 mmol) was added dropwise
into this solvent. The reaction continued at room temperature for
another 24 hours. Then the solvent was distilled by a heating
mantle. Collect the distillation between 65°C and 75 °C, given the
acryloyl chloride 1 4.3 g. (47.5 mmol, yield 69%). (Scheme S1)
Scheme S1. The synthesis of acryl chloride.
Synthesis of fluorescein-acryloyl chloride ester
Fluorescein (3.9g. 12 mmol) ,with a mole ratio 1:4 to acryloyl
chloride (4.3g), was dissolved in 20 mL methyl alcohol. The
reaction was conducted at room temperature for 48 hours.
Scheme S2. The synthesis of fluorescein-acryl chloride ester.
The volatiles were removed under reduced pressure using a rotary
evaporator. The residue was separated and purified by column
chromatography. The desired product 2 was obtained at a yield
83% (4.4g). (Scheme S2)
Synthesis of Fluorescien-cored PAMAM Dendrimer
Synthesis of Deprotected G3 PAMAM Dendrimer
Boc-PAMAM G3 with hydroxy-terminated was got from previous
work and deprotected in the way that has been reported. In brief,
Boc-PAMAM (10 g) was dissolved in methyl alcohol which was
saturated by HCl. After stirred for 24 hours, the volatiles were
removed under reduced pressure using a rotary evaporator. Then
the deprodected monolateral PAMAM 3 was purified using a
dialysis bag, dried under vacuum. (Scheme S3)
Scheme S3. The synthesis of HO-terminated G3 monolateral
PAMAM
Synthesis of Fluorescein-cored PAMAM Dendrimer (F-PAMAM)
Monolateral hydroxy-terminated G3 PAMAM 3(8 g, 2.28 mmol) was
dissolved in 30 mL methyl alcohol. FACE 2(0.5g, 1.14 mmol) ,with a
mole ratio 1:3 to 3, was added to the above solvent. This reaction
was conducted at a temperature around 30 degree with stirring for
another 7 days. (Scheme S4) The volatiles were removed under
reduced pressure using a rotary evaporator. The desired product 4
was further purified with a dialysis membrane, dried in vacuo. (7.5g,
yield 89%)
Scheme S4. The synthesis of F-PAMAM
Figure S1. The 1H NMR of fluorescein and F-PAMAM
Figure S2. The DLS results of F-PAMAM and siRNA-PAMAM.
Figure S3. The TEM image of siRNA-F-PAMAM.
Figure S4. Analysis of F-PAMAM/siRNA complex formation at
various N/P ratios by agarose gel electrophoresis using 0.8%
agarose in Tris-acetate running buffer as the control.