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: ;

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.