Supplementary Information Temperature-responsive Gene Silencing by a Smart Polymer

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1 Supplementary Information Temperature-responsive Gene Silencing by a Smart Polymer Mingming Wang, Yiyun Cheng * Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, China. * yycheng@mail.ustc.edu.cn Table of Contents Contents: Page(s): 1, Table of Contents S1 2, Experimental Section Materials S2 Synthesis of G5-PBA 30 S2 Synthesis of G5-PBA 30 -pnipam 35 S2 Preparation and characterization of the polymer/sirna complexes S3 EB displacement assay S3 Cell culture S3 Gene silencing experiments S3 Cellular uptake S4 CLSM studies S4 Cytotoxicity assay S5 3, Supplementary Figures Supplementary Figures S1-S8 S6-S9 S1

2 Experimental Section Materials. Ethylenediamine (EDA)-cored and amine-terminated G5 PAMAM dendrimer stored in water was purchased from Dendritech (Midland, MI). RBITC, N-hydroxysuccinimide activated pnipam (molecular weight 2000 Da), 4-(bromomethyl) phenylboronic acid and bpei with a molecular weight of 25 kda were purchased from Sigma-Aldrich (St. Louis, MO). Lipofectamine 2000 (Lipo 2000) was purchased from Invitrogen (Carlsbad, CA). The sirna targeting firefly luciferase (sense: 5 -CCCUAUUCUCCUUCUUCGCdTdT-3 ), scrambled sirna non-specific to any human gene (sinc) and FAM-siRNA (with a FAM dye conjugated at the 5 end of sirna) were obtained from GenePharma Co. Ltd. (Shanghai, China). All the chemicals were used without additional purification. Synthesis of G5-PBA 30. G5 PAMAM dendrimer and 4-(bromomethyl) phenylboronic acid were dissolved in dehydrated dimethyl sulphoxide. The molecular ratio of 4-(bromomethyl) phenylboronic acid to G5 dendrimer is 32. The reaction was carried out in a flask at 70 C for 24 h. After that, the mixture was intensively dialyzed against dimethyl sulphoxide and distilled water. The products were lyophilized to obtain light brown gels and characterized by 1 H NMR (Bruker, MHz). Synthesis of G5-PBA 30 -pnipam 35. N-hydroxysuccinimide activated pnipam dissolved in dehydrated DMSO was slowly added into G5-PBA 30 DMSO solution. The molecular ratio of pnipam to G5-PBA 30 is 36. The mixture was stirred at room temperature for 24 h and then intensively dialyzed against DMSO and distilled water. The purified samples were lyophilized and characterized by 1 H NMR (Bruker, MHz). G5-PBA 30 -pnipam 18, G5-PBA 30 -pnipam 46, G5-PBA 30 -pnipam 56, G5-pNIPAM 34 and bpei-pba 47 -pnipam 134 were synthesized by a similar procedure. The feeding ratio of N-hydroxysuccinimide activated pnipam to polymer is 24, 36, 48, 60 and 36, respectively. S2

3 Preparation and characterization of the polymer/sirna complexes. All the polymer/sirna polyplexes were freshly prepared before characterization or gene silencing experiments. Generally, the polymers were mixed with 0.5 µg sirna in diethylpyrocarbonate-treated water at different weight ratios (w/w). The samples were incubated at 37 C for 30 min. Sizes of the polymer/sirna complexes was measured by Zetasizer Nano ZS (Malvern Instrument) at 37 C and 4 C, respectively. Sizes of the complexes prepared at 37 C and 4 C were also characterized by TEM (JEOL JEM-2100). EB displacement assay and in vitro release profiles of siluc. 5 µg sirna (siluc) were labeled with 10 µg EB before adding to 50 µg G5-PBA 30 -PNIPAM 35 solution. The complex solution was incubated at 37 C or 4 C for 20 min. The EB fluorescence intensity of the complex solution was measured using a fluorescence spectrometer (Hitachi F-4500) under the excitation wavelength of 470 nm. G5 PAMAM dendrimer without modification was tested as the control material (equal molar ratio). To investigate the sirna release profiles in vitro, G5-PBA 30 -pnipam 35 /sirna complexes were dissolved in 1 ml PBS solution and incubated at 37 C with gentle shaking. At each sampling time, the complex solutions were incubated at 37 o C or 4 o C for 10 min, respectively. Subsequently, the samples were centrifuged for 15 min at 13.2 krpm. The sirna concentration in the supernatant was analyzed by measuring the UV absorbance of the solutions at a wavelength of 260 nm (NanoDrop TM 2000, Thermo Fisher Scientific Inc.). Three repeats were conducted for each sample. Cell culture. HeLa-Luc (ATCC) cells were cultured in DMEM media containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/ml streptomycin at 37 C in humidified atmosphere containing 5% CO 2. The cells were cultured for 24 h before gene silencing experiments. Gene silencing experiments. S3

4 The polymer/sirna complexes were diluted with 100 µl serum-free media and incubated at 37 C for 30 min. The sirna concentrations used in the gene silencing experiments are 50 nm, 20 nm, 10 nm respectively. Before incubation with the cells, the complex solution was further diluted with 150 µl serum-free media (37 C). The cells were then incubated with the complex solution at 37 C for 1 h, 4 C for 1 h, and then 37 C for 4 h. After that, the cells were replenished with 500 µl serum-containing media (10% FBS). The gene silencing experiments were further continued for 18 h. For the control experiments without cool treatment, the cells were incubated with complex solution at 37 C for 6 h, followed by the replenishment with 500 µl serum-containing media (37 C), and further incubated at 37 C for 18 h. G5 PAMAM, G5-PBA 30, and Lipo 2000 were tested as control materials. Luciferase expressions in the cells were analyzed according to the manufacturer s protocols (Promega). Protein concentration in each well was determined using a BCA Protein Assay Kit (TIANGEN, China). The data was normalized to relative luciferase light unit per mg protein (RLU/mg protein), and relative to that of untreated cells. Three repeats were conducted for each sample. Students t-test was used to analyze statistically significant differences. Cellular uptake. HeLa-Luc cells were treated with the polymer/fam-sirna complexes for 1 h at 37 C. The cells were washed with cold PBS buffer for three times and trypsinized. The mean fluorescence intensity of transfected cells was quantitatively analyzed using flow cytometry (BD FACSCalibur, San Jose). Three repeats were conducted for each complex. CLSM studies. The intracellular sirna release from the G5-PBA 30 -pnipam 35 upon temperature decrease is observed by CLSM. G5-PBA 30 -pnipam 35 was conjugated with RBITC and mixed with FAM-siRNA at 37 o C to form polymer/sirna complexes. Two plates of HeLa-Luc cells were incubated with the complexes for 1 h at 37 o C, and then one of plate was moved to 4 o C for another 1 h, and the other plate was kept at 37 o C for S4

5 the same period. After that, the nuclei of cells were stained with a blue fluorescent dye (Hoechst-33342) at 37 o C. Co-localizations of the green and red fluorescent signals in the cells were observed by CLSM (Leica SP5, Germany). Cytotoxicity assay. Viability of cells treated with the synthesized polymers was determined by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Generally, HeLa-Luc cells seeded in 96-well plates were incubated with the polymers at different molar concentrations for 24 h. Unmodified G5 PAMAM dendrimer and G5-PBA 30 were tested as controls. Five repeats were conducted for each sample. Five repeats were conducted for each sample and the data were given as means ± S.E.M. For AO/EB double-staining assay, the cells were treated with polymer/sirna complexes at the concentration equal to that in optimal gene silencing experiments. Cool treatment on the cells at 4 o C for 1 h was conducted as described in gene silencing experiments. The cells were then stained with AO (5 µg/ml) and EB (5 µg/ml) for 3 min, washed twice with cold PBS, and observed by a fluorescent microscope (Olympus, Japan). S5

6 Supplementary Figures Figure S1. 1H NMR spectrum of G5-PBA30. Figure S2. TEM images of the G5 PAMAM/siRNA complexes prepared at 4 oc and 37 oc. S6

7 Figure S3. Temperature-responsive luciferase gene silencing on HeLa-Luc cells mediated by G5-PBA 30 -pnipam 35 at different polymer/sirna weight ratios. Figure S4. Cytotoxicity of G5-PBA 30 -pnipam 18, G5-PBA 30 -pnipam 46, and G5-PBA 30 -pnipam 56 on HeLa-Luc cells at different molar concentrations. S7

8 Figure S5. 1 H NMR spectra for G5-PBA 30 -pnipam 18, G5-PBA 30 -pnipam 46, G5-PBA 30 -pnipam 56 and G5-pNIPAM 34. Figure S6. Temperature-responsive luciferase gene silencing on HeLa-Luc cells mediated by G5-PBA 30 -pnipam 18, G5-PBA 30 -pnipam 46, and G5-PBA 30 -pnipam 56 at optimal conditions (***p<0.005 according to relative luciferase activity). S8

9 Figure S7. Temperature-responsive luciferase gene silencing on HeLa-Luc cells mediated by G5-PBA 30 -pnipam 35 and Lipo 2000 at different sirna concentrations of 50nM, 20 nm and 10 nm respectively. **p<0.01, ***p<0.005 and n.s. non-statistical differences analyzed by Student s t-test. Figure S8. (a) Chemical structure of bpei-pba 47 -pnipam 134. (b) 1 H NMR spectrum of bpei-pba 47 -pnipam 134. (c) Temperature-responsive luciferase gene silencing on HeLa-Luc cells mediated by bpei-pba 47 -pnipam 134. S9