Star poly(β-amino esters) obtained from the combination of linear poly(β-amino esters) and polyethyleneimine

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1 Supporting information Star poly(β-amino esters) obtained from the combination of linear poly(β-amino esters) and polyethyleneimine Xiaobei Huang,, Dezhong Zhou,,* Ming Zeng, Fatma Alshehri, Xiaolin Li, Jonathan O Keeffe-Ahern, Yongsheng Gao, Luca Pierucci, Udo Greiser, Guangfu Yin,,* Wenxin Wang,* Materials. Branched polyethyleneimine (PEI, Mw=25 kda and Mw= 800 Da, respectively), 1,4-butanediol diacrylate (B4), 5-amino-1-pentanol (S5) and 3-morpholinosydnonimine (MPA) were purchased from Sigma-Aldrich. Solvents dimethyl sulfoxide (DMSO) diethyl ether were purchased from Fisher Scientific. Deuterated chloroform (CDCl 3 ) was purchased from Sigma-Aldrich. Picogreen was purchased from Life Technologies, Cy3 DNA labelling kit was purchased from Mirus and used as per protocol provided by the manufacturer. Cell culture media, trypsin-edta, fetal bovine serum (FBS) and Hank s balanced salt solution (HBSS) were purchased from Sigma-Aldrich and Life Technologies. Cell secreted Gaussia Princeps luciferase plasmid (pcmv-gluc) and Green Fluorescent Protein plasmid (pcmv-gfp) were obtained from New England Biolabs UK, and its expansion, isolation and purification were performed using the Giga-Prep (Qiagen) kits as per

2 protocol. BioLuxTM Gaussia luciferase Assay Kit (New England Biolabs) and Alamarblue (Invitrogen) were used according to protocols. 4,6-diamidino-2- phenylindole (DAPI) was purchased from Life Technologies. All reagents were used according to the manufacturers protocols. Synthesis of PAE 5K and PEI-g-PAE. B4 (7.128g, 36 mmol) and S5 (3.09 g, 30 mmol) were dissolved in DMSO (10 ml) and the reaction was performed under stirring at 90 C for 5 hours. Subsequently, the crude product was precipitated into 50 ml diethyl ether to remove the monomers to obtain the LPAE base polymer. For comparison, the LPAE base polymer was divided into two portions. One half was subjected to endcapping with 3-Morpholinopropylamine (MPA, 432 µg, 3 mmol) in DMSO (20mL) overnight at room temperature and the other half was diluted with DMSO (80 ml). In the next step, a solution of 0.18g of PEI (Mw=800, 0.3mmol) in DMSO (20 ml) was added dropwise under stirring at 25 C for 2hours. Then, the mixture was further stirred for another 2 days, MPA (432 mg, 3 mmol) was added to end-cap the acrylate terminated base polymer at room temperature overnight and the polymer was dialyzed with (MWCO=8000) against acetone. Finally, the product was dried under vacuum for 24 h. 1 H NMR Measurement. Chemical structure and composition of the polymers were confirmed by 1 H NMR. The polymers were dissolved in deuterated chloroform (CDCl 3 ), measurements were carried out on a Varian Inova 400 MHz spectrometer and reported in parts per million (ppm) relative to the response of the solvent (7.24 ppm).

3 Gel Permeation Chromatography (GPC). Molecular weight (Mw and Mn) and polydispersity index (PDI) of polymers were measured by GPC (Agilent 1260 Infinity Multi-Detector GPC). A volume of 50 µl of reaction solution was collected, diluted with 1 ml of DMF, filtered with a 0.2 µm filter, and then measured on a PL-GPC 50 Integrated GPC system equipped with a refractive index detector (RI), a viscometer detector (VS DP) and a dual angle light scattering detector (LS 15 and LS 90 ) at 50 C with DMF (plus 0.1% LiBr) as elution solution at a flow rate of 1 ml min -1. Cell Culture. The rat adipose-derived stem cells (radsc) and human cervical cancer cells (HeLa) were cultured in Dulbecco s modified Eagle Medium (DMEM) containing 10% FBS and 1% Penicillin/Streptomycin (P/S), at 37 C and 5% CO 2, in a humid incubator using standard cell culture techniques. Transfection Experiments. Cells were seeded in 96-well plates at a density of cells/well in 100 µl of media and cultured until 70-80% confluence. The radsc stem cells used for transfecting were below passage four. For polyplex preparation, PEI-g-PAE and PAE 5K were dissolved in DMSO to 100 mg/ ml stock solution, PEI 25k and PEI 800 were dissolved in DMSO to 1mg/ ml stock solution. 0.5 µg of DNA per well was employed. Then, according to polymer/dna weight (w/w) ratio (10:1 to 20:1 for PEI-g-PAE and PAE 5K, respectively or 1:1 for PEI), the solution was further diluted with commercially available sodium acetate buffer (ph 5.2, M). 10 µl polymer solution was added into 10 µl DNA solution, vortexed for 10 s and incubated for 10 min. Then, 80 µl cell culture media containing 10% FBS were added into the

4 solution of the polyplexes. The media in the wells of cell culture plates were removed quickly and the diluted polyplex solutions were added. In the next step, polymer solution was added into DNA, vortexed, allowed to form complexes for min, and then diluted with media to 100 µl. The media in the wells of cell culture plates were removed quickly, the diluted polyplex solution was added and the cells were further incubated for 48 hours post transfection. Reporter Protein Expression. To visualize the transfected cells with the fluorescence microscope, pcmv-gfp was used to prepare the polyplexes and perform transfection as mentioned above. 48 h after transfection, the media were removed and cells were washed with HBSS for three times and then observed under fluorescence microscope (Olympus IX81). Gaussia Luciferase Expression. pcmv-gluc was used to prepare the polyplexes and transfection as mentioned above. The amount of Gaussia luciferase (Gluciferase) protein expressed in each well was performed as per the provided protocol. The Gluciferase activity in the cell supernatant was directly plotted in relative light units (RLU). Alamarblue Assay. The cytotoxity of transfected cells was determined by the Alamarblue reduction method. In these gene transfection experiments, the cells were incubated for 48 h. The supernatant was entirely removed and then the cells were washed with HBSS, followed by the addition of 100 µl of 10% Alamarblue in HBSS. Two hours later, the solution from each well was transferred to a fresh flat bottomed

5 96-well plate for fluorescence measurements at 590 nm. Control cells without polyplex treatment were plotted as 100% viable. Polyplex Cellular Uptake. To investigate the cellular uptake efficiency of polyplexes, GFP DNA was labelled with a Cy3 (a red fluorescent dye) labelling kit as per recommended protocol. HeLa cells were seeded in 96 well plates at a density of cells per well. Preparation of polyplexes and transfection were performed as mentioned above. 4 hours post transfection, the cells were fixed with 4% paraformaldehyde after washing with PBS for three times. In the next step, the cells were stained with DAPI followed by visualization under the fluorescent microscope (Olympus IX81). DNA Binding Affinity. Picogreen assays were used to measure the DNA binding affinity of the HPAEs and corresponding LPAEs. Polymers were initially dissolved in DMSO to give stock solutions as mentioned above. According to the w/w ratio, polymer and DNA (2 µg for each sample) were diluted with 30 µl sodium acetate buffer. Then, polymer solution was added into the DNA solution, mixed by vortex for 10 seconds and incubated for 10 minutes. 60 µl Picogreen solution (80 µl Picogreen diluted with ml sodium acetate buffer) was added and incubated for another 5 minutes. In a black 96 well plate, 200 µl DMEM without FBS and 30 µl polyplex/picogreen solution were added. Finally, fluorescence measurements with an excitation at 490 nm and an emission at 535 nm were performed. Polyplex Sizes and Zeta Potential. To measure polyplex sizes and zeta potentials, 2 µg DNA was diluted in sodium acetate buffer used for each sample preparation. The

6 polymer solution was mixed with DNA solution according to the selected w/w ratio for 10 seconds (Vortex) and then incubated for 10 minutes. Polyplexes were then diluted with 1 ml of DMEM with 10% FBS. A Malvern Instruments Zetasizer (Nano-2590) with a scattering angle of 90 was used to measure polyplex sizes and zeta potentials. Statistics. All results were presented as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance (ANOVA) for multiple group comparisons and a two-tailed Student s t-test was used to compare two groups. A value of *p < 0.05 was considered statistically significant.

7 Figure S1. 1 H NMR spectrum of purified PEI-g-PAE in CDCl 3 at 400 MHz

8 Figure S2. DNA binding affinity of PEI-g-PAE, PAE 5K, PEI 800 and PEI 25K

9 Figure S3. The corresponding size and zeta potentials of the PEI-g-PAE /DNA and PEI 800/DNA and PEI 25K/DNA polyplexes

10 Figure S4. Cell viability of radsc cells after transfection with PEI-g-PAE, PAE 5K, PEI 800 and PEI 25K determined by Alamarblue assays Eq. S1. Number of arms,,,