Calcium Mediated Formation of Phosphorylcholine-based Polyplexes ...

Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2014

Supplementary Information

Calcium Mediated Formation of Phosphorylcholine-based Polyplexes for Efficient Knockdown of Epidermal Growth Factor Receptors (EGFR) in Hela Cells

Marya Ahmeda, Kazuhiko Ishiharab and Ravin Naraina* a

Department of Chemical and Materials Engineering, Alberta Glycomics Centre, University of Alberta, 116 St and 85 Ave, Edmonton, AB, T6G 2G6, Canada, bDepartment of Materials

Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

To whom correspondence should be addressed E-mail: [email protected] Phone: (780) 492-1736 Fax: (780) 492-2881


Experimental Procedures Materials and Methods: MPC was obtained from NOF Co, Tokyo, Japan, which was produced by the previous reported method. MTT assay kit to determine cell viability, Dulbecco’s phosphate buffer saline (DPBS), formalin (37 wt.), 4,4′-Azobis(4-cyanovaleric acid) (ACVA), tween-20, triton-X 100 and ethidium bromide were purchased from Sigma Aldrich. 3gluconamidopropyl methacrylamide (GAPMA), and 4-cyanopentanoic acid dithiobenzoate (CTP) were synthesized as described according to previous reports. HPLC grade water, acetone, and N,N’-Dimethylformamide (DMF) were purchased from Caledon Laboratories (Canada). Branched PEI (M w = 25 kDa) was purchased from Polysciences Inc. Cell Culture media DMEM/F12 with L -glutamine and sodium pyruvate), penicillin (10000 U/mL), and streptomycin (10 mg/mL), 0.25% trypsin-EDTA, Dulbecco’s modified Phosphate Buffer Saline (DPBS), OptiMEM (OMEM) and Fetal Bovine Serum (FBS) were from Invitrogen. Human epidermal growth factor receptor specific small interfering RNA (EGFR siRNA), control-siRNA-FITC conjugate, and primary antibody (rabbit polyclonal EGFR specific IgG) were obtained from Santa Cruz Biotechnology. Horse raddish peroxidase (HRP) conjugated, secondary antibody (Anti-rabbit IgG), HRP-stabilized (3,3´, 5,5´-tetramethylbenzidine) (TMB) substrate and blot qualified bovine serum albumin (BSA) were purchased from Promega. Calcium Chloride dihydrate (CaCl 2 .2H 2 O) Phosphoric acid, tris-base, sodium chloride, and potassium chloride were from Fisher Scientific. Synthesis of linear 2-Methacryloyloxyethyl Phosphorylcholine polymer Polymerization of PMPC was done via RAFT at 60°C. ACVA and CTP were used as the initiator and chain transfer agent, respectively. In a typical setup MPC (0.5 g, 1.69 mmol), CTP (


0.0027 g, 9×10-3 mmol, target DP n =175), and ACVA (0.00054 g, 1.9×10-3 mmol) were all dissolved in 1.5mL methanol and placed inside a 10mL schlenck tube. The solution was degassed in N 2 for 30 minutes and then placed in an oil bath at 60°C for 15 hours. The reaction was quenched using liquid nitrogen and precipitated in a large amount of acetone. The polymers were dissolved in water and were freeze dried overnight. Molecular weight M n and molecular weight distribution (M w /M n ) were obtained from aqueous gel permeation chromatography (GPC), using a Viscotek conventional GPC instrument connect to two waters Ultrahydrogel linear WAT011545 columns (pore size, blend; exclusion limit of 7.0×106) and with a Viscotek model 250 dual detector. An acidic buffer of 0.50M sodium acetate/0.50M acetic acid was used as eluent. Calibration of the GPC was done by six monodisperse polyethylene oxide standards (M p – 1.01×103- 1.01×105 g mol-1). Synthesis of Linear P(MPC 19 -st-GAPMA 51 ): Copolymerization of MPC with GAPMA was done via RAFT at 70°C. In a typical setup, MPC (0.5 g, 1.69 mmol), GAPMA (0.27g, 0.84 mmol) CTP (0.0027 g, 9×10-3 mmol, target DP n =175), and ACVA (0.00054 g, 1.9×10-3 mmol) were all dissolved in 3 mL water and 0.6 mL of DMF, in a 10mL schlenck tube. The solution was degassed in N 2 for 30 minutes and then placed in an oil bath at 70°C for 15 hours. The reaction was quenched using liquid nitrogen and precipitated in a large amount of acetone. Molecular weight M n and molecular weight distribution (M w /M n ) were obtained from aqueous gel permeation chromatography (GPC), using a Viscotek conventional GPC instrument connect to two waters Ultrahydrogel linear WAT011545 columns (pore size, blend; exclusion limit of 7.0×106) and with a Viscotek model 250 dual detector. An acidic buffer of 0.50M sodium acetate/0.50M acetic acid was used as eluent. Calibration of the GPC was done by six monodisperse polyethylene oxide standards (M p – 1.01×103- 1.01×105 g mol-1).


Inversion of PMPC Charges: Homopolymers of PMPC were dissolved in deionized water or 10 mM CaCl 2 .2H 2 O solution at 20 mg/mL concentration. The charges of PMPC were determined using Brookhaven zeta potential instrument. Formulation of Polyplexes. The homopolymers of PMPC (in water and in 10 mM CaCl 2 .2H 2 O solution) were complexed with 1.2 µg β-galactosidase plasmid in OMEM or water at varying polymer\plasmid ratios. The mixture was incubated at room temperature for 30 minutes and was analyzed by Brookhaven DLS and zeta potential instrument for their net size and charges, respectively. Agarose Gel Electrophoresis The polyplexes of formed with β-galactosidase plasmid with MPC based polymers (in water or 10mM CaCl 2 .2H 2 O solution) were formulated in OMEM at varying polymer/plasmid ratios, as described above. The polyplexes were loaded in 1% agarose gel containing 1µg/mL ethidium bromide in 1X tris acetate/EDTA buffer. The gel was run for 45 minutes at 130 V. The gel was illuminated with UV light and the DNA bands were visualized using UV transilluminator (Alpha Innotech; San Leandro, CA). Formulation of MPC-siRNA complexes. EGFR-siRNA(133 ng, 0.1 nmoles) was combined with homopolymers of MPC (in water or OMEM media) at varying weight/weight ratio and the mixture was incubated at 25 ºC for 30 min. Sizes and charges of PMPC-siRNA complexes were analyzed using Brookhaven DLS and zeta potential instrument. The net charge of complexes was studied in deionized water. Transmission Electron Microscopy (TEM): Size and morphology of complexes formed by MPC based homopolymers/copolymer and siRNA were analyzed at room temperature by Transmission Electron Microscopy on Philips transmission electron microscope operated at 80


kV and fitted with a CCD camera. A droplet of a properly diluted siRNA complexes (in water) was placed on the TEM carbon coated copper grid and allowed to air dry before it was stained with phosphotungstic acid (PTA) (1 wt% aqueous solution) for 1 min. The sample was then allowed to dry overnight prior to observation. Cell Culture Hela cells were maintained in DMEM/F12 media supplemented with 10% FBS and 1% antibiotic, in a humidified atmosphere and 5% CO 2 at 37 °C. Upon 80% confluency, Hela cells were trypsinized with 0.25% trypsin with EDTA, were cultured twice a week and were seeded in 96 well tissue culture plates. EGFR siRNA Knockdown Hela cells were seeded in 96 well tissue culture plates at the cell density of 5000 cells/well. The cells were allowed to adhere overnight. The media was then changed and 50 µL of OMEM (in the presence or absence of serum proteins) was added per well. The PMPC/P(MPC 19 -stGAPMA 51 )-siRNA complexes were formulated in OMEM as described above. 50 µL of complexation mixture containing 133ng of siRNA or control siRNA and varying amount of polymer was then added per well. The cells were incubated for 6 hours and the media was then changed and 100 µL of fresh serum containing media was then added. The cells were allowed to grow for another 48 hours followed by their analysis for siRNA knockdown efficiency. In-Cell Enzyme Linked Immunosorbent Assay (ELISA) All the buffers were prepared in house using previously reported protocol and are mentioned below. 1X TBS of pH 7.4 (0.1M NaCl, 2mM KCl, and 20mM tris-base), permeabilization buffer (10% tritonX-100 in 1X TBS (1:99 dilution)) wash buffer (10% tween in 1X TBS (1:99 dilution)), blocking buffer (2 wt. % BSA in 1X TBS), quenching solution (10% H 2 O 2 in 1X TBS), and primary anti-body dilution buffer (1:1 (v/v) ratio of blocking buffer and wash buffer)


were prepared. 48 hours post-siRNA delivery the media was removed and cells were fixed in 96 well plate using 3.7% formalin in 1X TBS at room temperature for 15 minutes. The cells were washed twice with 1X TBS and were permeabilized for 15 minutes using permeabilization buffer. The cells were then washed once with 1X TBS and were quenched for 20 minutes using quenching solution. The cells were washed once with TBS and blocking solution was then added for 30 minutes. The primary antibody rabbit polyclonal EGFR-IgG was diluted in dilution buffer (1:250 dilutions) and 50 µL of this solution was added per well. 50 µL of dilution buffer alone was added per well for the negative control. The plate was sealed and was incubated at room temperature for 1 hour. The wells were then washed with 100 µL of wash buffer 3X, followed by the addition of 100 µL of HRP-conjugated secondary antibody solution (1:10,000 dilution in wash buffer). The plate was sealed and was incubated at room temperature for 30 minutes. The plate was then washed with 200 µL of wash buffer per well three times, followed by the addition of 100 µL of TMB substrate per well. The plate was incubated in the dark for 20 minutes (formation of blue precipitates). 0.1 M phosphoric acid solution (100 µL/well) was then added and plate was incubated for another 20 minutes. The yellow color formed (indicating the presence of EGFR receptors) was recorded using TECAN microplate reader at λ = 420 nm. Nonspecific interactions (in the absence of primary antibody) are subtracted from positive controls and treated samples. %EGFR receptors on treated cells are calculated = (treated samples/positive control)*100 Janus Green Assay of Cell Viability Janus green solution was prepared by dissolving 0.3% janus green dye in distilled deionized water. The plate content (after siRNA analysis) was removed and plate was washed with distilled deionized water 3X. 100 µL of Janus green solution was added per well and plate was incubated


at room temperature for 5 minutes. The plate was washed 3-5X with distilled water followed by the addition of 0.5 M HCl solution for 10 minutes. The plate was read at 570 nm using TECAN microplate reader. % Cell viability was obtained = (treated cells-negative control)/(positive control-negative control)


Scheme S1. Chemical structures of monomers, chain transfer agent (CTP) and initiator (ACVA).

O

HN

O

O

O O P O HO HO

O

OH HN OH

O

+ N

HO

GAPMA S

MPC

CN S

COOH

N

HOOC NC

CTP

ACVA

CN N

COOH


Scheme S2. RAFT Synthesis of MPC homopolymer and copolymer.


Table S1. Molecular weights and compositions of the polymers as determined by GPC and 1HNMR. Polymer Composition

GPC M n (g/mol)

M w /M n

P(MPC) 91

27,000

1.23

P(MPC) 257

76,000

1.19

P(MPC 19 -st-GAPMA 51 )

22,000

1.37


Figure S1: DLS and zeta potential data of polyplexes in water. P(MPC) 257 -pDNA complexes were prepared at w/w ratio of 2000 and P(MPC 19 -st-GAPMA 51 )-pDNA complexes were prepared at w/w ratio of 667.


Figure S2: TEM images of polyplexes after staining with 1% PTA solution in water. P(MPC) 91 siRNA complexes were prepared at w/w ratio of 206 and P(MPC 19 -st-GAPMA 51 )-siRNA complexes were prepared at w/w ratio of 750.


Figure S3: Agarose gel electrophoresis showing complexation of control siRNA with 10 mM CaCl 2 solution at varying weight by weight ratios of Ca2+ to siRNA.


Figure S4: Relative % cell viability of MPC-siRNA complexes, at varying w/w ratios and 0.1 nmoles siRNA concentration, 72 hours post-transfection, as determined by Janus green assay.