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Supplementary Materials and Methods
Materials for Polymer Synthesis. Chemicals and all materials were supplied by Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Cyano-4-(ethylsulfanylthiocarbonyl)sulfanyl pentanoic acid (ECT) was synthesized as described previously (20). Biotin-dPEGTM3-NH3+TFA- was obtained from Quanta Biodesign (Powell, Ohio). 2,2′-Azobis(4-methoxy-2.4-dimethyl valeronitrile) (V-70) was obtained from Wako Chemicals (Richmond, VA).
Synthesis of N-hydroxy succinimide activated ECT. A solution of ECT (2 g, 7.59 mmol) and N-Hydroxysuccinimide (NHS) (2.6206 g, 22.77 mmol) in chloroform (200 mL) was purged by nitrogen for 1 hour and cooled over an ice bath. N,N′-Dicyclohexylcarbodiimide (6.2642 g, 30.36 mmol) was slowly added while vigorously stirring the mixture. The reaction remained in the ice bath for 1 hour, then removed and allowed to react at room temperature for 22 hours. The reaction mixture was filtered and the organic solvent was removed by vacuum. The NHS activated ECT was redissolved in chloroform (200 mL) and propargylamine (0.1808 g, 3.28 mmol) was then slowly added. The reaction mixture was stirred at room temperature for 18 hours. Chloroform (100 mL) was added and the mixture was extracted with an aqueous solution of hydrochloric acid (1/10 v/v, 5 x 100 mL), 10 wt% sodium hydroxide (5 x 100 mL) and ddH2O water (2 x 100 mL). The chloroform solution was dried over magnesium sulfate. After solvent removal by vacuum, three successive column chromatographies (Silica gel 60) were performed (ethyl acetate/hexanes (75%/25%), ethyl acetate (100%), chloroform/methanol (98%/2%). After drying under vacuum, a viscous orange-red oil was obtained (yield 92%).1H NMR 300MHz (CDCl3, RT, ppm) (see structure below for proton assignment): 1.35 (t, 3 H, CH3); 1.89 (s, 3 H, CH3); 2.2-2.6 (m, 5 H, CH2CH2, CH); 3.34 (q, 2 H, CH2); 4.00 (m, 2 H, CH2); 5.93 (b,1 H, NH).
Synthesis of BIOTIN-PEO3-ECT. The synthesis of the biotinylated RAFT chain transfer agent was accomplished according to a related previously described procedure (22-23). BrieflyBiotin-dPEGTM3-NH3+TFA- (2.0 g, 3.567 mmol) and triethylamine (720 mg, 7.1 mmol) in 200 mL chloroform was added dropwise in three equal portions 20 minutes apart to a stirred solution of N-hydroxy succinimide activated ECT (1.41 g, 3.92 mmol) in 350 mL chloroform. The reaction was allowed to stir at room temperature for 18 hours. The chloroform was then removed by rotary evaporation and the product isolated by column chromatography (silica gel stationary phase, 9:1 v/v CH2Cl2:CH3OH). 1H NMR: (CDCl3) δ 1.38 t (SCH2CH3); δ 1.93 s (CCNCH3); δ 2.3–2.65 m (CH2CH2); δ 3.35 q (SCH2CH3 convoluted). 4.35-4.6 (m, CHSCH2); δ 2.75-2.9 (dd, CHCH); δ 5.15-5.79 (S, NHCONH); δ 2.85-291 (d, CHCHCH); δ 2.72-2.74 (dd, CHCHCH2); δ 4.28-4.54 (SCH2CH); δ 3.14 (dt, CH2CHS); δ 2.23 (t, COCH2CH2CH2CH2); δ 1.44 (dt, CH2CHS); δ 1.68 (overlapping multiplet, COCH2CH2CH2CH2); δ 3.5-3.6 (CH2CH2CH2(OCH2CH2)3, multiplets).
HABA assay. A modification of the HABA (4-hydroxyazobenzene-2’carboxylic acid) assay was used to measure available biotin sites on polymeric micelles. HABA dye bound to streptavidin (HABA-SA) absorbs light at 500 nm wavelength (A500). Biotin competitively displaces HABA from HABA-SA resulting in reduced A500. A competitive HABA assay was used whereby mAb-SA bearing polymeric micelles were formed by incubating polymer with siRNA 30 minutes prior to addition of mAb-SA at varying concentrations. This mixture was then added to HABA-SA solution containing 265 M HABA and 10.9 M streptavidin. The solution was vortexed briefly then incubated for 15 minutes. The A500 was measured and absorbance difference was calculated by subtracting A500 of polyplex solution from A500 of HABA-SA solution. Unbound biotin sites on polymeric micelles displace HABA from HABA-SA resulting in reduced A500 reading. Conversely, excess unbound mAb-SA binds to free HABA and is measured by increased A500.
Complexation of siRNA by polymer. Binding of siRNA by polymer was shown via agarose gel electrophoresis demonstrating retardation of siRNA migration after incubation with polymer. Briefly, 200 ng of siRNA was incubated either alone or with increasing concentrations of polymer with or without mAb-SA conjugate for 30 minutes at room temperature. Complexes were loaded into a 2% agarose gel and run for 1.5 hours at 60V in 1X TAE buffer, pH 7.4 and subsequently stained in Sybr Green II RNA Gel Stain (Invitrogen, 1:5000 dilution in TAE buffer).
Binding of mAb-SA conjugate to polymer. Protein electrophoresis under non-denaturing conditions was used to evaluate binding of mAb-SA conjugate to polymer. 8.4 picomoles of HD39-SA conjugate was incubated for 30 minutes in the presence of 211 picomoles of polymer alone or polymer complexed to 20 picomoles of siRNA. Complexes were diluted 1:1 with 2X Tris Glycine Native Sample Buffer then loaded into a 3-8% NuPage Tris Acetate gel (Invitrogen) and run for 1 hour at 150V in 1X Tris Glycine Native Running Buffer (Invitrogen). The gel was subsequently stained with Coomassie Blue protein stain.
Polymeric micelle internalization and microscopy in HeLa-R cells. HeLa-R cellswere plated in 12 well plates at 50,000 cells per well in 500 l of media the day before transfection. Media was changed on the day of transfection. Complexes were formed by incubating non-biotinylated polymer with Silencer Cy3-labeled Negative control siRNA #1 (AM4621; Ambion) at a 5:1 polymer to siRNA molar ratio and added to HeLa-R cells for a final siRNA dose of 100 nM. Treated cells were trypsinized after 24 hours of treatment then cytospun on slides, dried, then fixed with 10% neutral buffered formalin and rinsed in PBS prior to being coverslipped with Prolong Gold antifade reagent with DAPI (Invitrogen). Random fields were imaged using a Nikon Eclipse E800 wide-field fluorescence microscope fitted with 100x/1.30 PlanFluor numerical aperture objective. Images were collected with MetaMorph software (Molecular Devices). Scale bars were added with public domain software ImageJ.
Cell viability assessment. HeLaR CD22 cells were plated the day before treatment in white 96-well assay plateswith clear bottoms at a density of 3,000 cells per well in 100 l of culture media. On the day of treatment, the media was changed and cells treated in quadruplicate with polymeric micelles at 15 nM siRNA final dose in 100 l volume. Cells were harvested 24 hours after treatment and luminescence readings obtained according to the CellTiter-Glo kit manufacturer’s protocol (Promega). Briefly, the plate was equilibrated to room temperature for 30 minutes prior to the addition of 100 l of CellTiter-Glo reagent to each well. Plates were protected from light then mixed thoroughly to lyse cells. After 10 minute incubation at room temperature, luminescence was measured using a Centro LB 960 microplate luminometer (Berthold Technologies).
Transfections and qRT-PCR for NAC1 and Mcl-1. Transfections for NAC1 and Mcl-1 were performed as described for GAPD siRNA in the Methods section. Sequences for the siRNAs used are as follows: Myeloid Cell Leukemia sequence 1, Mcl-1 (sense strand 5’-CCAGUAUACUUCUUAGAAATT-3’; Ambion); Nucleus Accumbens Associated 1, NAC1 (sense strand 5’-CGAGAAAUUGCACAACCGATT-3’; Ambion). RNA was extracted using the RNeasy Mini Kit (Qiagen). Quantitative RT-PCR was performed and analyzed as described in the Methods section. FAM-labeled probe and primers were obtained as 20X primer-probe mixes from Applied Biosystems: Mcl-1 (Catalog#Hs00172036_m1), NAC1 (Catalog#Hs00369413_m1).
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