Supporting Information

Siegwart et al. 10.1073/pnas.1106379108 Additional Experimental Details TDA 305-040 triple detector, VE 7510 degasser, VE 1122 pump, Materials. Oligo(ethylene glycol) methacrylate 300 (OEOMA300, and VE 5200 autosampler. In the case of homo- and diblock poly(ethylene glycol) methyl ether methacrylate, Mn ¼ 300 g∕ copolymers, samples were eluted though three GPMWxL col- mol), oligo(ethylene glycol) methacrylate 475 (OEOMA475, poly umns (Viscotek) in series at a flow rate of 1 mL∕ min in 0.05M (ethylene glycol) methyl ether methacrylate, Mn ¼ 475 g∕mol), gly- sodium nitrate (aq.). The columns and detectors were both cidyl methacrylate (GMA), 2-hydroxyethyl methacrylate (HEMA), thermostated at 40 °C. For the HT analysis of core-shell nano- 2-carboxyethyl acrylate (CEA), methacrylic acid (MAA), 2-(di- particles, the flow injection polymer analysis (FIPA) technique methylamino)ethyl methacrylate (DMAEMA), and methyl metha- was utilized, eluting polymers through a W-100-1078 ViscoGEL crylate (MMA) were purchased from Aldrich and passed through a FIPA (Viscotek) column. A single PEO standard with known short column containing basic aluminum oxide to remove the MW, concentration, intrinsic viscosity, and dn/dc was used for inhibitor before use. Oligo(ethylene glycol) methacrylate 1100 triple detection calibration in both cases. Block copolymers K (OEOMA1100, poly(ethylene glycol) methyl ether methacrylate, and L were analyzed using two A-MBHMW-3078 columns for Mn ¼ 1100 g∕mol) (Aldrich) was slightly heated to melt and then anionic polymers (Viscotek) under normal mode with linear passed through a short column containing basic alumina to remove poly(acrylic acid) standards and RI detection at 30 °C. The flow the inhibitor before use. 2,2′-Azobis(2-methylpropionitrile) (AIBN) rate was 1 mL∕ min and the mobile phase was 5% ammonium (Aldrich, 98%) and 4,4′-azobis(4-cyanovaleric acid) (V-501) (Wako hydroxide (aq.). Block copolymer M was analyzed using two Pure Chemical Industries) were recrystallized from methanol before C-MBHMW-3078 columns for cationic polymers (Viscotek) use. N,N-Dimethylformamide (DMF) (Aldrich, 99.8%, anhydrous), under normal mode with linear poly(2-vinylpyridine) standards diethyl ether (Aldrich, 99%), toluene (Aldrich, 99.8%, anhydrous), and RI detection at 30 °C. The flow rate was 1 mL∕ min and the S-(thiobenzoyl)thioglycolic acid (Aldrich, 99%), ethanol (Pharma- mobile phase was 3% acetic acid (aq.). For organic soluble homo- Aaper, 100%), [3-(methacryloylamino)propyl]dimethyl(3-sulfopro- and diblock copolymers, GPC was performed using a Waters pyl)ammonium hydroxide inner salt) (Zwit) (Aldrich), dimethyl system equipped with a 2400 differential refractometer, 515 sulfoxide (DMSO) (Aldrich, anhydrous), tetrahydrofuran (THF) pump, and 717-plus autosampler. The flow rate was 1 mL∕ min (Aldrich, anhydrous), dichloromethane (DCM) (Aldrich), trifluor- and the mobile phase was tetrahydrofuran (THF). The Styragel oacetic acid (TFA) (Aldrich), cholesteryl chloroformate (Aldrich), columns (Waters) and detector were thermostated at 35 °C. N-boc-ethylene diamine (Aldrich), and fluorescein isothiocyanate Linear polystyrene standards were used for calibration. (FITC) (Aldrich) were used as received. Unless labeled as anhy- drous, all solvents used for polymerizations were bubbled with Chemical Structure Drawing and Physical Property Modeling. Chemi- for at least 5 hr before use. Unless otherwise noted, all numbered cal structures were drawn using ChemDraw and ChemAxon amines and other chemicals were obtained from commercial MarvinSketch. HT reactions with the library of amines were sources (Aldrich, Alfa Aesar, TCI-America, and Fluka) and used modeled using ChemAxon Reactor. A single nucleophilic nitro- without further purification. Cumyl dithiobenzoate was synthesized gen from each amine was reacted in silico with one equivalent following a procedure from the literature.(1) of GMA. JChem for Excel (version 5.3.1) enabled rapid modeling of microspecies pKas. 1 Instrumentation. H NMR was performed on a Bruker Avance- 400 spectrometer. Transmission electron (TEM) was In Vitro siRNA Transfection Assay. HeLa cells, stably expressing performed using a JEOL JEM200CX TEM operated at an accel- both firefly (Photinus pyralis) and Renilla (Renilla reniformis) erated voltage of 200 kV. For sample preparation, a droplet of luciferase were seeded (15;000 cells∕well) into each well of an nanoparticle solution was placed on a carbon film covered TEM opaque white 96-well plate (Corning) and allowed to attach over- grid. The that was placed underneath the grid wicked night in growth medium composed of 90% phenol red-free the water and allowed the particles to be deposited on the grid. DMEM and 10% FBS. Cells were transfected with 50 ng of fire- Because the density of the cross-linked core was generally much fly-specific siLuc complexed with nanoparticle at a nanoparticle/ higher than the uncross-linked shell, sufficient contrast was ob- siRNA ratio of 50∶1 (wt∕wt). Transfections were performed in tained without any staining. AFM was performed using a Veeco triplicate. Working dilutions of each nanoparticle were prepared Nanoscope IV scanning probe (SPM) controller with in 25 mM sodium acetate buffer (pH 5.2). The diluted nano- a Dimension 3100 SPM. Samples were prepared by dropping particle was added to diluted siRNA in a well of a 96-well plate 30 μLofa5 mg∕mL nanoparticle suspension in purified water and vigorously mixed using a tip. The mixtures were onto mica. Excess solvent was removed by wicking using a piece incubated at room temperature for 20 min to allow for complex of filter paper. The surface was washed several times with water formation. The growth medium was removed from the cells and and then allowed to dry before imaging. Particle size (diameter, fresh media was added. Nanoparticle/siRNA complexes (contain- nm) and surface charge (zeta potential) measurements were ing 50 ng siRNA and 2.5 μg nanoparticle) were applied, followed made using a ZetaPALS dynamic light scattering (DLS) instru- by gentle pipette mixing. Cells were incubated for 24 h at 37 °C, ment (Brookhaven Instruments). Experiments were performed 5% CO2 and then firefly and Renilla luciferase activity was ana- in PBS, and viscosity and refraction indices were set equal to lyzed using Dual-Glo assay kits (Promega). those specific to water. Average electrophoretic mobilities were measured at 25 °C using PALS zeta potential analysis software In Vivo Factor VII Silencing in Mice. All procedures used in animal and the Smoluchowsky model for aqueous suspensions. Zeta studies were approved by the Institutional Animal Care and Use potential (mV) is expressed as an average of 9 runs standard Committee and were consistent with local, state, and federal deviation. regulations as applicable. C57BL/6 mice (Charles River Labs) were used for siRNA silencing experiments. 2′-O-methyl sugar Gel Permeation Chromatography (GPC). For water soluble polymers, modified siRNAs (Alnylam) were used to prevent activation of GPC was performed using a Viscotek system equipped with a the Toll-like receptor 7 immune response and confer enzymatic

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 1of9 resistance. Nanoparticles were purified by dialysis into sterile The contents were stirred to dissolve the macroCTA and initiator. PBS. Prior to injection, complexes were diluted in PBS at siRNA The solution was bubbled with argon for 30 min to deoxygenate concentrations such that each mouse was administered a dose the reaction. The flask was lowered into a preheated oil bath set of 0.01 mL∕g body weight. Formulations were administered in- at 65 °C. The reaction was stopped after 15 min by exposure to travenously via tail vein injection. After 48 h, body weight gain/ air. The polymer was isolated by dialysis using a MWCO 3500 loss was measured and mice were anaesthetized by isofluorane membrane for 24 hr, changing the water three times. The dried inhalation for blood sample collection by retro-orbital eye bleed. block copolymer was obtained by lyphilization. Mn ¼ 15200, Serum was isolated with serum separation tubes (Becton Dickin- PDI ¼ 1.31 (THF GPC). son) and Factor VII protein levels were analyzed by chromogenic assay (Biophen FVII, Aniara Corporation). A standard curve Synthesis of Precursor Block Copolymer B, polyððoligoðethylene Þ Þ b ð Þ Þ Pðð Þ b was constructed using samples from PBS-injected mice and rela- oxide 5methacrylate 56- - glycidyl methacrylate 8 OEO5MA 56- - Þ tive Factor VII activity was determined by comparing treated GMA8 . 152.3 mg cumyl dithiobenzoate (0.56 mmol), 20 mL OEO- groups to an untreated PBS control. MA300 (70 mmol), 18.4 mg AIBN (0.112 mmol), and 30 mL toluene were added to a 100 mL Schlenk flask equipped with a stir In Vitro pDNA Transfection Assay. HeLa cells (ATTC) were seeded bar. The contents were stirred to dissolve the CTA and initiator. (15;000 cells∕well) into each well of an opaque white 96-well The solution was bubbled with argon for 30 min to deoxygenate plate and allowed to attach overnight in growth medium com- the reaction. The flask was lowered into a preheated oil bath set posed of 90% phenol red-free DMEM and 10% FBS. Cells were at 65 °C. The reaction was stopped after 3 hr by exposure to air. transfected with 150 ng of Gwiz (luciferase) pDNA (Aldevron) The polymer was isolated by dialysis using a MWCO 3500 mem- complexed with nanoparticle at a nanoparticle/pDNA ratio of brane for 24 hr, changing the water three times. The dried macro- 50∶1 (wt∕wt). Transfections were performed in triplicate. Work- CTA was obtained by lyphilization (yield ¼ 1.72 g). Mn ¼ 17100, ing dilutions of each nanoparticle were prepared in 25 mM PDI ¼ 1.32 (THF GPC). sodium acetate buffer (pH 5.2). The diluted nanoparticle was 897 mg PððOEO5MAÞ56 macroCTA (52.4 μmol), 1.73 mg added to diluted pDNA in a well of a 96-well plate and vigorously AIBN (10.5 μmol), 720 μL GMA (5.27 mmol), and 1.2 mL DMF mixed using a pipette tip. The mixtures were incubated at room were added to a 10 mL Schlenk flask equipped with a stir bar. temperature for 20 min to allow for complex formation. The The contents were stirred to dissolve the macroCTA and initiator. growth medium was removed from the cells and fresh media was The solution was bubbled with argon for 30 min to deoxygenate added. Nanoparticle/pDNA complexes (containing 150 ng pDNA the reaction. The flask was lowered into a preheated oil bath set and 7.5 μg nanoparticle) were applied, followed by gentle pipette at 65 °C. The reaction was stopped after 30 min by exposure to mixing. Cells were incubated for 24 hr day at 37 °C, 5% CO2 and air. The polymer was isolated by dialysis using a MWCO 3500 then luciferase activity was analyzed using ONE-Glo assay kits membrane for 24 hr, changing the water three times. The dried (Promega). block copolymer was obtained by lyphilization (yield ¼ 0.9477 g). Mn ¼ 18300, PDI ¼ 1.37 (THF GPC). Microscopy (Fig. 6 C and D). 50,000 Hela cells/well were plated in chambered glass coverslips and allowed to grow for 1 d. Cells Synthesis of Precursor Block Copolymer C, polyððoligoðethylene 50 μ ∕ Þ Þ b ð Þ Þ Pðð Þ b were then exposed to g mL of either FITC-labeled C80 or oxide 5methacrylate 12- - glycidyl methacrylate 135 OEO5MA 12- - Þ C227 for 1 hr, followed by incubation with Hoescht (2 μg∕ml) for GMA135 . 76 mg cumyl dithiobenzoate (0.279 mmol), 10 mL OEO- nuclear stain. The cells were washed with PBS and imaged using a MA300 (35 mmol), 9.2 mg AIBN (56 μmol), and 16 mL toluene Perkin Elmer Spinning Disk Confocal Microscope. 3D z-stacks were added to a 50 mL Schlenk flask equipped with a stir bar. The were captured and processed utilizing the Ultra View ERS soft- contents were stirred to dissolve the CTA and initiator. The solu- ware. Images are shown as a top view 3D image. The scale bar tion was bubbled with argon for 30 min to deoxygenate the reac- and any further modifications were made using Image-J software. tion. The flask was lowered into a preheated oil bath set at 65 °C. The reaction was stopped after 80 min by exposure to air. The Mouse Fluorescence Imaging. Female SKH1 hairless mice were polymer was isolated by dialysis using a MWCO 1000 membrane injected via the tail vein with either PBS (negative control) or na- for 48 hr, changing the water three times. The dried macroCTA noparticles complexed with Cy5-labeled siRNA (Integrated DNA was obtained by lyphilization (yield ¼ 1.5 g). Mn ¼ 3970, PDI ¼ Technologies). When imaging organs, mice were euthanized after 1.29 (THF GPC). 2 hr post-delivery, and the heart, lungs, liver, spleen, and kidneys 1.38 g PððOEO5MAÞ135 macroCTA (0.34 mmol), 11.3 mg were removed for imaging with an IVISR Spectrum system (Caliper AIBN (69 μmol), 3.8 mL GMA (34.5 mmol), and 2 mL toluene Life Sciences). Excitation and emission wavelengths used for Cy5 were added to a 10 mL Schlenk flask equipped with a stir bar. The imaging on the IVIS were 640 nm and 680 nm, respectively. contents were stirred to dissolve the macroCTA and initiator. The solution was bubbled with argon for 30 min to deoxygenate Synthesis of Precursor Block Copolymer A, polyððoligoðethylene the reaction. The flask was lowered into a preheated oil bath Þ Þ b ð Þ Þ Pðð Þ oxide 5 methacrylate 47- - glycidyl methacrylate 6 OEO5MA 47 set at 65 °C. The reaction was stopped after 60 min by exposure b Þ - -GMA6 . 152.3 mg cumyl dithiobenzoate (0.56 mmol), 20 mL to air. The polymer was precipitated in diethyl ether, filtered, and OEOMA300 (70 mmol), 18.4 mg AIBN (0.112 mmol), and dried under vacuum overnight (yield ¼ 1.36 g). Mn ¼ 23050, 30 mL toluene were added to a 100 mL Schlenk flask equipped PDI ¼ 1.35 (THF GPC). with a stir bar. The contents were stirred to dissolve the CTA and initiator. The solution was bubbled with argon for 30 min to Synthesis of Precursor Block Copolymer D, polyððoligoðethylene Þ Þ b ð Þ Þ Pðð Þ deoxygenate the reaction. The flask was lowered into a preheated oxide 5methacrylate 56- - glycidyl methacrylate 112 OEO5MA 56- b Þ Pðð Þ μ oil bath set at 65 °C. The reaction was stopped after 2 hr by -GMA93 . 822 mg OEO5MA 56 macroCTA (48.1 mol), exposure to air. The polymer was isolated by dialysis using a 1.58 mg AIBN (9.67 μmol), 660 μL GMA (4.83 mmol), and MWCO 3500 membrane for 24 hr, changing the water three 1.1 mL DMF were added to a 10 mL Schlenk flask equipped with times. The dried macroCTA was obtained by lyphilization a stir bar. The contents were stirred to dissolve the macroCTA (yield ¼ 0.42 g). Mn ¼ 14400, PDI ¼ 1.26 (THF GPC). and initiator. The solution was bubbled with argon for 30 min 354 mg PððOEO5MAÞ47 macroCTA (24.9 μmol), 0.82 mg to deoxygenate the reaction. The flask was lowered into a pre- AIBN (4.98 μmol), 340 μL GMA (2.49 mmol), and 570 μL DMF heated oil bath set at 65 °C. The reaction was stopped after were added to a 5 mL pear shaped flask equipped with a stir bar. 1.5 hr by exposure to air. The polymer was isolated by dialysis

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 2of9 using a MWCO 3500 membrane for 24 hr, changing the water 50 μL GMA (0.366 mmol), 50 μL HEMA (0.412 mmol), and three times. The dried block copolymer was obtained by lyphili- 0.5 mL DMF were added to a 5 mL pear shaped flask equipped zation (yield ¼ 1.02 g). Mn ¼ 30400, PDI ¼ 1.98 (THF GPC). with a stir bar. The contents were stirred to dissolve the macro- CTA and initiator. The solution was bubbled with argon for Synthesis of Precursor Block Copolymer E, polyððoligoðethylene 30 min to deoxygenate the reaction. The flask was lowered into Þ Þ b ð Þ Þ Pðð Þ oxide 5methacrylate 50- - glycidyl methacrylate 178 OEO5MA 50- a preheated oil bath set at 65 °C. The reaction was stopped after b Þ -GMA178 . 76 mg cumyl dithiobenzoate (0.279 mmol), 10 mL OEO- 45 min by exposure to air. The polymer was precipitate twice in MA300 (35 mmol), 9.2 mg AIBN (56 μmol), and 16 mL toluene diethyl ether, filtered, and dried under vacuum overnight were added to a 50 mL Schlenk flask equipped with a stir bar. (yield ¼ 0.17 g). Mn ¼ 34300, PDI ¼ 1.68 (THF GPC). The contents were stirred to dissolve the CTA and initiator. The solution was bubbled with argon for 44 min to deoxygenate the re- Synthesis of precursor block copolymer H, polyððoligoðethylene Þ Þ b ð Þ Þ Pðð Þ action. The flask was lowered into a preheated oil bath set at 65 °C. oxide 9methacrylate 48- - glycidyl methacrylate 17 OEO9MA 48- b Þ μ The reaction was stopped after 5 hr by exposure to air. The polymer -GMA17 . 24 mg cumyl dithiobenzoate (88.4 mol), 5 mL OEO- was isolated by dialysis using a MWCO 3500 membrane for 24 hr, MA475 (11.05 mmol), 2.9 mg AIBN (17.6 μmol), and 8 mL changing the water three times. The dried macroCTA was toluene were added to a 25 mL Schlenk flask equipped with a obtained by lyphilization (yield ¼ 6.5 g). Mn ¼ 15150,PDI¼ 1.28 stir bar. The contents were stirred to dissolve the CTA and initia- (THF GPC). tor. The solution was bubbled with argon for 30 min to deoxygen- 2gPððOEO5MAÞ50 macroCTA (0.132 mmol), 4.3 mg AIBN ate the reaction. The flask was lowered into a preheated oil bath (26.2 μmol), 1.87 mL GMA (13.2 mmol), and 2 mL toluene were set at 65 °C. The reaction was stopped after 1 hr by exposure to added to a 25 mL Schlenk flask equipped with a stir bar. The air. The polymer was isolated by dialysis using a MWCO 3500 contents were stirred to dissolve the macroCTA and initiator. membrane for 24 hr, changing the water three times. The dried The solution was bubbled with argon for 30 min to deoxygenate macroCTA was obtained by lyphilization (yield ¼ 0.86 g). the reaction. The flask was lowered into a preheated oil bath set Mn ¼ 22800, PDI ¼ 1.24 (THF GPC). at 65 °C. The reaction was stopped after 75 min by exposure to air. 769 mg PððOEO9MAÞ48 macroCTA (33.7 μmol), 1.1 mg AIBN The polymer was precipitated in diethyl ether, filtered, and dried (6.74 μmol), 460 μL GMA (3.37 mmol), and 828 μL DMF were under vacuum overnight (isolated yield ¼ 0.72 g). Mn ¼ 40530, added to a 5 mL pear shaped flask equipped with a stir bar. The PDI ¼ 1.49 (THF GPC). contents were stirred to dissolve the macroCTA and initiator. The solution was bubbled with argon for 30 min to deoxygenate Synthesis of Precursor Block Copolymer F, polyððoligoðethylene the reaction. The flask was lowered into a preheated oil bath set Þ Þ b ð Þ Þ Pðð Þ oxide 5methacrylate 106- - glycidyl methacrylate 478 OEO5MA 106- at 65 °C. The reaction was stopped after 20 min by exposure to b Þ -GMA478 . 38.1 mg cumyl dithiobenzoate (0.14 mmol), 5 mL air. The polymer was precipitated in diethyl ether, filtered, and OEOMA300 (17.5 mmol), 4.6 mg AIBN (27.9 μmol), and dried under vacuum overnight (yield ¼ 975 mg). Mn ¼ 25300, 8 mL toluene were added to a 25 mL Schlenk flask equipped with PDI ¼ 1.24 (THF GPC). a stir bar. The contents were stirred to dissolve the CTA and in- itiator. The solution was bubbled with argon for 30 min to deox- Synthesis of Precursor Block Copolymer I, polyððoligoðethylene Þ Þ b ð Þ Þ Pðð Þ b ygenate the reaction. The flask was lowered into a preheated oil oxide 23methacrylate 9- - glycidyl methacrylate 3 OEO23MA 9- - Þ μ bath set at 65 °C. The reaction was stopped after 17.3 hr by ex- GMA3 . 8.7 mg cumyl dithiobenzoate (32 mol), 4 mL OEO- posure to air. The polymer was isolated by dialysis using a MA1100 (4 mmol), 1.05 mg AIBN (6.4 μmol), and 6.4 mL DMF MWCO 3500 membrane for 24 hr, changing the water three were added to a 25 mL Schlenk flask equipped with a stir bar. The times. The dried macroCTA was obtained by lyphilization contents were stirred to dissolve the CTA and initiator. The (yield ¼ 2.5 g). Mn ¼ 32050, PDI ¼ 1.3 (THF GPC). solution was bubbled with argon for 30 min to deoxygenate the 2.35 g PððOEO5MAÞ106 macroCTA (73.3 μmol), 2.4 mg AIBN reaction. The flask was lowered into a preheated oil bath set at (14.6 μmol), 2.5 mL GMA (18.3 mmol), and 4.15 mL DMF were 65 °C. The reaction was stopped after 2 hr by exposure to air. The added to a 25 mL Schlenk flask equipped with a stir bar. The polymer was isolated by dialysis using a MWCO 3500 membrane contents were stirred to dissolve the macroCTA and initiator. for 24 hr, changing the water three times. The dried macroCTA The solution was bubbled with argon for 30 min to deoxygenate was obtained by lyphilization (yield ¼ 1.862 g). Mn ¼ 9616, the reaction. The flask was lowered into a preheated oil bath set at PDI ¼ 1.14 (THF GPC). 65 °C. The reaction was stopped after 75 min by exposure to air. 200 mg PððOEO23MAÞ9 macroCTA (6.5 μmol), 0.21 mg AIBN The polymer was precipitate twice in diethyl ether, filtered, and (1.3 μmol) (note: added as a stock solution in DMF), 86 μL GMA dried under vacuum overnight (yield ¼ 1.43 g). Mn ¼ 100300, (650 μmol), and 550 μL DMF were added to a 5 mL pear shaped PDI ¼ 2.56 (THF GPC). flask equipped with a stir bar. The contents were stirred to dissolve the macroCTA and initiator. The solution was bubbled Synthesis of precursor block copolymer G, polyððoligoðethylen with argon for 30 min to deoxygenate the reaction. The flask was Þ Þ b ðð Þ − r − eoxide 5methacrylate 90- - hydroxyethyl methacrylate 23 lowered into a preheated oil bath set at 65 °C. The reaction was ð Þ ÞÞ Pðð Þ Þ b ð r ÞÞ glycidyl methacrylate 17 OEO5MA 90 - - HEMA23- -GMA17 . stopped after 40 min by exposure to air. The polymer was preci- 45 mg cumyl dithiobenzoate (0.165 mmol), 6 mL OEOMA300 pitated in diethyl ether, filtered, and dried under vacuum over- (21 mmol), 5.5 mg AIBN (33.5 μmol), and 9.5 mL toluene were night (yield ¼ 119 mg). Mn ¼ 10040, PDI ¼ 1.2 (THF GPC). added to a 25 mL Schlenk flask equipped with a stir bar. The con- tents were stirred to dissolve the CTA and initiator. The solution Synthesis of Precursor Block Copolymer J, polyððoligoðethylene Þ Þ b ð Þ Þ Pðð Þ was bubbled with argon for 35 min to deoxygenate the reaction. oxide 23methacrylate 18- - glycidyl methacrylate 168 OEO23MA 18- b Þ μ The flask was lowered into a preheated oil bath set at 65 °C. -GMA168 . 10.38 mg cumyl dithiobenzoate (38.1 mol), 5 mL The reaction was stopped after 20 hr by exposure to air. Unreacted OEOMA1100 (4.77 mmol), 1.25 mg AIBN (7.63 μmol), and monomer and solvent was removed on a rotavapor. The polymer 8 mL DMF were added to a 25 mL Schlenk flask equipped with was isolated by dialysis using a MWCO 6000–8000 membrane for a stir bar. The contents were stirred to dissolve the CTA and 24 hr, changing the water three times. The dried macroCTA was initiator. The solution was bubbled with argon for 27 min to deox- obtained by lyphilization. Mn ¼ 27400,PDI¼ 1.43 (THF GPC). ygenate the reaction. The flask was lowered into a preheated oil 100 mg PððOEO5MAÞ90 macroCTA (3.65 μmol), 0.12 mg bath set at 65 °C. The reaction was stopped after 105 min by AIBN (0.73 μmol) (note: added as a stock solution in DMF), exposure to air. The polymer was isolated by dialysis using a

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 3of9 MWCO 10000 membrane for 24 hr, changing the water three DMF were added to a 25 mL Schlenk flask equipped with a stir times. The dried macroCTA was obtained by lyphilization bar. The contents were stirred to dissolve the CTA and initiator. (yield ¼ 0.602 g). Mn ¼ 20605, PDI ¼ 1.14 (THF GPC). The solution was bubbled with argon for 30 min to deoxygenate 592 mg PððOEO23MAÞ18 macroCTA (28.73 μmol), 0.94 mg the reaction. The flask was lowered into a preheated oil bath set AIBN (5.72 μmol), 588 μL GMA (4.3 mmol), 300 μL DMF at 65 °C. The reaction was stopped after 45 min by exposure to and 1.05 mL toluene were added to a 5 mL pear shaped flask air. The polymer was isolated by dialysis using a MWCO 3500 equipped with a stir bar. The contents were stirred to dissolve membrane for 24 hr, changing the water three times. The dried the macroCTA and initiator. The solution was bubbled with argon macroCTA was obtained by lyphilization (yield ¼ 261 mg). for 30 min to deoxygenate the reaction. The flask was lowered Mn ¼ 6700, PDI ¼ 1.6 (THF GPC). into a preheated oil bath set at 65 °C. The reaction was stopped 246 mg PðDMAEMA43Þ macroCTA (36.7 μmol), 1.2 mg AIBN after 60 min by exposure to air. The polymer was precipitated in (7.3 μmol), 0.5 mL GMA (3.66 mmol), 10.8 mL DMF were added diethyl ether, filtered, and dried under vacuum overnight to a 25 mL pear shaped flask equipped with a stir bar. The con- (yield ¼ 770 mg). Mn ¼ 34300, PDI ¼ 1.56 (THF GPC). tents were stirred to dissolve the macroCTA and initiator. The solution was bubbled with argon for 30 min to deoxygenate Synthesis of Precursor Block Copolymer K, polyðð2-carboxyethyl the reaction. The flask was lowered into a preheated oil bath Þ b ð Þ Þ Pð b Þ acrylate 104- - glycidyl methacrylate 126 CEA104- -GMA126 . set at 65 °C. The reaction was stopped after 20 min by exposure 715.6 mg S-(Thiobenzoyl)thioglycolic acid (3.37 mmol), 5 mL to air. The polymer was isolated by dialysis using a MWCO 3500 CEA (168.5 mmol), 110 mg V-501 (0.671 mmol), and 30 mL membrane for 24 hr, changing the water three times. The dried DMF were added to a 100 mL Schlenk flask equipped with a stir block copolymer was obtained by lyphilization (yield ¼ 320 mg). bar. The contents were stirred to dissolve the CTA and initiator. Mn ¼ 13550, PDI ¼ 1.35 (water GPC, cationic mode). The solution was bubbled with argon for 35 min to deoxygenate the reaction. The flask was lowered into a preheated oil bath set at Synthesis of Precursor Block Copolymer N, polyð½3- 65 °C. The reaction was stopped after 5.75 hr by exposure to air. ðmethacryloylaminoÞpropyldimethylð3-sulfopropylÞ Þ b ð Þ Þ The polymer was isolated by dialysis using a MWCO 3500 mem- ammonium hydroxideinner salt 57- - glycidyl methacrylate 371 . brane for 24 hr, changing the water three times. The dried macro- PðZwit57-b-GMA371Þ. 96.8 mg S-(Thiobenzoyl)thioglycolic acid CTA was obtained by lyphilization (yield ¼ 2.1 g). Mn ¼ 15100, (0.455 mmol), 10 g Zwit (34.2 mmol), 25.5 mg V-501 (91 μmol), PDI ¼ 1.34 (water GPC, anionic columns). 15 mL water, and 2 mL methanol were added to a 25 mL Schlenk 1gPðCEA104Þ macroCTA (28.7 μmol), 6.8 mg AIBN flask equipped with a stir bar. The CTA and V-501 were dissolved (41.4 μmol), 4 mL GMA (29.3 mmol), 8 mL DMF were added in the methanol and added to the monomer dissolved in water. to a 25 mL Schlenk flask equipped with a stir bar. The contents The solution was bubbled with argon for 40 min to deoxygenate were stirred to dissolve the macroCTA and initiator. The solution the reaction. The flask was lowered into a preheated oil bath set was bubbled with argon for 30 min to deoxygenate the reaction. at 65 °C. The reaction was stopped after 2 hr. The polymer was iso- The flask was lowered into a preheated oil bath set at 65 °C. The lated by dialysis using a MWCO 3500 membrane for 24 hr, chan- reaction was stopped after 12 min by exposure to air. The polymer ging the water three times. The dried macroCTA was obtained by was isolated by dialysis using a MWCO 3500 membrane for 24 hr, lyphilization (yield ¼ 1.393 g). Mn ¼ 16600,PDI¼ 1.25 (water changing the water three times. The dried block copolymer was ob- GPC, normal mode). tained by lyphilization (yield ¼ 1.26 g). Mn ¼ 34200,PDI¼ 1.48 1.392 g PðZwit57Þ macroCTA (83.9 μmol) was dissolved in (water GPC, anionic columns). 2.2 mL water in a 10 mL Schlenk flask. 4.7 mg V-501 (16.77 μmol) was dissolved in 300 μL methanol and added to the monomer Synthesis of Precursor Block Copolymer L, polyððmethacrylic solution. Finally, 1.14 mL GMA (8.39 mmol) was added. The con- Þ b ð Þ Þ Pð b Þ acid 232- - glycidylmethacrylate 639 MAA232- -GMA639 . 333.7 mg tents were stirred and bubbled with argon for 30 min to deoxygen- S-(Thiobenzoyl)thioglycolic acid (1.57 mmol), 10 mL MAA ate the reaction. The flask was lowered into a preheated oil bath set (117.9 mmol), 51.6 mg AIBN (0.314 mmol), and 15 mL DMF at 65 °C. The reaction was stopped after 13 min by exposure to were added to a 50 mL Schlenk flask equipped with a stir bar. air. The polymer was isolated by dialysis using a MWCO 3500 The contents were stirred to dissolve the CTA and initiator. membrane for 24 hr, changing the water three times. The dried The solution was bubbled with argon for 35 min to deoxygenate block copolymer was obtained by lyphilization (yield ¼ 1.2 g). the reaction. The flask was lowered into a preheated oil bath set Mn ¼ 69400,PDI¼ 1.6 (water GPC, normal mode). at 65 °C. The reaction was stopped after 29 min by exposure to air. The polymer was isolated by dialysis using a MWCO 3500 Synthesis of Precursor Block Copolymer O, polyððmethyl Þ b ð Þ Þ Pð b Þ membrane for 24 hr, changing the water three times. The dried methacrylate 146- - glycidylmethacrylate 25 MMA146- -GMA25 . macroCTA was obtained by lyphilization (yield ¼ 255 mg). 127 mg cumyl dithiobenzoate (0.467 mmol), 10 mL MMA Mn ¼ 49400, PDI ¼ 1.8 (water GPC, anionic columns). (93.9 mmol), 15.4 mg AIBN (93.8 μmol), and 15 mL toluene were 116 mg PðMAA232Þ macroCTA (2.35 μmol), 1.8 mg AIBN added to a 50 mL Schlenk flask equipped with a stir bar. The (10.9 μmol), 0.75 mL GMA (5.49 mmol), 1.5 mL ethanol were contents were stirred to dissolve the CTA and initiator. The solu- added to a 5 mL pear shaped flask equipped with a stir bar. tion was bubbled with argon for 30 min to deoxygenate the reac- The contents were stirred to dissolve the macroCTA and initiator. tion. The flask was lowered into a preheated oil bath set at 65 °C. The solution was bubbled with argon for 30 min to deoxygenate The reaction was stopped after 20 hr by exposure to air. The poly- the reaction. The flask was lowered into a preheated oil bath set mer was isolated by precipitation in diethyl ether, filtered, and at 65 °C. The reaction was stopped after 40 min by exposure to dried under vacuum (yield ¼ 2.59 g). Mn ¼ 14600, PDI ¼ 1.24 air. The polymer was isolated by dialysis using a MWCO 3500 (THF GPC). membrane for 24 hr, changing the water three times. The dried 2.14 g PðMMAÞ146 macroCTA (0.1466 mmol), 4.8 mg AIBN block copolymer was obtained by lyphilization (yield ¼ 103 mg). (29.3 μmol), 3 mL GMA (22 mmol), and 4.8 mL toluene were Mn ¼ 110800, PDI ¼ 1.95 (water GPC, anionic columns). added to a 10 mL Schlenk flask equipped with a stir bar. The contents were stirred to dissolve the macroCTA and initiator. Synthesis of precursor block copolymer M, polyðð2-ðdimethylaminoÞ The solution was bubbled with argon for 30 min to deoxygenate Þ b ð Þ Þ Pð b ethyl methacrylate 43- - glycidyl methacrylate 23 DMAEMA43- - the reaction. The flask was lowered into a preheated oil bath set Þ GMA23 . 32.2 mg cumyl dithiobenzoate (0.118 mmol), 4 mL at 65 °C. The reaction was stopped after 35 min by exposure to DMAEMA (23.7 mmol), 3.9 mg AIBN (23.7 μmol), and 6 mL air. The polymer was precipitated in diethyl ether, filtered, and

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 4of9 dried under vacuum overnight (yield ¼ 2.26 g). Mn ¼ 18200, for 20 hr. 214 μL water was added, and the nanoparticles were PDI ¼ 1.12 (THF GPC). dialyzed using a 3500 MWCO microdialysis cup against 2 L PBS for 1.5 hr. ð Þ Synthesis of Precursor Polymer P, poly glycidyl methacrylate 25 Pð Þ μ 100 ∕ GMA25 . 79.7 mg cumyl dithiobenzoate (0.293 mmol), 4 mL Synthesis of C227-FITC. Block C (20 Lofa mg mL solution in MMA (29.32 mmol), 9.6 mg AIBN (58.6 μmol), and 6 mL toluene DMSO, 2 mg polymer), amine 227 (22 μLofa100 mg∕mL solu- were added to a 25 mL Schlenk flask equipped with a stir bar. The tion in DMSO), and FITC (2 μLofa100 mg∕mL solution) were contents were stirred to dissolve the CTA and initiator. The solu- added to a 1 mL glass vial equipped with a tumbling stir bar. It tion was bubbled with argon for 34 min to deoxygenate the reac- was heated and stirred on the deck of robot at 52.5 °C for 20 hr. tion. The flask was lowered into a preheated oil bath set at 65 °C. 206 μL water was added, and the nanoparticles were dialyzed The reaction was stopped after 2.5 hr by exposure to air. The using a 3500 MWCO microdialysis cup against 2 L PBS for 1.5 hr. polymer was isolated by precipitation in diethyl ether, filtered, and dried under vacuum (yield ¼ 155 mg). Mn ¼ 3120,PDI¼ 1.27 Synthesis of D222-FITC. Block D (40 μLofa50 mg∕mL solution in (THF GPC). DMSO, 2 mg polymer), amine 222 (18.34 μLofa100 mg∕mL solution in DMSO), and FITC (2 μLofa100 mg∕mL solution) Synthesis of 2,2′-disulfanediyldiethanamine hydrochloride (amine were added to a 1 mL glass vial equipped with a tumbling stir bar. 312). 2 g Mercaptoethylamine (25.9 mmol, 2.0 eq.) was dissolved It was heated and stirred on the deck of robot at 52.5 °C for 20 hr. in 6 mL water in a 100 mL round bottom flask equipped with a stir 190 μL water was added, and the nanoparticles were dialyzed bar. This mixture was cooled down with an ice/salt bath. Then a using a 3500 MWCO microdialysis cup against 2 L PBS for 1.5 hr. cooled solution of hydrogen peroxide (26 mL of a 1 M H2O2 so- lution (aq.), 26 mmol, 2.0 eq.) was added drop-wise while stirring. Synthesis of I126-FITC. Block I (100 μLofa23.8 mg∕mL solution in After this reaction mixture did not decolorize a violet I2∕KI DMSO, 2.38 mg polymer), 126 (16.6 μLofa3 mg∕mL solution in solution anymore, 1 M HCl was added to lower the pH below DMSO, 2.38 μmol), and FITC (0.84 μLofa100 mg∕mL solution) 4. The solvent was evaporated and the residual solid was recrys- were added to a 1 mL glass vial equipped with a tumbling stir bar. tallized from ethanol affording 2,2′-disulfanediyldiethanamine It was heated and stirred on the deck of robot at 55 °C for 24 hr. hydrochloride (2.37 mg, 10.5 mmol, 81.1%). 1H-NMR (400 MHz, 800 μL water was added, and the nanoparticles were dialyzed D2O): δ 3.17 (t, 4H), 2.93 (t, 4H). using a 3500 MWCO microdialysis cup against 2 L water.

Synthesis of N0-cholesteryloxycarbonyl-1,2-diaminoethane (amine Synthesis of F126-FITC. Block F (50 μLofa100 mg∕mL solution in 318). A modified procedure from reference (2) was followed. DMSO, 5 mg polymer), 126 (40.8 μLofa3 mg∕mL solution in 2.17 g Cholesteryl chloroformate (4.83 mmol) and 1.09 g DMSO), and FITC (2.1 μLofa100 mg∕mL solution) were added N-boc-ethylene diamine (6.8 mmol) were reacted in 25 mL of to a 1 mL glass vial equipped with a tumbling stir bar. It was anhydrous THF with 1.5 mL triethylamine at room temperature heated and stirred on the deck of robot at 55 °C for 24 hr. for 24 hr. The product was washed three times with 100 mL 1M 800 μL water was added, and the nanoparticles were dialyzed HCl (sat. NaCl) and extracted into DCM. The organic layer was using a 3500 MWCO microdialysis cup against 2 L water. collected and dried over MgSO4. The boc-protected cholesterol derivative was recrystallized using cold ethanol. Tocleave the pro- Synthesis of T110-FITC. In a 1.5 ml Eppendorf tube, a solution of tecting group, 1 g was dissolved in 2.5 mL DCM and 2.5 mL TFA block T (50 μLofa0.10 mg∕μL solution in DMSO, 5.0 mg, was added dropwise. The colorless solution became dark yellow. 0.46 μmol, 8.30 μmol of epoxides, 1.0 eq.) was added to a solution It was allowed to react for 40 min and then washed. The final of 110 (1.1 μLofa0.7 mg∕μL solution in DMSO, 0.77 mg, product was isolated as a white solid using flash chromatography. 4.15 μmol, 0.50 eq.) and FITC (1.7 μLofa0.1 mg∕μL solution in DMSO, 0.17 mg, 0.44 μmol, 0.05 eq.). This reaction mixture Synthesis of C80-Cholesterol. Block C (40 μLofa100 mg∕mL was heated at 55 °C for 26 hr. It was then precipitated by the ad- solution in DMSO, 4.0 mg polymer, 1.735 × 10−7 mol C, 2.8301 × dition of water, centrifuged, dried under vacuum, and redispersed 10−5 mol epoxide groups) was added to a 1 mL glass vial in PBS at a concentration of 0.1 mg∕μL. equipped with a tumbling stir bar. N0-cholesteryloxycarbonyl- 1,2-diaminoethane (76.7 μLofa17.4 mg∕mL solution in ethanol, Synthesis of T126-FITC. In a 1.5 ml Eppendorf tube, a solution of 2.8301 × 10−6 mol, 10 mol% compared to epoxide groups) was block T (50 μLofa0.10 mg∕μL solution in DMSO, 5.0 mg, added and the mixture was gently vortexed for 30 seconds. Amine 0.46 μmol, 8.30 μmol of epoxides, 1.00 eq.) was added to a solu- 80 (26.02 μLofa100 mg∕mL solution in DMSO, 26.02 mg, tion of solution of 126 (1.0 μLofa1.0 mg∕μL solution in DMSO, 2.547 × 10−5 mol, 90 mol% compared to epoxide groups) was 1.0 mg, 4.15 μmol, 0.50 eq.) and FITC (1.7 μLofa0.1 mg∕μL added and the mixture was gently vortexed for 30 seconds. The solution in DMSO, 0.17 mg, 0.44 μmol, 0.05 eq.). This reaction reaction was heated and stirred on the deck of the robot at 52.5 °C mixture was heated at 55 °C for 26 hr. It was then precipitated for 24 hr. The contents were transferred to 3500 MWCO micro- by the addition of water, centrifuged, dried under vacuum, and dialysis cups, washing each vial with 158 μL PBS to transfer all redispersed in PBS at a concentration of 0.1 mg∕μL. of the nanoparticles, and dialyzed against 2 L PBS for 1.5 hr. The incorporation of cholesterol and 80 amine was quantified by General Work-Up of Nanoparticles after HT Synthesis on the Robot. 1H NMR using d-DMSO as the solvent. A comparison of the area After the reaction was completed, DMSO and unreacted amines under the peak at δ ¼ 0.65 (s, 3H, H-18′) for cholesterol with the were removed via evaporation. The dried nanoparticles were re- peak at δ ¼ 2.2 (t, 2H, ðCH3Þ2-N-CH2-CH2) for amine 80 showed dispersed in purified water or 25 mM sodium acetate buffer. the 9.5% of the epoxides had been opened by N0-cholesterylox- ycarbonyl-1,2-diaminoethane and 90.5% by amine 80. Peptides. Peptides (amines 308, 309, 310, 316, 317) were synthe- sized by the MIT Koch Institute Biopolymers and Proteomics Synthesis of C80-FITC. Block C (20 μLofa100 mg∕mL solution in core facility. DMSO, 2 mg polymer), amine 80 (14.3 μLofa100 mg∕mL solu- tion in DMSO), and FITC (1.4 μLofa100 mg∕mL solution) pKa Modeling. were added to a 1 mL glass vial equipped with a tumbling stir Models were generated and pKa calculations were performed bar. It was heated and stirred on the deck of robot at 52.5 °C using J Chem for Excel, version 5.3.1 (http://www.chemaxon.

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 5of9 com). We prepared a database containing the structure of the 102 of 100 μL were taken by argon-filled syringe for kinetic analysis. amines in the library. A single nucleophilic nitrogen from each The reaction was eventually stopped by exposure to air. The re- amine was reacted in silico with one equivalent of glycidyl metha- action mixture was concentrated on a rotavapor and then preci- cylate. The pKa values of the nitrogen atoms in these structures pitated in diethyl ether, centrifuged, filtered and dried under were calculated. Based upon the modeled pKa values, the likely vacuum to afford PGMA as a pink powder. protonation state of each amine derivative was calculated at two pH values by solving the Henderson–Hasselbalch equation Kinetic Study for the Chain Extension of OEOMA300 from PGMA   MacroCTAs. 1 mg AIBN (6.34 μmol, 0.2 molar eq.) was added ½A− to a dry 10 mL Schlenk flask and dried under vacuum for pH ¼ pKa þ ½HA 30 min before back-filling the flask with argon. 186.4 mg PGMA39 macroCTA (31.72 μmol, 1.0 eq.), dissolved in where [A−] is the concentration of the amine conjugate base and 2.25 mL toluene (2.5∶1 (v∕v) vs. monomer), and 900 μL OEO- [HA] is the concentration of the protonated form of the amine. MA300 (31.75 mmol, 100 eq.) were added via syringe. The con- This equation was manipulated to provide “Y” as follows: tents were stirred and bubbled with argon for 30 min to deoxygenate the reaction. The flask was lowered into a preheated 10pH oil bath set at 65 °C and stirred under argon. Samples of 100 μL ½A−1 were taken by argon-filled syringe for kinetic analysis. The reac- ½ Y ¼ HA tion was eventually stopped by exposure to air. The reaction mix- 10−pH þ 10−pH ½A− ture was concentrated on a rotavapor and then precipitated in ½HA diethyl ether, centrifuged, filtered and dried under vacuum to af- ford P(GMA-b-OEOMA). Y represents the percent concentration of the protonated form of Pð Þ each ionizable nitrogen atom in the amine derivatives. The total Kinetic Study for the Chain Extension of GMA from OEO5MA Macro- charge was calculated for each model compound at the pH of CTA. 1 mg AIBN (6.34 μmol, 0.2 molar eq.) was added to a 10 mL siRNA complexation (pH 7.4) and at a value selected to repre- Schlenk flask and dried under vacuum for 30 min before back- sent the endosomal compartment (pH 5.2). The difference be- filling the flask with argon. 250 mg PðOEO5MAÞ25 macroCTA tween these two values (Δ charge) was assumed to be (31.45 μmol, 1.0 eq.), dissolved in 660 μL toluene (1.5∶1 (v∕v) representative of the buffering capacity of the species within of monomer), and 430 μL GMA (3.15 mmol, 100 eq.) were added the endosomal compartment. The resulting data were compared via syringe. The contents were stirred and bubbled with argon for with dye exclusion data (RiboGreen assay) and with in vitro siR- 30 min to deoxygenate the reaction. The flask was lowered into a NA knockdown efficacy. preheated oil bath set at 65 °C and stirred under argon. Samples of 100 μL were taken by argon-filled syringe for kinetic analysis. Kinetic Study for the Homopolymerization of OEOMA300. 5.5 mg The reaction was eventually stopped by exposure to air. The re- AIBN (33.49 μmol, 0.2 molar eq.) was added to a dry 10 mL action mixture was concentrated on a rotavapor and then preci- Schlenk flask and dried under vacuum for 30 min before back- pitated in diethyl ether, centrifuged, filtered and dried under filling the flask with argon. 45.5 mg Cumyl dithiobenzoate vacuum to afford PðOEO5MA-b-GMAÞ. (167.47 μmol, 1.0 eq.), dissolved in 9 mL toluene (1.5∶1 (v∕v) vs. monomer), and 6 mL OEOMA300 (20.93 mmol, 125 eq.) were Model Reaction: Ring Opening of Epoxide Groups Inside the Polymer added via syringe. The contents were stirred and bubbled with Chain of PGMA Using Sodium Azide. In a 10 ml reaction vial argon for 30 min to deoxygenate the reaction. The flask was low- equipped with a stir bar, 10 mg PGMA39 (1.7 μmol polymer, ered into a preheated oil bath set at 65 °C and stirred under ar- 66.36 μmol of epoxides, 1.0 eq.) was dissolved in 1.3 mL DMF gon. Samples of 100 μL were taken by argon-filled syringe for with 12.52 mg ammonium sulfate (101.67 μmol, 1.5 eq.). Then kinetic analysis. The reaction was eventually stopped by exposure 13.22 mg sodium azide (203.4 μmol, 3.0 eq.) was added. The re- to air. The polymer was isolated by dialysis using a MWCO 3500 action mixture was stirred for 30 h at 55 °C. The modified poly- membrane for 24 hr, changing the water three times. The dried mer was purified by precipitating in water, centrifuging, filtering polymer was obtained by lyphilization. and drying in vacuum.

Kinetic Study for the Homopolymerization of Glycidyl Methacrylate. Model Reaction: Ring Opening of Epoxide Groups Inside the Polymer 2.4 mg AIBN (14.61 μmol, 0.2 molar eq.) was added to a dry Chain of PGMA Using Piperidine. In a 10 ml reaction vial equipped 10 mL Schlenk flask and dried under vacuum for 30 min before with a stir bar, 10 mg PGMA39 (1.7 μmol polymer, 66.36 μmol of back-filling the flask with argon. 19.9 mg Cumyl dithiobenzoate epoxides, 1.0 eq.) was dissolved in 1.3 mL dimethyl formamide (73.16 μmol, 1.0 eq.), dissolved in 1.5 mL toluene (1.5∶1 (v∕v) vs. with 12.52 mg ammonium sulfate (101.67 μmol, 1.5 eq.). Then monomer), and 1 mL GMA (7.33 mmol, 100 eq.) were added via 20.1 μL piperidine (203.4 μmol, 3.0 eq.) was added. The reaction syringe. The contents were stirred and bubbled with argon for mixture was stirred for 30 h at 55 °C. The modified polymer was 30 min to deoxygenate the reaction. The flask was lowered into purified by precipitating in water, centrifuging, filtering and dry- a preheated oil bath set at 65 °C and stirred under argon. Samples ing in vacuum.

1. Perrier S, Barner-Kowollik C, Quinn J, Vana P, Davis T (2002) Origin of inhibition effects 2. Waterhouse J, et al. (2005) Synthesis and application of integrin targeting lipopeptides in the reversible addition fragmentation chain transfer (RAFT) polymerization of in targeted gene delivery. Chem Bio Chem 6:1212–1223. methyl acrylate. Macromolecules 35:8300–8306.

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 6of9 Fig. S1. Scale diagram illustrating relationship between (calculated) buffering capacity of amines and efficacy of nanoparticle formulations. (Area A) Modeled amine derivatives with insignificant buffering capacity. (Area B) Count of nanoparticles among all blocks which enabled >30% luciferase silencing. (Area C) Modeled amine derivatives expected to gain charge during expected pH drop within endosomal compartment.

Fig. S2. (A) First-order kinetic plot for the controlled homopolymerization of GMA by RAFT (molar ratios: ½GMA0∕½AIBN0∕½CTA0Þ¼100∶0.2∶1). The linear relationship indicates a constant concentration of radicals, and therefore a living polymerization. (B) Plot of molecular weight (Mn) versus monomer conversion along with the theoretical growth of molecular weight for the controlled homopolymerization of GMA by RAFT (molar ratios: ½GMA0∕½AIBN0∕½CTA0Þ¼ 1 100∶0.2∶1). (C) Polydispersity remained low during the polymerization of GMA by RAFT. (D) H-NMR spectrum of PGMA39.(E) GPC traces for the RAFT chain extension of PGMA39 (black) to PðGMA39-b-ðOEO5MAÞ22Þ (red). (F) Linear chain growth of POEOMA block during the chain extension of OEOMA300 from PGMA by RAFT (molar ratios: ½OEOMA3000∕½AIBN0∕½PGMA macro-CTA0Þ¼100∶0.2∶1). (G) Polydispersity remained constant during RAFT chain extension OEOMA300 from PGMA. (H) GPC traces for the RAFT chain extension of PðOEO5MAÞ25 (black) to PððOEO5MAÞ25-b-GMA18Þ (red). (I) Chain growth of the PGMA block during the chain extension of GMA from PðOEO5MAÞ by RAFT (molar ratios: ½GMA0∕½AIBN0∕½POEO5MA macro-CTA0Þ¼100∶0.2∶1Þ.(J) Polydispersity 1 remained constant during the chain extension of GMA from PðOEO5MAÞ by RAFT. (K) H-NMR spectrum of PððOEO5MAÞ44-b-GMA87Þ.(L) Synthetic scheme for 1 the model ring opening reactions. H-NMR spectra of (M) PGMA39-azide and (N) PGMA39-piperidine. For the reaction with sodium azide, a shift could be detected for all the epoxide signals a, b, b’, c and c’, although some unopened epoxides remained. For the reaction with piperidine, a shift could be detected for all the epoxide signals a, b, b’, c and c’ including the new piperidine signals f and f’. No unopened epoxides were observed.

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 7of9 Fig. S3. AFM images of (A) R126 (d ¼ 22.9 nm by AFM, d ¼ 21.4 nm by DLS), (B) S126 (d ¼ 25.4 nm by AFM, d ¼ 21.5 nm by DLS), and (C) FF110 (d ¼ 34 nm by AFM). TEM image of (D) C80 (d ¼ 36 nm by DLS) and (E) C227 (d ¼ 40 nm by DLS).

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 8of9 Fig. S4. Cellular internalization of all C-based nanoparticles after 1 hr of incubation is demonstrated by HT automated confocal microscopy. HeLa cells were exposed to all C-based nanoparticles complexed with Alexa-594 siRNA (50∶1,wt∕wt). Twenty different fields were imaged and a representative image of nanoparticle/siRNA complex is presented (Alexa 594 is pseudocolored green).

Other Supporting Information Files Table S1 (PDF) Table S2 (PDF) Table S3 (PDF) Table S4 (PDF) Table S5 (PDF)

Siegwart et al. www.pnas.org/cgi/doi/10.1073/pnas.1106379108 9of9