Biodegradable Block Copolyelectrolyte Hydrogels for Tunable ...
Supporting Information for
Biodegradable Block Copolyelectrolyte Hydrogels for Tunable Release of Therapeutics and Topical Antimicrobial Skin Treatment Robert J. Ono,†,§ Ashlynn L. Z. Lee,‡,§ Willy Chin,‡ Wei Sheng Goh,‡ Amelia Y. L. Lee,‡ Yi Yan Yang‡,* and James L. Hedrick†,* † ‡
IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, United States
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore Email: [email protected]; [email protected]
Table of Contents General Considerations
S2
Experimental Procedures and NMR Spectra
S4
Additional Rheology Data
S12
Cytotoxicity Data
S12
References
S13
S1
General Considerations Materials. Dichloromethane and tetrahydrofuran were dried using activated alumina columns and stored over molecular sieves (3 Å). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was dried over CaH2 and distilled prior to use. MTC-OCH2BnCl,1 MTC-OBn,2 MPA-OBn,2 MTCOtBuAc,3 MPA-OCH2Tol,4 and 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea (TU)5 were synthesized according to previously reported procedures. All other materials were used as received. Methods. 1H NMR spectra were obtained on a Bruker Avance 400 instrument at 400 MHz. Chemical shifts are reported in delta (δ) units and expressed in parts per million (ppm) downfield from tetramethylsilane using the residual solvent as an internal standard. For 1H NMR: CDCl3, 7.26 ppm; DMSO-d6, 2.50 ppm. For 13C NMR: CDCl3, 77.1 ppm. Gel permeation chromatography (GPC) was performed in tetrahydrofuran (THF) using a Waters system equipped with four 5 μm Waters columns (300 mm × 7.7 mm) with an increasing pore size (100, 1000, 105, 106 Å) connected in series, a Waters 410 differential refractometer, and a 996 photodiode array detector. Retention times are corrected to account for differences in column-todetector path lengths. The system was calibrated with polystyrene standards. Rheological experiments. Blank and antimicrobial composite hydrogels were prepared by dissolving the triblock copolymer (13.0 wt%) or a mixture of the triblock copolymer and a thiouronium-containing cationic polycarbonate (0.1 and 1 wt.%) in DI water at 25 °C. As for the diclofenac and vancomycin-loaded hydrogels, the triblock copolymer (7.5 wt.%) was mixed with 3.75 wt.% PC-P(TMA)-PC or 0.5 wt.% PC-P(Acid)-PC for the loading of diclofenac and vancomycin respectively. The rheological analysis of the hydrogels was performed on an ARESG2 rheometer (TA Instruments, U.S.A.) equipped with a plate-plate geometry of 8 mm diameter. Measurements were taken by equilibrating the gels at 25 °C between the plates at a gap of 1.0 mm. The data were collected under a controlled strain of 3.0 % and a frequency scan of 1.0 to 100 rad/s. Gelation properties of the polymer solutions was monitored by measuring the shear storage modulus (G’), as well as the loss modulus (G’’), at each point. To investigate the shearthinning properties, the viscosity of the hydrogels was monitored as function of shear rate from 0.1 to 10 s-1. Killing efficiency test. E. coli, P. aeruginosa and S. aureus were cultured in MHB at 37 °C under constant shaking of 300 rpm, while C. albicans was cultured in YMB at room temperature under constant shaking of 50 rpm. Prior to the experiment, the microbes were first inoculated overnight to enter into log growth phase. Cationic polycarbonate-containing hydrogels were prepared using 13.0 wt.% of the triblock copolymer PC-PEG-PC and varying contents of PC-P(Thiouronium)PC. 50 µL of the hydrogels were aliquoted into 96-well microplate and an equal volume of microbe suspension (3 x 105 CFU/mL) was added subsequently. Prior to this, the concentration of microbe solution was adjusted to obtain an optical density (O.D.) reading of approximately 0.07 at 600 nm wavelength on a microplate reader (TECAN, Switzerland), which corresponds to S2
the concentration of McFarland 1 solution (3 x 108 CFU/mL). The culture plate was kept either at 37 °C for bacterial samples or room temperature for C. albicans under constant shaking of 300 or 50 rpm for 24 h respectively. After treatment, the samples were diluted in a series of tenfolds and spread onto agar plates. The plates were incubated for 24 h or 72 h at 37 °C or room temperature respectively for bacterial samples and C. albicans. The number of colony-forming units (CFU) was counted. Samples that were treated with blank PC-PEG-PC hydrogel was used as negative control, and each test was carried out in triplicates. The colony left after treatment was normalized against that of the negative control and expressed as the killing efficiency of the sample. Cytotoxicity test. Human dermal fibroblasts (HDF) were seeded onto a 96-well microplate at a density of 20,000 cells per well and incubated overnight at 37°C. The medium was removed and 90 µL of colorless DMEM was added to each well, followed by 10 µL of the hydrogels. The plate was then incubated for 24 h at 37°C. CellTitre-blue (Promega, USA) and DMEM were then mixed at a volume ratio of 2:3. After 24 h of treatment, 100 µL of this mixture was then added to each well and the cells were left to incubate in the dark at 37°C for 4 h. Subsequently, the fluorescence intensities at Ex/Em 560/590 nm were measured. Cells that were untreated were used as control. The readings were then expressed as a percentage of cell viability of the control group.
S3
Experimental Procedures Scheme S1. Synthesis of PC-PEG-PC Triblock Copolymer.
Synthesis of PC-PEG-PC. In a nitrogen filled glovebox, a 20 mL glass vial was charged with azeotropically dried poly(ethylene glycol) (Mn = 18500; 2.03 g, 0.140 mmol), MTC-OBn (0.549 g, 2.2 mmol), TU (33 mg, 0.09 mmol), a Teflon-coated stir bar, and dry DCM (4 mL). After the solids dissolved, DBU (0.2M premade solution in DCM; 0.45 mL, 0.09 mmol) was added to start the polymerization. After stirring for 30 minutes at room temperature, an excess of benzoic acid (30 mg, 0.24 mmol) was added to quench the catalyst and stop the polymerization. The crude reaction mixture was then precipitated into diethyl ether (40 mL). Three cycles of centrifugation/decantation of the supernatant, followed by drying under reduced pressure, afforded the desired polymer PC-PEG-PC as a white solid (2.49 g, 97% yield). 1H NMR (400 MHz, CDCl3): δ 7.32-7.26 (br, 128H, Ph-H), 5.13 (s, 40H, Ph-CH2), 4.26 (s, 72H, -OCOOCH2- and OCH2CCH3-), 3.64 (s, 1680H, PEG -OCH2CH2-), 1.23 (s, 56H, -CH3). Mn,NMR: 23.5 kDa.
Figure S1. 1H NMR Spectrum of PC-PEG-PC (CDCl3). S4
Scheme S2. Synthesis of Cationic Triblock Copolymers.
Scheme S3. Synthesis of Anionic Triblock Copolymers.
S5
Synthesis of PC-P(BnCl)-PC. The following procedure is representative. In a nitrogen filled glovebox, a 20 mL glass vial was charged with MTC-BnCl (1.2 g, 4.0 mmol), MPA-OCH2Tol (24 mg, 0.11 mmol, initiator), 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea (TU, 74 mg, 0.20 mmol), a Teflon-coated stir bar, and dry DCM (4 mL). After the solids dissolved, 1,8diazabicyclo[5.4.0]undec-7-ene (DBU, 30 mg, 0.2 mmol) was added to start the polymerization. The reaction mixture was stirred for 30 minutes at room temperature, at which point a small aliquot (0.1 mL) of the crude mixture was removed for 1H NMR and GPC analysis. A solution of MTC-OBn (0.5 g, 2.0 mmol) in DCM (2 mL) was then added to the reaction mixture, and stirring was continued for another 30 minutes. An excess of benzoic acid (30 mg, 0.24 mmol) was added to quench the catalyst and stop the polymerization. The crude reaction mixture was then precipitated into cold methanol (20 mL). Two cycles of centrifugation/decantation of the supernatant, followed by drying under reduced pressure, afforded the desired intermediate polymer PC-P(BnCl)-PC as a white solid (1.4 g, 82% yield). 1H NMR (400 MHz, CDCl3): 7.38-7.29 (m, 200H, Ph-H and -CH2Ph-H-CH2Cl), 5.15 (s, 83H, -O-CH2-Ph and -OCH2-BnCl), 4.57 (s, 64H, -CH2-Cl), 4.25 (br, 163H, -OCOOCH2- and -OCH2CCH3-), 2.33 (s, 3H, initiator -PhCH3), 1.25 (s, 125H, -CCH3). Mn,NMR: 13.2 kDa; Mn,GPC: 14.4 kDa, Ð = 1.10.
Figure S2. 1H NMR Spectrum of PC-P(BnCl)-PC (CDCl3).
S6
Synthesis of PC-P(Thiouronium)-PC. The following procedure is representative. To a 20 mL glass vial was added polymer PC-P(BnCl)-PC (700 mg, 1.83 mmol BnCl groups), thiourea (290 mg, 3.8 mmol), and DMF (5 mL). The reaction mixture was stirred for 18 hours at room temperature, transferred directly to a dialysis membrane (1000 Da molecular weight cutoff (MWCO)), and dialyzed against a 3:1 v/v 2-propanol:acetonitrile mixture for 18 hours. Concentration under reduced pressure afforded the desired polymer PC-P(Thiouronium)-PC as a white solid (872 mg, 99% yield). 1H NMR (400 MHz, DMSO-d6): 9.35 (br, 3H, -NH and -NH2), 7.44-7.30 (m, 4.7H, Ph-H and -CH2-Ph-H-CH2-), 5.10 (s, 2H, -O-CH2-Ph and -O-CH2-Ph-), 4.55 (s, 1.6H, -CH2-S-), 4.24 (m, 5H, -OCOOCH2- and -OCH2CCH3-), 1.19 (s, 3.4H, -CCH3). Mn,NMR: 16.1 kDa.
Figure S3. 1H NMR Spectrum of PC-P(Thiouronium)-PC (DMSO-d6). 2-propanol (*); DMF ( / ).
S7
Synthesis of PC-P(TMA)-PC. The quaternization of PC-P(BnCl)-PC with trimethylamine (TMA) was carried out in a pressure safe Schlenk tube owing to the gaseous nature of TMA. PCP(BnCl)-PC (0.554 g, 1.45 mmol BnCl groups) was dissolved in DMF (5 mL). The reaction vessel was sealed with a rubber septum and cooled in a -78 °C bath, at which point TMA gas (4 mL, 43 mmol) was condensed into the vessel. After warming to ambient temperature, the reaction mixture was subsequently warmed to 50 °C and stirred overnight. Following quaternization, the crude mixture was subjected to dialysis against a 3:1 v/v 2propanol:acetonitrile mixture. After replenishing with fresh dialysate three times, the contents of the dialysis membrane was concentrated under reduced pressure to afford the desired polymer PC-P(TMA)-PC as a white solid (0.523 g, 82% yield). 1H NMR (400 MHz, DMSO-d6): 7.587.30 (m, 192H, Ph-H and -CH2-Ph-H-CH2-), 5.17-5.10 (m, 90H, , -O-CH2-Ph and -O-CH2-Ph- ), 4.72 (br, 72H, -CH2-N-), 4.27 (br, 167H, -OCOOCH2- and -OCH2CCH3-), 3.09 (br s, 316H, N(CH3)3), 1.21 (s, 127H, -CCH3). Mn,NMR: 15.9 kDa.
Figure S4. 1H NMR Spectrum of PC-P(TMA)-PC (DMSO-d6). 2-propanol (*).
S8
Synthesis of PC-P(t-Bu)-PC. In a nitrogen filled glovebox, a 20 mL glass vial was charged with MTC-OtBuAc (1.50 g, 5.47 mmol), MPA-OCH2Tol (33 mg, 0.14 mmol, initiator), TU (81 mg, 0.22 mmol), a Teflon-coated stir bar, and dry DCM (5.5 mL). After the solids dissolved, DBU (33 mg, 0.22 mmol) was added to start the polymerization. The reaction mixture was stirred for 90 minutes at room temperature, at which point a small aliquot (0.1 mL) of the crude mixture was removed for 1H NMR and GPC analysis. A solution of MTC-OBn (0.685 g, 2.74 mmol) in DCM (2.7 mL) was then added to the reaction mixture, and stirring was continued for another 30 minutes. An excess of benzoic acid (50 mg, 0.41 mmol) was added to quench the catalyst and stop the polymerization. The crude reaction mixture was subjected to dialysis against a 3:1 v/v 2propanol:acetonitrile solvent mixture. Concentration under reduced pressure afforded the desired intermediate polymer PC-P(tBu)-PC as a white solid (1.79 g, 82% yield). 1H NMR (400 MHz, CDCl3): δ 7.30 (br, 106H, Ph-H), 5.13 (s, 40H, -OCH2-Ph), 4.52 (s, 74H, -OCH2-CO2tBu), 4.34 (m, 241H, -OCOOCH2- and -OCH2CCH3-), 2.33 (s, 3H, initiator -PhCH3), 1.45 (s, 391H, C(CH3)3), 1.32 (s, 137H, -CCH3 P(tBu) block), 1.24 (m, 70H, -CCH3 PC block). Mn,NMR: 13.8 kDa. Mn,GPC: 6.25 kDa, Ð = 1.27.
Figure S5. 1H NMR Spectrum of PC-P(tBu)-PC (CDCl3). BA = benzoic acid; DCM (*).
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Synthesis of PC-P(Acid)-PC. A 20 mL glass vial was charged with PC-P(tBu)-PC (1.79 g, 4.4 mmol tBu ester groups), a Teflon-coated stir bar, and dry DCM (4 mL). Trifluoroacetic acid (TFA; 1.79 g, 15.7 mmol) was added, and the reaction mixture was stirred for 72 h at room temperature. After removing the solvent under reduced pressure, the crude material was triturated in diethyl ether (20 mL) to afford the desired polymer PC-P(Acid)-PC as a white solid (1.3 g, 84% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.31 (br s, 80H, Ph-H), 5.10 (s, 33H, OCH2-Ph), 4.61 (s, 81H, -OCH2-CO2H), 4.26 (m, 200H, -OCOOCH2- and -OCH2CCH3-), 2.28 (s, 3H, initiator -PhCH3), 1.21 (s, 169H, -CCH3). Mn,NMR: 11.9 kDa.
Figure S6. 1H NMR Spectrum of PC-P(Acid)-PC (DMSO-d6). Diethyl ether (*).
S10
Formation of diclofenac-loaded hydrogels and in vitro drug release. Diclofenac-loaded hydrogels were formed by loading 0.75 wt.% diclofenac in the absence or presence of 3.75 wt.% of PC-P(TMA)-PC and 7.5 wt.% PC-PEG-PC. The diluent used was HPLC grade water. Briefly, diclofenac was first dissolved in HPLC grade water at 0.75 wt.%. Subsequently, this solution was added to PC-P(TMA)-PC and PC-PEG-PC and placed in an ultrasound bath for 30 min for hydrogel formation. The release of diclofenac from the hydrogels was studied by placing 0.2 mL of the hydrogel containing in a dialysis membrane tube with MWCO of 1 kDa (Spectrum Laboratories, U.S.A.). This was then immersed in 20 mL of the release medium phosphatebuffered saline (PBS, pH 7.4). The samples were kept shaking on an orbital shaker at 100 rpm at 37 °C. At designated time intervals, the release medium was removed and replaced with fresh PBS. The collected medium was analyzed for its drug content via HPLC by mixing with the HPLC mobile in the volume ratio of 1:3. The samples were analyzed under the following conditions: Column temperature 28 oC, sample temperature 20 oC, mobile phase: A – acetonitrile, B – 0.0025M sodium acetate at 70%A / 30%B with pH adjusted to 4.0 using glacial acetic acid; detection: 276 nm; flow rate: 1 mL/min. Formation of vancomycin-loaded hydrogels and in vitro drug release. Vancomycin-loaded hydrogels were formed by loading 0.5 wt.% vancomycin in the absence or presence of 0.5 wt.% of PC-P(Acid)-PC and 7.5 wt.% PC-PEG-PC. The diluent used was 10 mM phosphate buffer (pH 7). Briefly, vancomycin was first dissolved in HPLC grade water at 1.0 wt.% while PCP(Acid)-PC was dissolved in 20 mM phosphate buffer (pH 7) at 1.0 wt.% via ultrasonication. Subsequently, vancomycin was slowly added to PC-P(Acid)-PC, dropwise and vortexing with every addition to prevent precipitation. This resultant solution was then added to PC-PEG-PC and placed in an ultrasound bath for 30 min for hydrogel formation. The release of vancomycin from the hydrogels was studied by placing 0.2 mL of the vancomycin-loaded hydrogel in a dialysis membrane tube with MWCO of 2 kDa (Spectrum Laboratories, U.S.A.). This was then immersed in 20 mL of the release medium phosphate-buffered saline (PBS, pH 7.4). The samples were kept shaking on an orbital shaker at 100 rpm at 37 °C. At designated time intervals, the release medium was removed and replaced with fresh PBS. The collected medium was analyzed for its drug content via HPLC by mixing with the HPLC mobile phase in the volume ratio of 1:3. The samples were analyzed under the following conditions: Column temperature 28 oC, sample temperature 20 oC, mobile phase: A – acetonitrile, B – water and C – 85% phosphoric acid at 8.5% A / 91.5% B / 0.125% C with pH adjusted to 3.0 using triethylamine; detection: 230 nm; flow rate: 1 mL/min.
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Additional Rheology Data
Figure S7. Viscosity as a function of shear rate for PC-PEG-PC (7.5 wt.%) hydrogel loaded with polyelectrolytes and/or drug listed in Table 2.
Cytotoxicity Data Table S1. Compositions of polymers and drugs in hydrogels used in cytotoxicity test. Sample Polymer 1
2
3
4
5
6
7
7.5
7.5
7.5
7.5
7.5
7.5
7.5
PC-P(Acid)-PC
0
0.5
0
0.5
0
0
0
PC-P(TMA)-PC
0
0
0
0
3.75
0
3.75
Vancomycin
0
0
0.5
0.5
0
PC-PEG-PC
Diclofenac
0 0 0 0 0 *Values listed are in wt.%.
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0
0
0.75
0.75
Figure S8. Viability of human dermal fibroblast (HDF) cells after 24 h treatment with hydrogels. The compositions used are listed in Table S1. References 1. Chin, W.; Yang, C.; Ng, V. W. L.; Huang, Y.; Cheng, J.; Tong, Y. W.; Coady, D. J.; Fan, W.; Hedrick, J. L.; Yang. Y. Y. Macromolecules 2013, 46, 8797-8807 2. Pratt, R. C.; Nederberg, F.; Waymouth, R. M.; Hedrick, J. L. Chem. Commun. 2008, 114116. 3. Bartolini, C.; Mespouille, L.; Verbruggen, I.; Willem, R.; Dubois, P. Soft Matter 2011, 7, 9628. 4. Ono, R. J.; Liu, S. Q.; Venkataraman, S.; Chin, W.; Yang,Y. Y.; Hedrick, J. L. Macromolecules 2014, 47, 7725–7731. 5. Pratt, R.C.; Lohmeijer, B.G.G.; Long, D.A.; Dove, A.P.; Li, H.; Waymouth, R.M.; Hedrick, J.L. Macromolecules 2006, 39, 7863.
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Biodegradable Block Copolyelectrolyte Hydrogels for Tunable Release of Therapeutics and Topical Antimicrobial Skin Treatment Robert J. Ono,†,§ Ashlynn L. Z. Lee,‡,§ Willy Chin,‡ Wei Sheng Goh,‡ Amelia Y. L. Lee,‡ Yi Yan Yang‡,* and James L. Hedrick†,* † ‡
IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120, United States
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore Email: [email protected]; [email protected]
Table of Contents General Considerations
S2
Experimental Procedures and NMR Spectra
S4
Additional Rheology Data
S12
Cytotoxicity Data
S12
References
S13
S1
General Considerations Materials. Dichloromethane and tetrahydrofuran were dried using activated alumina columns and stored over molecular sieves (3 Å). 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was dried over CaH2 and distilled prior to use. MTC-OCH2BnCl,1 MTC-OBn,2 MPA-OBn,2 MTCOtBuAc,3 MPA-OCH2Tol,4 and 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea (TU)5 were synthesized according to previously reported procedures. All other materials were used as received. Methods. 1H NMR spectra were obtained on a Bruker Avance 400 instrument at 400 MHz. Chemical shifts are reported in delta (δ) units and expressed in parts per million (ppm) downfield from tetramethylsilane using the residual solvent as an internal standard. For 1H NMR: CDCl3, 7.26 ppm; DMSO-d6, 2.50 ppm. For 13C NMR: CDCl3, 77.1 ppm. Gel permeation chromatography (GPC) was performed in tetrahydrofuran (THF) using a Waters system equipped with four 5 μm Waters columns (300 mm × 7.7 mm) with an increasing pore size (100, 1000, 105, 106 Å) connected in series, a Waters 410 differential refractometer, and a 996 photodiode array detector. Retention times are corrected to account for differences in column-todetector path lengths. The system was calibrated with polystyrene standards. Rheological experiments. Blank and antimicrobial composite hydrogels were prepared by dissolving the triblock copolymer (13.0 wt%) or a mixture of the triblock copolymer and a thiouronium-containing cationic polycarbonate (0.1 and 1 wt.%) in DI water at 25 °C. As for the diclofenac and vancomycin-loaded hydrogels, the triblock copolymer (7.5 wt.%) was mixed with 3.75 wt.% PC-P(TMA)-PC or 0.5 wt.% PC-P(Acid)-PC for the loading of diclofenac and vancomycin respectively. The rheological analysis of the hydrogels was performed on an ARESG2 rheometer (TA Instruments, U.S.A.) equipped with a plate-plate geometry of 8 mm diameter. Measurements were taken by equilibrating the gels at 25 °C between the plates at a gap of 1.0 mm. The data were collected under a controlled strain of 3.0 % and a frequency scan of 1.0 to 100 rad/s. Gelation properties of the polymer solutions was monitored by measuring the shear storage modulus (G’), as well as the loss modulus (G’’), at each point. To investigate the shearthinning properties, the viscosity of the hydrogels was monitored as function of shear rate from 0.1 to 10 s-1. Killing efficiency test. E. coli, P. aeruginosa and S. aureus were cultured in MHB at 37 °C under constant shaking of 300 rpm, while C. albicans was cultured in YMB at room temperature under constant shaking of 50 rpm. Prior to the experiment, the microbes were first inoculated overnight to enter into log growth phase. Cationic polycarbonate-containing hydrogels were prepared using 13.0 wt.% of the triblock copolymer PC-PEG-PC and varying contents of PC-P(Thiouronium)PC. 50 µL of the hydrogels were aliquoted into 96-well microplate and an equal volume of microbe suspension (3 x 105 CFU/mL) was added subsequently. Prior to this, the concentration of microbe solution was adjusted to obtain an optical density (O.D.) reading of approximately 0.07 at 600 nm wavelength on a microplate reader (TECAN, Switzerland), which corresponds to S2
the concentration of McFarland 1 solution (3 x 108 CFU/mL). The culture plate was kept either at 37 °C for bacterial samples or room temperature for C. albicans under constant shaking of 300 or 50 rpm for 24 h respectively. After treatment, the samples were diluted in a series of tenfolds and spread onto agar plates. The plates were incubated for 24 h or 72 h at 37 °C or room temperature respectively for bacterial samples and C. albicans. The number of colony-forming units (CFU) was counted. Samples that were treated with blank PC-PEG-PC hydrogel was used as negative control, and each test was carried out in triplicates. The colony left after treatment was normalized against that of the negative control and expressed as the killing efficiency of the sample. Cytotoxicity test. Human dermal fibroblasts (HDF) were seeded onto a 96-well microplate at a density of 20,000 cells per well and incubated overnight at 37°C. The medium was removed and 90 µL of colorless DMEM was added to each well, followed by 10 µL of the hydrogels. The plate was then incubated for 24 h at 37°C. CellTitre-blue (Promega, USA) and DMEM were then mixed at a volume ratio of 2:3. After 24 h of treatment, 100 µL of this mixture was then added to each well and the cells were left to incubate in the dark at 37°C for 4 h. Subsequently, the fluorescence intensities at Ex/Em 560/590 nm were measured. Cells that were untreated were used as control. The readings were then expressed as a percentage of cell viability of the control group.
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Experimental Procedures Scheme S1. Synthesis of PC-PEG-PC Triblock Copolymer.
Synthesis of PC-PEG-PC. In a nitrogen filled glovebox, a 20 mL glass vial was charged with azeotropically dried poly(ethylene glycol) (Mn = 18500; 2.03 g, 0.140 mmol), MTC-OBn (0.549 g, 2.2 mmol), TU (33 mg, 0.09 mmol), a Teflon-coated stir bar, and dry DCM (4 mL). After the solids dissolved, DBU (0.2M premade solution in DCM; 0.45 mL, 0.09 mmol) was added to start the polymerization. After stirring for 30 minutes at room temperature, an excess of benzoic acid (30 mg, 0.24 mmol) was added to quench the catalyst and stop the polymerization. The crude reaction mixture was then precipitated into diethyl ether (40 mL). Three cycles of centrifugation/decantation of the supernatant, followed by drying under reduced pressure, afforded the desired polymer PC-PEG-PC as a white solid (2.49 g, 97% yield). 1H NMR (400 MHz, CDCl3): δ 7.32-7.26 (br, 128H, Ph-H), 5.13 (s, 40H, Ph-CH2), 4.26 (s, 72H, -OCOOCH2- and OCH2CCH3-), 3.64 (s, 1680H, PEG -OCH2CH2-), 1.23 (s, 56H, -CH3). Mn,NMR: 23.5 kDa.
Figure S1. 1H NMR Spectrum of PC-PEG-PC (CDCl3). S4
Scheme S2. Synthesis of Cationic Triblock Copolymers.
Scheme S3. Synthesis of Anionic Triblock Copolymers.
S5
Synthesis of PC-P(BnCl)-PC. The following procedure is representative. In a nitrogen filled glovebox, a 20 mL glass vial was charged with MTC-BnCl (1.2 g, 4.0 mmol), MPA-OCH2Tol (24 mg, 0.11 mmol, initiator), 1-(3,5-bis(trifluoromethyl)-phenyl)-3-cyclohexyl-2-thiourea (TU, 74 mg, 0.20 mmol), a Teflon-coated stir bar, and dry DCM (4 mL). After the solids dissolved, 1,8diazabicyclo[5.4.0]undec-7-ene (DBU, 30 mg, 0.2 mmol) was added to start the polymerization. The reaction mixture was stirred for 30 minutes at room temperature, at which point a small aliquot (0.1 mL) of the crude mixture was removed for 1H NMR and GPC analysis. A solution of MTC-OBn (0.5 g, 2.0 mmol) in DCM (2 mL) was then added to the reaction mixture, and stirring was continued for another 30 minutes. An excess of benzoic acid (30 mg, 0.24 mmol) was added to quench the catalyst and stop the polymerization. The crude reaction mixture was then precipitated into cold methanol (20 mL). Two cycles of centrifugation/decantation of the supernatant, followed by drying under reduced pressure, afforded the desired intermediate polymer PC-P(BnCl)-PC as a white solid (1.4 g, 82% yield). 1H NMR (400 MHz, CDCl3): 7.38-7.29 (m, 200H, Ph-H and -CH2Ph-H-CH2Cl), 5.15 (s, 83H, -O-CH2-Ph and -OCH2-BnCl), 4.57 (s, 64H, -CH2-Cl), 4.25 (br, 163H, -OCOOCH2- and -OCH2CCH3-), 2.33 (s, 3H, initiator -PhCH3), 1.25 (s, 125H, -CCH3). Mn,NMR: 13.2 kDa; Mn,GPC: 14.4 kDa, Ð = 1.10.
Figure S2. 1H NMR Spectrum of PC-P(BnCl)-PC (CDCl3).
S6
Synthesis of PC-P(Thiouronium)-PC. The following procedure is representative. To a 20 mL glass vial was added polymer PC-P(BnCl)-PC (700 mg, 1.83 mmol BnCl groups), thiourea (290 mg, 3.8 mmol), and DMF (5 mL). The reaction mixture was stirred for 18 hours at room temperature, transferred directly to a dialysis membrane (1000 Da molecular weight cutoff (MWCO)), and dialyzed against a 3:1 v/v 2-propanol:acetonitrile mixture for 18 hours. Concentration under reduced pressure afforded the desired polymer PC-P(Thiouronium)-PC as a white solid (872 mg, 99% yield). 1H NMR (400 MHz, DMSO-d6): 9.35 (br, 3H, -NH and -NH2), 7.44-7.30 (m, 4.7H, Ph-H and -CH2-Ph-H-CH2-), 5.10 (s, 2H, -O-CH2-Ph and -O-CH2-Ph-), 4.55 (s, 1.6H, -CH2-S-), 4.24 (m, 5H, -OCOOCH2- and -OCH2CCH3-), 1.19 (s, 3.4H, -CCH3). Mn,NMR: 16.1 kDa.
Figure S3. 1H NMR Spectrum of PC-P(Thiouronium)-PC (DMSO-d6). 2-propanol (*); DMF ( / ).
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Synthesis of PC-P(TMA)-PC. The quaternization of PC-P(BnCl)-PC with trimethylamine (TMA) was carried out in a pressure safe Schlenk tube owing to the gaseous nature of TMA. PCP(BnCl)-PC (0.554 g, 1.45 mmol BnCl groups) was dissolved in DMF (5 mL). The reaction vessel was sealed with a rubber septum and cooled in a -78 °C bath, at which point TMA gas (4 mL, 43 mmol) was condensed into the vessel. After warming to ambient temperature, the reaction mixture was subsequently warmed to 50 °C and stirred overnight. Following quaternization, the crude mixture was subjected to dialysis against a 3:1 v/v 2propanol:acetonitrile mixture. After replenishing with fresh dialysate three times, the contents of the dialysis membrane was concentrated under reduced pressure to afford the desired polymer PC-P(TMA)-PC as a white solid (0.523 g, 82% yield). 1H NMR (400 MHz, DMSO-d6): 7.587.30 (m, 192H, Ph-H and -CH2-Ph-H-CH2-), 5.17-5.10 (m, 90H, , -O-CH2-Ph and -O-CH2-Ph- ), 4.72 (br, 72H, -CH2-N-), 4.27 (br, 167H, -OCOOCH2- and -OCH2CCH3-), 3.09 (br s, 316H, N(CH3)3), 1.21 (s, 127H, -CCH3). Mn,NMR: 15.9 kDa.
Figure S4. 1H NMR Spectrum of PC-P(TMA)-PC (DMSO-d6). 2-propanol (*).
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Synthesis of PC-P(t-Bu)-PC. In a nitrogen filled glovebox, a 20 mL glass vial was charged with MTC-OtBuAc (1.50 g, 5.47 mmol), MPA-OCH2Tol (33 mg, 0.14 mmol, initiator), TU (81 mg, 0.22 mmol), a Teflon-coated stir bar, and dry DCM (5.5 mL). After the solids dissolved, DBU (33 mg, 0.22 mmol) was added to start the polymerization. The reaction mixture was stirred for 90 minutes at room temperature, at which point a small aliquot (0.1 mL) of the crude mixture was removed for 1H NMR and GPC analysis. A solution of MTC-OBn (0.685 g, 2.74 mmol) in DCM (2.7 mL) was then added to the reaction mixture, and stirring was continued for another 30 minutes. An excess of benzoic acid (50 mg, 0.41 mmol) was added to quench the catalyst and stop the polymerization. The crude reaction mixture was subjected to dialysis against a 3:1 v/v 2propanol:acetonitrile solvent mixture. Concentration under reduced pressure afforded the desired intermediate polymer PC-P(tBu)-PC as a white solid (1.79 g, 82% yield). 1H NMR (400 MHz, CDCl3): δ 7.30 (br, 106H, Ph-H), 5.13 (s, 40H, -OCH2-Ph), 4.52 (s, 74H, -OCH2-CO2tBu), 4.34 (m, 241H, -OCOOCH2- and -OCH2CCH3-), 2.33 (s, 3H, initiator -PhCH3), 1.45 (s, 391H, C(CH3)3), 1.32 (s, 137H, -CCH3 P(tBu) block), 1.24 (m, 70H, -CCH3 PC block). Mn,NMR: 13.8 kDa. Mn,GPC: 6.25 kDa, Ð = 1.27.
Figure S5. 1H NMR Spectrum of PC-P(tBu)-PC (CDCl3). BA = benzoic acid; DCM (*).
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Synthesis of PC-P(Acid)-PC. A 20 mL glass vial was charged with PC-P(tBu)-PC (1.79 g, 4.4 mmol tBu ester groups), a Teflon-coated stir bar, and dry DCM (4 mL). Trifluoroacetic acid (TFA; 1.79 g, 15.7 mmol) was added, and the reaction mixture was stirred for 72 h at room temperature. After removing the solvent under reduced pressure, the crude material was triturated in diethyl ether (20 mL) to afford the desired polymer PC-P(Acid)-PC as a white solid (1.3 g, 84% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.31 (br s, 80H, Ph-H), 5.10 (s, 33H, OCH2-Ph), 4.61 (s, 81H, -OCH2-CO2H), 4.26 (m, 200H, -OCOOCH2- and -OCH2CCH3-), 2.28 (s, 3H, initiator -PhCH3), 1.21 (s, 169H, -CCH3). Mn,NMR: 11.9 kDa.
Figure S6. 1H NMR Spectrum of PC-P(Acid)-PC (DMSO-d6). Diethyl ether (*).
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Formation of diclofenac-loaded hydrogels and in vitro drug release. Diclofenac-loaded hydrogels were formed by loading 0.75 wt.% diclofenac in the absence or presence of 3.75 wt.% of PC-P(TMA)-PC and 7.5 wt.% PC-PEG-PC. The diluent used was HPLC grade water. Briefly, diclofenac was first dissolved in HPLC grade water at 0.75 wt.%. Subsequently, this solution was added to PC-P(TMA)-PC and PC-PEG-PC and placed in an ultrasound bath for 30 min for hydrogel formation. The release of diclofenac from the hydrogels was studied by placing 0.2 mL of the hydrogel containing in a dialysis membrane tube with MWCO of 1 kDa (Spectrum Laboratories, U.S.A.). This was then immersed in 20 mL of the release medium phosphatebuffered saline (PBS, pH 7.4). The samples were kept shaking on an orbital shaker at 100 rpm at 37 °C. At designated time intervals, the release medium was removed and replaced with fresh PBS. The collected medium was analyzed for its drug content via HPLC by mixing with the HPLC mobile in the volume ratio of 1:3. The samples were analyzed under the following conditions: Column temperature 28 oC, sample temperature 20 oC, mobile phase: A – acetonitrile, B – 0.0025M sodium acetate at 70%A / 30%B with pH adjusted to 4.0 using glacial acetic acid; detection: 276 nm; flow rate: 1 mL/min. Formation of vancomycin-loaded hydrogels and in vitro drug release. Vancomycin-loaded hydrogels were formed by loading 0.5 wt.% vancomycin in the absence or presence of 0.5 wt.% of PC-P(Acid)-PC and 7.5 wt.% PC-PEG-PC. The diluent used was 10 mM phosphate buffer (pH 7). Briefly, vancomycin was first dissolved in HPLC grade water at 1.0 wt.% while PCP(Acid)-PC was dissolved in 20 mM phosphate buffer (pH 7) at 1.0 wt.% via ultrasonication. Subsequently, vancomycin was slowly added to PC-P(Acid)-PC, dropwise and vortexing with every addition to prevent precipitation. This resultant solution was then added to PC-PEG-PC and placed in an ultrasound bath for 30 min for hydrogel formation. The release of vancomycin from the hydrogels was studied by placing 0.2 mL of the vancomycin-loaded hydrogel in a dialysis membrane tube with MWCO of 2 kDa (Spectrum Laboratories, U.S.A.). This was then immersed in 20 mL of the release medium phosphate-buffered saline (PBS, pH 7.4). The samples were kept shaking on an orbital shaker at 100 rpm at 37 °C. At designated time intervals, the release medium was removed and replaced with fresh PBS. The collected medium was analyzed for its drug content via HPLC by mixing with the HPLC mobile phase in the volume ratio of 1:3. The samples were analyzed under the following conditions: Column temperature 28 oC, sample temperature 20 oC, mobile phase: A – acetonitrile, B – water and C – 85% phosphoric acid at 8.5% A / 91.5% B / 0.125% C with pH adjusted to 3.0 using triethylamine; detection: 230 nm; flow rate: 1 mL/min.
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Additional Rheology Data
Figure S7. Viscosity as a function of shear rate for PC-PEG-PC (7.5 wt.%) hydrogel loaded with polyelectrolytes and/or drug listed in Table 2.
Cytotoxicity Data Table S1. Compositions of polymers and drugs in hydrogels used in cytotoxicity test. Sample Polymer 1
2
3
4
5
6
7
7.5
7.5
7.5
7.5
7.5
7.5
7.5
PC-P(Acid)-PC
0
0.5
0
0.5
0
0
0
PC-P(TMA)-PC
0
0
0
0
3.75
0
3.75
Vancomycin
0
0
0.5
0.5
0
PC-PEG-PC
Diclofenac
0 0 0 0 0 *Values listed are in wt.%.
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0
0
0.75
0.75
Figure S8. Viability of human dermal fibroblast (HDF) cells after 24 h treatment with hydrogels. The compositions used are listed in Table S1. References 1. Chin, W.; Yang, C.; Ng, V. W. L.; Huang, Y.; Cheng, J.; Tong, Y. W.; Coady, D. J.; Fan, W.; Hedrick, J. L.; Yang. Y. Y. Macromolecules 2013, 46, 8797-8807 2. Pratt, R. C.; Nederberg, F.; Waymouth, R. M.; Hedrick, J. L. Chem. Commun. 2008, 114116. 3. Bartolini, C.; Mespouille, L.; Verbruggen, I.; Willem, R.; Dubois, P. Soft Matter 2011, 7, 9628. 4. Ono, R. J.; Liu, S. Q.; Venkataraman, S.; Chin, W.; Yang,Y. Y.; Hedrick, J. L. Macromolecules 2014, 47, 7725–7731. 5. Pratt, R.C.; Lohmeijer, B.G.G.; Long, D.A.; Dove, A.P.; Li, H.; Waymouth, R.M.; Hedrick, J.L. Macromolecules 2006, 39, 7863.
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