Synthesis of bioreducible core crosslinked star polymers with

N,N'-bis(acryloyl)cystamine crosslinker via aqueous ethanol

dispersion RAFT polymerization

Qijing Chen, Fei Han, Chao Lin, Xuejun Wen, and Peng Zhao*

Shanghai East Hospital, The Institute for Translational Medicine, The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Tongji University, Shanghai 200092, People’s Republic of China.

Correspondence: zp@tongji.edu.cn

Supplementary Information

Synthesis of L-PPEGMA arm polymers.

The preparation procedure of L-PPEGMA4 was as following. CTA (2.774 g, 6.7 mmol), PEGMA (10 g, 33 mmol) and DMF (0.244 g, 3.3 mmol, internal standard) were dissolved in 25 mL of dioxane. The solution was degassed with nitrogen at 0 °C for 40 min before immersion into a preheated oil bath at 70 °C. After the temperature was stabilized, a degassed solution of AIBN (54.7 mg, 0.33 mmol) in dioxane was injected via a microsyringe. The polymerization was conducted for 6 h and was stopped at 78% monomer conversion as determined by 1H NMR. The solution was precipitated into hexane for purification. The precipitation was re-dissolved by THF and precipitated into hexane again and the purification process was done for three times until the unreacted monomer was removed completely. After drying under vacuum, 8.68 g of a yellow solid was obtained in 68% yield. Mn,th = 1600 g mol-1, Mn = 2200 g mol-1 (RI-GPC), Đ = 1.49 (RI-GPC). 1H NMR (500 MHz, CDCl3): 4.04 ppm (s, –COOCH2–), 3.78–3.49 ppm (m, –O(CH2)2O–), 3.32 ppm (s, –OCH3), 1.98–1.66 ppm (m, backbone –CH2–), 1.05–0.72 ppm (s, –CH3).

Scheme S1 RAFT synthesis of L-PPEGMA arm polymers.

Synthesis of L-PDMA76 arm polymers. CTA (1.1 g, 3.1 mmol), DMA (25 g, 0.247 mol) and trioxane (2.86 g, 31 mmol, internal standard) were dissolved in 50 mL of dioxane. The solution was degassed with nitrogen at 0 °C for 40 min before immersion into a preheated oil bath at 70 °C. After the temperature was stabilized, a degassed solution of AIBN (0.102 g, 0.6 mmol) in dioxane was injected via a microsyringe. The polymerization was conducted for 6 h and was stopped at 95.6% monomer conversion as determined by 1H NMR. The solution was precipitated into diethyl ether for purification. The precipitation was re-dissolved by THF and precipitated into diethyl ether again and the purification process was done for three times until the unreacted monomer was removed completely. After drying under vacuum, 24.5 g of a yellow solid was obtained in 94% yield. Mn,th = 7900 g mol-1, Mn = 7100 g mol-1 (RI-GPC), Đ = 1.23 (RI-GPC). 1H NMR (500 MHz, CDCl3): 3.26–2.75 ppm (m, –N(CH3)2), 2.75–2.45 ppm (m, –(CO)CHCH2–), 1.91–1.15 (m, –CHCH2–).

Scheme S2 RAFT synthesis of PDMA76 arm polymer.

Synthesis of L-PDMAEMA59 arm polymers. CTA (0.283 g, 0.79 mmol), DMAEMA (10 g, 63.6 mmol) and trioxane (0.716 g, 7.9 mmol, internal standard) were dissolved in 20 mL of dioxane. The solution was degassed with nitrogen at 0 °C for 40 min before immersion into a preheated oil bath at 70 °C. After the temperature was stabilized, a degassed solution of AIBN (13.2 mg, 0.08 mmol) in dioxane was injected via a microsyringe. The polymerization was conducted for 22 h and was stopped at 74% monomer conversion as determined by 1H NMR. The solution was precipitated into hexane for purification. The precipitation was re-dissolved by THF and precipitated into hexane again and the purification process was done for three times until the unreacted monomer was removed completely. After drying under vacuum, 7.4 g of a yellow solid

Scheme S3 RAFT synthesis of PDMAEMA59 arm polymer.

was obtained in 72% yield. Mn,th = 11900 g mol-1, Mn = 8900 g mol-1 (RI-GPC), Đ = 1.19 (RI-GPC). 1H NMR (500 MHz, CDCl3): 4.05 ppm (m, –COOCH2–), 2.55 ppm (m, –CH2N–), 2.26 ppm (s, –N(CH3)2), 1.97–1.78 ppm (m, backbone –CH2–), 1.09–0.82 ppm (m, backbone –CH3).

Synthesis of S-PPEGMA4 CCS polymers. The macro-CTA of L-PPEGMA4 (4.050 g, 2.53 mmol) arm polymer, crosslinker BAC (3.9 g, 0.015 mmol) and V50 (0.1415 g, 0.49 mmol) were dissolved in a water–ethanol (50/50 v/v) solution of 40 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 98% yield, which was purified by dialysis (MWCO 2.5 kg mol-1) of its aqueous solution. After the dialysis, S-PPEGMA4 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 40.2 kg mol-1, Đ = 1.2.

Synthesis of S-PPEGMA10 CCS polymers. The macro-CTA of L-PPEGMA10 (0.4 g, 0.117 mmol) arm polymer, crosslinker BAC (0.188 g, 0.7 mmol) and V50 (6.6 mg, 0.024 mmol) were dissolved in a water–ethanol (70/30 v/v) solution of 4 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 94% yield, which was purified by dialysis (MWCO 2.5 kg mol-1) of its aqueous solution. After the dialysis, S-PPEGMA10 was obtained by freeze-drying of the dialysis solution.

RI-GPC: Mn = 56.2 kg mol-1, Đ = 1.16.

Synthesis of S-PPEGMA21 CCS polymers. The macro-CTA of L-PPEGMA21 (0.4 g, 0.06 mmol) arm polymer, crosslinker BAC (0.095 g, 0.36 mmol) and V50 (3.4 mg, 0.012 mmol) were dissolved in a water–ethanol (75/25 v/v) solution of 4 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 92% yield, which was purified by repeated ultrafiltration of its aqueous solutions using Amicon Ultra-15 (MWCO 30 kg mol-1) filtration tubes at a speed of 4000 rpm. After the dialysis, S-PPEGMA21 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 72.3 kg mol-1, Đ = 1.16.

Synthesis of S-PPEGMA4-D144 CCS polymers. The macro-CTA of L-PPEGMA4 (0.4 g, 0.25 mmol) arm polymer, crosslinker BAC (0.39 g, 1.5 mmol), DMAEMA (0.395 g, 2.5 mmol) and V50 (0.1415 g, 0.49 mmol) were dissolved in a water–ethanol (80/20 v/v) solution of 40 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 91% yield, which was purified by dialysis (MWCO 2.5 kg mol-1) of its aqueous solution. After the dialysis, S-PPEGMA4-D144 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 57.1 kg mol-1, Đ = 1.23.

Synthesis of S-PPEGMA10-D121 CCS polymers. The macro-CTA of L-PPEGMA10 (0.4 g, 0.117 mmol) arm polymer, crosslinker BAC (0.188 g, 0.7 mmol), DMAEMA (0.187 g, 1.17 mmol) and V50 (6.6 mg, 0.024 mmol) were dissolved in a water–ethanol (85/15 v/v) solution of 4 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 90% yield, which was purified by dialysis (MWCO 2.5 kg mol-1) of its aqueous solution. After the dialysis, S-PPEGMA10-D121 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 64.7 kg mol-1, Đ = 1.19.

Synthesis of S-PPEGMA21-D57 CCS polymers. The macro-CTA of L-PPEGMA21 (0.4 g, 0.06 mmol) arm polymer, crosslinker BAC (0.159 g, 0.6 mmol), DMAEMA (0.096 g, 0.6 mmol) and V50 (3.4 mg, 0.012 mmol) were dissolved in a water–ethanol (85/15 v/v) solution of 4 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 92% yield, which was purified by repeated ultrafiltration of its aqueous solutions using Amicon Ultra-15 (MWCO 30 kg mol-1) filtration tubes at a speed of 4000 rpm. After the dialysis, S-PPEGMA21-D57 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 62.8 kg mol-1, Đ = 1.15.

Synthesis of S-PDMA76 CCS polymers. The macro-CTA of L-PDMA76 (0.4 g, 0.05 mmol) arm polymer, crosslinker BAC (0.199 g, 0.75 mmol) and V50 (2.8 mg, 0.01 mmol) were dissolved in the water–ethanol (80/20 v/v) solution of 4 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in

75% yield, which was purified by repeated ultrafiltration of its aqueous solutions using Amicon Ultra-15 (MWCO 30 kg mol-1) filtration tubes at a speed of 4000 rpm. After the dialysis, S-PDMA76 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 130.5 kg mol-1, Đ = 1.18.

Synthesis of S-PDMAEMA59 CCS polymers. The macro-CTA of L-PDMAEMA59 (0.4 g, 0.033 mmol) arm polymer, crosslinker BAC (0.088 g, 0.33 mmol) and V50 (0.002 g, 0.007 mmol) were dissolved in the acidic water–ethanol (80/20 v/v) solution of 4 mL. After degassed with nitrogen in an ice/water bath for 40 min, the mixture was immersed into a preheated oil bath at 70 °C. The polymerization was allowed to continue for 1 h under protection of nitrogen. The CCS polymer was synthesized in 82% yield, which was purified by repeated ultrafiltration of its aqueous solutions using Amicon Ultra-15 (MWCO 30 kg mol-1) filtration tubes at a speed of 4000 rpm. After the dialysis, S-PDMAEMA59 was obtained by freeze-drying of the dialysis solution. RI-GPC: Mn = 81.8 kg mol-1, Đ = 1.15.Calculation of arm numbers of the CCS polymers.For PPEGMA, PDMA and PDMAEMA CCS polymers, the arm numbers were calculated as following.Firstly, weight fraction of the arm (Xarm) in the star was calculated by the equation (1)

X arm=marm∗r arm, conversion

marm∗r arm, conversion+mBAC ,conversion∗rBAC , conversion

(1)

where m is the reaction mass and arm conversion (rarm,conversion) is the star yield determined by RI-GPC. Unreacted BAC (mBAC, unreaction) is determined by firstly centrifuging the reaction solution after stored at 4 °C for 48 hours, then filtering the unreacted BAC deposition and finally weighting

the dried deposition powder. The BAC conversion was determined by (1−mBAC ,unreation

mBAC) .

Secondly, the arm number Narm is calculated by the equation (2)N arm∗M w , arm=Xarm∗M w , star (2)where the CCS polymer absolute molecular weight (Mw,arm) and arm polymer absolute molecular weight (Mw,star) were determined by GPC with triple detectors.

For PPEGMA-based cationic CCS polymers, the arm numbers were calculated as following.Firstly, weight fraction of the arm (Xarm) in the star was calculated by the equation (1)

X arm=marm∗rarm ,conversion

marm∗r arm, conversion+mBAC∗r BAC, conversion+mDMAEMA∗r DMAEMA ,conversion

(1)

where m is the reaction mass and arm conversion (rarm,conversion) is determined by RI-GPC. Unreacted BAC (mBAC, unreaction) is determined by firstly centrifuging the reaction solution after stored at 4 °C for 48 hours, then filtering the unreacted BAC deposition and finally weighting the dried deposition powder. The BAC conversion (rBAC,conversion) was determined by

(1−mBAC ,unreation

mBAC) . Conversion of DMAEMA (rDMAEMA,conversion) was determined by GC.

Secondly, the arm number Narm is calculated by the equation (2)N arm∗M w , arm=Xarm∗M w , star (2)where the CCS polymer molecular weight (Mw,arm) and arm polymer molecular weight (Mw,star) were determined by triple-detection GPC.

Fig. S1 GPC traces of L-PPEGMA arm polymers.

8000 16000 24000 32000 40000Molecular weight (g/mol)

L-PDMAEMA59

L-PDMA76

Fig. S2 GPC traces of L-PDMAEMA59 and L-PDMA76 arm polymers.

Fig. S3 GPC curves of S-PPEGMA10 polymers prepared in anhydrous ethanol with different reaction times and the arm polymer concentration of 5%.

Fig. S4 Triple-detection GPC curves of S-PPEGMA10 polymer synthesized in water/ethanol mixture with Vw = 90%.

Fig. S5 Photo of S-PPEGMA10 prepared in water/ethanol mixture with Vw of 80% and 90%, respectively.

Fig. S6 Triple-detection GPC curves of S-PPEGMA10 prepared in 75% water-based solution with

the arm concentration of 10% w/v.

Fig. S7 Triple-detection GPC curves of S-PPEGMA10 prepared in 75% water-based solution with

the arm concentration of 15% w/v.

Arm polymerVw

(%)

Arm polymer

concentration (w/v)Time (min) Yield (%)a

Mn

(kg/mol)b

Ðc

L-PPEGMA10 30 5 60 0 0 ―d

L-PPEGMA10 40 5 60 0 0 ―

L-PPEGMA10 50 5 60 <50% ― ―

L-PPEGMA10 60 5 60 65% ― ―

L-PPEGMA10 70 5 60 94% 56.2 1.16

L-PPEGMA10 80 5 60 94% 56.2 1.16

L-PPEGMA10 90 5 60 98% 83.6 1.2

L-PPEGMA10 70 5 10 0 0 ―

L-PPEGMA10 70 5 20 70% 45.9 1.38

L-PPEGMA10 70 5 30 94% 56.2 1.16

L-PPEGMA10 70 5 40 94% 56.2 1.16

L-PPEGMA10 70 5 60 94% 56.2 1.16

L-PPEGMA10 70 10 60 94% 57.6 1.26

L-PPEGMA10 70 15 60 94% 57.8 1.26

aCCS yield, determined by GPC with RI detector, CCS yield = ACCS/(ACCS + Alow molecular weight

polymers), where A is the integral area. bNumber-average molecular weight, determined by GPC with RI

detector, calibrated with linear PS as standards. cPolydispersity as a value of Mw/Mn, determined by GPC with

RI detector. dNo value was determined.

Table S1 Summary of CCS polymers preparation using L-PPEGMA10 as the arm.

Fig. S8 GPC traces of S-PPEGMA21-D cationic CCS polymers prepared in water/ethanol mixtures with Vw = 90%.

Fig. S9 DLS results of S-PPEGMA-D CCS polymers.

Fig. S10 GPC curves of S-PDMAEMA59 prepared in acidic water-ethanol solutions with the Vw of 50% and 70%, respectively.

Fig. S11 Degradation behavior of S-PDMAEMA59 polymer in 10 mM DTT solutions characterized by GPC.

Fig. S12 DLS results of S-PDMA76 reaction solution (50 mg/mL, Vw = 80%) before and after degradation in DTT.

S-PPEGMA10, Reaction solution: Vw = 30%, Conversion of BAC = 4%

S-PPEGMA10, Reaction solution: Vw = 50%, Conversion of BAC = 54%

0 h

1 h

0 h

DMF

S-PPEGMA10, Reaction solution: Vw = 80%, Conversion of BAC = 100%

Fig. S13 1H NMR spectra in methanol-d4 of S-PPEGMA10 prepared in water-ethanol solutions with Vw = 30%, 50% and 80% before and after reaction for 1 h, and for Vw = 30% and 50%, the ethanol is ethanol-d6.

1 h

0 h

1 h