Supporting Information
Supporting Information 3D Tissue Engineered Supramolecular Hydrogels for Controlled Chondrogenesis of Human Mesenchymal Stem Cells
Hyuntae Jung,† Ji Sun Park,⊥ Junseok Yeom,§ Narayanan Selvapalam,‡ Kyeng Min Park,‡ Kyunghoon Oh,‡ Jeong-A Yang,§ Keun Hong Park,⊥,* Sei Kwang Hahn,†,§,* and Kimoon Kim †, ‡,*
†
School of Interdisciplinary Bioscience and Bioengineering, §Department of Materials Science and
Engineering, ‡Center for Self-assembly and Complexity, Institute for Basic Science, Department of Chemistry, Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Korea ⊥
Department of Biomedical Science, College of Life Science, CHA University, Bundang-gu,
Seongnam, 463-840, Korea
Synthesis and characterization of monohydroxy CB[6]. A slurry of acidic CB[6] (10 g, 10.0 mmol) and K2SO4 (10.48 g, 60.0 mmol) were taken together into a 1 L round bottom flask. Water (700 mL) was added and sonicated until complete dissolution. To this, K2S2O8 (2.57 g, 4.56 mmol) was added to the reaction mixture, which was heated at 85oC under N2 atmosphere for 12 h and then cooled to room temperature. The obtained precipitate was removed by filtration. The filtrate was then concentrated to
1
dryness using a rotary evaporator. The resulting material was then dissolved in 200 mL of HCl by sonication, and insoluble salt was eliminated by centrifugation. To the filtrate, methanol (400 mL) was added and the precipitate was filtered and dried in vacuum. 1H NMR (500 MHz, D2O with NaCl) 5.72(d, 2 H), 5.64 (d, 8 H), 5.52 (s, 9 H), 5.45 (d, 4 H), 5.30 (s, 1 H), 4.50 (d, 2 H), 4.26 (d, 10 H). MALDI-TOF, m/z [M+ + Na] calcd for C36H36N24O13Na, 1035.3; found 1035.2, [M+ + K] calcd for C36H36N24O13K, 1051.3; found 1051.2. Synthesis and characterization of monoallyloxy CB[6]. Monohydroxy CB[6] (0.5 g, 0.5 mmol) was dissolved in anhydrous DMSO (200 mL). NaH (240 mg, 5.0 mmol) was separately taken in a 500 mL round bottom flask and dried in vacuum. Then, monohydroxy CB[6] in DMSO was added to the flask and stirred with heating at 50°C for 5 h. After cooling to room temperature, allyl bromide (0.6 g, 5.0 mmol) was added to the reaction mixture at 0°C and stirred at room temperature for 12 h. The reaction mixture was poured into 2 L beaker and mixed with ether (1.5 L) using a glass rod. The ether layer was decanted once it was settled down. This step was repeated one more time to get a semi-solid precipitate. To the rest of the semi-solid, acetonitrile (50 mL) was added and stirred with a glass rod to get a precipitate. The precipitate was collected by centrifugation, washed with acetonitrile twice, and finally dried in vacuum to get a pale yellow solid (400 mg). Pure monoallyloxy CB[6] was isolated using a neutral alumina column (22 cm L × 3 cm D) with elution of acetonitrile and 5% increment in water (400 mL in total) until products were eluted and the fractions were collected by gravity. The fractions were examined using MALDI-TOF to identify the product. Monoallyloxy CB[6] came out at ratios of 55 to 60% water and 45 to 40% acetonitrile. Monoallyloxy CB[6] was recovered from the particular fractions by the elimination of acetonitrile followed by lypholization. 1H NMR (500 MHz, D2O) 6.02 (m, 1 H), 5.82 (d, 2 H, J = 16.0 Hz), 5.75 (d, 8 H, J = 16.0 Hz), 5.60 (m, br, 11 H), 5.52 (d, 1
2
H), 5.42 (d, 1 H, J = 17.0 Hz), 5.34 (d, 1 H, J = 11.0 Hz), 4.48 (d, 2 H, J = 16.0 Hz), 4.32 (m, 10 H), 4.05 (s, 2 H). MALDI-TOF, m/z [M+ + Na] calcd for C39H40N24O13Na, 1073.3; found 1073.5, [M+ + K] calcd for C39H40N24O13K, 1073.3; found 1073.5. Synthesis and characterization of monoCB[6]-HA and DAH-HA. Mono-functionalized CB[6]-HA (monoCB[6]-HA) was synthesized by thiol-ene “click” reaction between monoallyloxy CB[6] and thiol-functionalized HA (HS-HA, MW = 100 kDa). The photoreactions were performed in a quartz tube by UV irradiation in a RMR-600 photochemical reactor (Rayonet, Branford, CT) equipped with four 254 nm lamps and four 300 nm lamps. HS-HA (300 mg) was dissolved in 60 mL of distilled water with tris(2-carboxyethyl)phosphine (TCEP, 0.5 molar ratio of HA repeating unit). After adding monoallyloxy CB[6] (0.5 molar ratio of HA repeating unit) to the solution, the mixed solution was sonicated until complete dissolution and photoreacted for 3 h. MonoCB[6]-HA conjugate was purified by dialysis against water for 4 days. DAH-HA (MW = 230 kDa), a counterpart to monoCB[6]-HA for the hydrogel formation, was synthesized and characterized as reported elsewhere.22 The modification degree of monoallyoxy CB[6] or DAH to HA was analyzed by 1H NMR (DRX-500, Bruker, Germany). SEM image analysis of monoCB[6]/DAH-HA hydrogel. The monoCB[6]/DAH-HA hydrogel was equilibrated in distilled water for 24 h at room temperature and then quickly frozen below its freezing point using liquid nitrogen as the cryogen. The frozen hydrogels were cryofractured to obtain crosssectional images of the hydrogels, showing the interior structure. The frozen hydrogels were fractured with a sharp scalpel. The sample was then transferred to a freeze dryer and freeze-dried until all the water was sublimated. The swollen freeze-dried sample was mounted onto an aluminum stud and sputter-coated with gold/palladium for 60 sec. The hydrogels were observed with a field emission scanning electron microscope (Phillips XL30S FEG) at 5.0 kV.
3
Modular modification of monoCB[6]/DAH-HA hydrogel with FITC-CB[6]. A solution of FITCCB[6] (25 µL, 60 µM) was added to a monoCB[6]/DAH-HA hydrogel (600 µL, 2.0 wt%), which was kept in a humid chamber at room temperature for 2 h. The hydrogel was then immersed in PBS (20 mL) and the PBS was exchanged every 8 h for a day to remove any unbound FITC-CB[6]. A part of hydrogel, a few hundred µm in diameter, was extracted to confirm the successful modification of monoCB[6]/DAH-HA hydrogel with FITC-CB[6] under a fluorescence microscope with I3 filters (excitation 450 ~ 490 nm and emission > 515 nm) for FITC. As a control, the same experiment was performed with carboxyfluorescein (CF). The relative fluorescence intensity (%) of the FITCCB[6]@monoCB[6]/DAH-HA hydrogel was monitored for 256 h (n = 3). Synthesis and characterization of dexamethasone-21-hemiesters. Dexamethasone (2.5 mmol), excess succinic anhydride (42 mmol), and 4-dimethylaminopyridine (2.6 mmol) were dissolved in 80 mL of anhydrous acetone and allowed to react for 24 h at room temperature. After removal of the solvent by evaporation under reduced pressure, the crude product was recrystallized from ethanol:water (3:7). 1H NMR (500 MHz, DMSO) 5.42 (s, 1 H), 5.23 (d, 1 H), 5.14 (d, 1 H), 4.76 (d, 1 H), 4.28 (s, 1 H), 2.84 (s, 1 H), 2.25-2.62 (m, 10 H, J = 16.0 Hz), 1.62-1.74 (m, 3 H, J = 16.0 Hz), 1.50 (s, 3 H), 1.38 (s, 1 H), 1.18 (s, 1 H), 0.86 (s, 3 H), 0.72 (s, 3 H). MALDI-TOF, m/z [M+ + H] calcd for C26H34FO8, 493.2; found 493.1, [M+ + Na] calcd for C26H33FO8Na, 516.2; found 516.1. Synthesis and characterization of dexamethasone-CB[6] conjugate. Dexamethasone-21-hemiesters (1.0 mmol), dicyclohexylcarbodiimide (DCC, 1.5 mmol), and N-hydroxy-succinimide (NHS, 1.5 mmol) were dissolved in 1 mL of anhydrous DMSO and stirred until forming urea. After filtration to remove the urea, amineCB[6] (0.5 mmol) was added and allowed to react for 2 days. The Dexa-CB[6] was obtained by freeze-drying after purification by sequential dialysis against DMSO and water using a
4
dialysis membrane (MWCO 1000) for 1 day. 1H NMR (500 MHz, D2O) 6.42 (d, 1 H), 6.16 (s, 1 H), 5.60 (m, br, 11 H), 3.68-4.46 (m, br, 24 H), 3.16 (m, br, 22 H), 2.75 (m, br, 44 H), 2.42 (m, br, 22 H), 1.65-2.06 (m, br, 22 H). MALDI-TOF, m/z [M+ + H] calcd for C122H200FN36O31S12 3067.2; found 3067.4, [M+ – m + H] (m = C5H12NOS) C117H188FN35O30S11, 2933.1; found 2933.4, [M+ - 2m + H] calcd for C112H176FN34O29S10, 2799.1; found 2799.3, [M+ - 3m + H] calcd for C107H164FN33O28S9, 2665.0; found 2665.2, [M+ - 4m + H] calcd for C102H152FN32O27S8, 2530.9; found 2530.2.
5
(a)
(b)
6
5
4
3
2
1
Figure S1. (a) Synthetic scheme and (b) 1H NMR spectra of monoCB[6]-HA conjugate.
6
(a)
(b)
Figure S2. Characterization of monoCB[6]/DAH-HA hydrogels. (a) Phase transition from sol to gel upon mixing (1) monoCB[6]-HA with (2) DAH-HA to form (3) monoCB[6]/DAH-HA hydrogel. (b) SEM image of monoCB[6]/DAH-HA hydrogel coated with Pt (scale bar = 20 µm). (a) Day 3
Day 10
Day 7
(b)
Relative cell number (%)
300 250 200 150 100 50 0
Day 3
Day 7
Day 10
Figure S3. (a)The viability of hMSCs encapsulated in monoCB[6]/DAH-HA hydrogel. The hMSCs were stained with calcein AM for live cells and imaged under a fluorescence microscope (scale bar = 20 µm). (b) The quantified data for cell numbers in monoCB[6]/DAH-HA hydrogel (n = 3).
7
(a)
(b)
Figure S4. Modular modification of monoCB[6]/DAH-HA hydrogels with FITC-CB[6] by highly selective host-guest interaction. (a) Fluorescence images of monoCB[6]/DAH-HA hydrogels treated with FITC-CB[6] (top) or carboxyfluorescein (CF, bottom). (b) The relative fluorescence intensity (%) of FITC-CB[6]@monoCB[6]/DAH-HA hydrogel and CF@monoCB[6]/DAH-HA hydrogel in PBS with increasing time (mean ± SD, n = 3).
Figure S5. Synthetic scheme of Dexa-CB[6] conjugate.
8
+ TGF-β3 Control
+ Dexa
+ Dexa-CB[6]
Figure S6. In vivo formation of monoCB[6]/DAH-HA hydrogels encapsulating hMSCs. Equal volumes of monoCB[6]-HA mixed with hMSCs (2.0 wt%, 5 × 106 cells in 0.2 mL) and DAH-HA (2.0 wt%, 0.2 mL) in PBS were subcutaneously injected to the back of six-week old Balb/c nude mice (left), with free dexamethasone (middle) or Dexa-CB[6] (right). After 4 weeks post-injection, the mice were sacrificed to obtain photographs of the skin flaps showing the appearance of the tissue surrounding the treated site.
9
Hyuntae Jung,† Ji Sun Park,⊥ Junseok Yeom,§ Narayanan Selvapalam,‡ Kyeng Min Park,‡ Kyunghoon Oh,‡ Jeong-A Yang,§ Keun Hong Park,⊥,* Sei Kwang Hahn,†,§,* and Kimoon Kim †, ‡,*
†
School of Interdisciplinary Bioscience and Bioengineering, §Department of Materials Science and
Engineering, ‡Center for Self-assembly and Complexity, Institute for Basic Science, Department of Chemistry, Division of Advanced Materials Science, Pohang University of Science and Technology (POSTECH), San 31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Korea ⊥
Department of Biomedical Science, College of Life Science, CHA University, Bundang-gu,
Seongnam, 463-840, Korea
Synthesis and characterization of monohydroxy CB[6]. A slurry of acidic CB[6] (10 g, 10.0 mmol) and K2SO4 (10.48 g, 60.0 mmol) were taken together into a 1 L round bottom flask. Water (700 mL) was added and sonicated until complete dissolution. To this, K2S2O8 (2.57 g, 4.56 mmol) was added to the reaction mixture, which was heated at 85oC under N2 atmosphere for 12 h and then cooled to room temperature. The obtained precipitate was removed by filtration. The filtrate was then concentrated to
1
dryness using a rotary evaporator. The resulting material was then dissolved in 200 mL of HCl by sonication, and insoluble salt was eliminated by centrifugation. To the filtrate, methanol (400 mL) was added and the precipitate was filtered and dried in vacuum. 1H NMR (500 MHz, D2O with NaCl) 5.72(d, 2 H), 5.64 (d, 8 H), 5.52 (s, 9 H), 5.45 (d, 4 H), 5.30 (s, 1 H), 4.50 (d, 2 H), 4.26 (d, 10 H). MALDI-TOF, m/z [M+ + Na] calcd for C36H36N24O13Na, 1035.3; found 1035.2, [M+ + K] calcd for C36H36N24O13K, 1051.3; found 1051.2. Synthesis and characterization of monoallyloxy CB[6]. Monohydroxy CB[6] (0.5 g, 0.5 mmol) was dissolved in anhydrous DMSO (200 mL). NaH (240 mg, 5.0 mmol) was separately taken in a 500 mL round bottom flask and dried in vacuum. Then, monohydroxy CB[6] in DMSO was added to the flask and stirred with heating at 50°C for 5 h. After cooling to room temperature, allyl bromide (0.6 g, 5.0 mmol) was added to the reaction mixture at 0°C and stirred at room temperature for 12 h. The reaction mixture was poured into 2 L beaker and mixed with ether (1.5 L) using a glass rod. The ether layer was decanted once it was settled down. This step was repeated one more time to get a semi-solid precipitate. To the rest of the semi-solid, acetonitrile (50 mL) was added and stirred with a glass rod to get a precipitate. The precipitate was collected by centrifugation, washed with acetonitrile twice, and finally dried in vacuum to get a pale yellow solid (400 mg). Pure monoallyloxy CB[6] was isolated using a neutral alumina column (22 cm L × 3 cm D) with elution of acetonitrile and 5% increment in water (400 mL in total) until products were eluted and the fractions were collected by gravity. The fractions were examined using MALDI-TOF to identify the product. Monoallyloxy CB[6] came out at ratios of 55 to 60% water and 45 to 40% acetonitrile. Monoallyloxy CB[6] was recovered from the particular fractions by the elimination of acetonitrile followed by lypholization. 1H NMR (500 MHz, D2O) 6.02 (m, 1 H), 5.82 (d, 2 H, J = 16.0 Hz), 5.75 (d, 8 H, J = 16.0 Hz), 5.60 (m, br, 11 H), 5.52 (d, 1
2
H), 5.42 (d, 1 H, J = 17.0 Hz), 5.34 (d, 1 H, J = 11.0 Hz), 4.48 (d, 2 H, J = 16.0 Hz), 4.32 (m, 10 H), 4.05 (s, 2 H). MALDI-TOF, m/z [M+ + Na] calcd for C39H40N24O13Na, 1073.3; found 1073.5, [M+ + K] calcd for C39H40N24O13K, 1073.3; found 1073.5. Synthesis and characterization of monoCB[6]-HA and DAH-HA. Mono-functionalized CB[6]-HA (monoCB[6]-HA) was synthesized by thiol-ene “click” reaction between monoallyloxy CB[6] and thiol-functionalized HA (HS-HA, MW = 100 kDa). The photoreactions were performed in a quartz tube by UV irradiation in a RMR-600 photochemical reactor (Rayonet, Branford, CT) equipped with four 254 nm lamps and four 300 nm lamps. HS-HA (300 mg) was dissolved in 60 mL of distilled water with tris(2-carboxyethyl)phosphine (TCEP, 0.5 molar ratio of HA repeating unit). After adding monoallyloxy CB[6] (0.5 molar ratio of HA repeating unit) to the solution, the mixed solution was sonicated until complete dissolution and photoreacted for 3 h. MonoCB[6]-HA conjugate was purified by dialysis against water for 4 days. DAH-HA (MW = 230 kDa), a counterpart to monoCB[6]-HA for the hydrogel formation, was synthesized and characterized as reported elsewhere.22 The modification degree of monoallyoxy CB[6] or DAH to HA was analyzed by 1H NMR (DRX-500, Bruker, Germany). SEM image analysis of monoCB[6]/DAH-HA hydrogel. The monoCB[6]/DAH-HA hydrogel was equilibrated in distilled water for 24 h at room temperature and then quickly frozen below its freezing point using liquid nitrogen as the cryogen. The frozen hydrogels were cryofractured to obtain crosssectional images of the hydrogels, showing the interior structure. The frozen hydrogels were fractured with a sharp scalpel. The sample was then transferred to a freeze dryer and freeze-dried until all the water was sublimated. The swollen freeze-dried sample was mounted onto an aluminum stud and sputter-coated with gold/palladium for 60 sec. The hydrogels were observed with a field emission scanning electron microscope (Phillips XL30S FEG) at 5.0 kV.
3
Modular modification of monoCB[6]/DAH-HA hydrogel with FITC-CB[6]. A solution of FITCCB[6] (25 µL, 60 µM) was added to a monoCB[6]/DAH-HA hydrogel (600 µL, 2.0 wt%), which was kept in a humid chamber at room temperature for 2 h. The hydrogel was then immersed in PBS (20 mL) and the PBS was exchanged every 8 h for a day to remove any unbound FITC-CB[6]. A part of hydrogel, a few hundred µm in diameter, was extracted to confirm the successful modification of monoCB[6]/DAH-HA hydrogel with FITC-CB[6] under a fluorescence microscope with I3 filters (excitation 450 ~ 490 nm and emission > 515 nm) for FITC. As a control, the same experiment was performed with carboxyfluorescein (CF). The relative fluorescence intensity (%) of the FITCCB[6]@monoCB[6]/DAH-HA hydrogel was monitored for 256 h (n = 3). Synthesis and characterization of dexamethasone-21-hemiesters. Dexamethasone (2.5 mmol), excess succinic anhydride (42 mmol), and 4-dimethylaminopyridine (2.6 mmol) were dissolved in 80 mL of anhydrous acetone and allowed to react for 24 h at room temperature. After removal of the solvent by evaporation under reduced pressure, the crude product was recrystallized from ethanol:water (3:7). 1H NMR (500 MHz, DMSO) 5.42 (s, 1 H), 5.23 (d, 1 H), 5.14 (d, 1 H), 4.76 (d, 1 H), 4.28 (s, 1 H), 2.84 (s, 1 H), 2.25-2.62 (m, 10 H, J = 16.0 Hz), 1.62-1.74 (m, 3 H, J = 16.0 Hz), 1.50 (s, 3 H), 1.38 (s, 1 H), 1.18 (s, 1 H), 0.86 (s, 3 H), 0.72 (s, 3 H). MALDI-TOF, m/z [M+ + H] calcd for C26H34FO8, 493.2; found 493.1, [M+ + Na] calcd for C26H33FO8Na, 516.2; found 516.1. Synthesis and characterization of dexamethasone-CB[6] conjugate. Dexamethasone-21-hemiesters (1.0 mmol), dicyclohexylcarbodiimide (DCC, 1.5 mmol), and N-hydroxy-succinimide (NHS, 1.5 mmol) were dissolved in 1 mL of anhydrous DMSO and stirred until forming urea. After filtration to remove the urea, amineCB[6] (0.5 mmol) was added and allowed to react for 2 days. The Dexa-CB[6] was obtained by freeze-drying after purification by sequential dialysis against DMSO and water using a
4
dialysis membrane (MWCO 1000) for 1 day. 1H NMR (500 MHz, D2O) 6.42 (d, 1 H), 6.16 (s, 1 H), 5.60 (m, br, 11 H), 3.68-4.46 (m, br, 24 H), 3.16 (m, br, 22 H), 2.75 (m, br, 44 H), 2.42 (m, br, 22 H), 1.65-2.06 (m, br, 22 H). MALDI-TOF, m/z [M+ + H] calcd for C122H200FN36O31S12 3067.2; found 3067.4, [M+ – m + H] (m = C5H12NOS) C117H188FN35O30S11, 2933.1; found 2933.4, [M+ - 2m + H] calcd for C112H176FN34O29S10, 2799.1; found 2799.3, [M+ - 3m + H] calcd for C107H164FN33O28S9, 2665.0; found 2665.2, [M+ - 4m + H] calcd for C102H152FN32O27S8, 2530.9; found 2530.2.
5
(a)
(b)
6
5
4
3
2
1
Figure S1. (a) Synthetic scheme and (b) 1H NMR spectra of monoCB[6]-HA conjugate.
6
(a)
(b)
Figure S2. Characterization of monoCB[6]/DAH-HA hydrogels. (a) Phase transition from sol to gel upon mixing (1) monoCB[6]-HA with (2) DAH-HA to form (3) monoCB[6]/DAH-HA hydrogel. (b) SEM image of monoCB[6]/DAH-HA hydrogel coated with Pt (scale bar = 20 µm). (a) Day 3
Day 10
Day 7
(b)
Relative cell number (%)
300 250 200 150 100 50 0
Day 3
Day 7
Day 10
Figure S3. (a)The viability of hMSCs encapsulated in monoCB[6]/DAH-HA hydrogel. The hMSCs were stained with calcein AM for live cells and imaged under a fluorescence microscope (scale bar = 20 µm). (b) The quantified data for cell numbers in monoCB[6]/DAH-HA hydrogel (n = 3).
7
(a)
(b)
Figure S4. Modular modification of monoCB[6]/DAH-HA hydrogels with FITC-CB[6] by highly selective host-guest interaction. (a) Fluorescence images of monoCB[6]/DAH-HA hydrogels treated with FITC-CB[6] (top) or carboxyfluorescein (CF, bottom). (b) The relative fluorescence intensity (%) of FITC-CB[6]@monoCB[6]/DAH-HA hydrogel and CF@monoCB[6]/DAH-HA hydrogel in PBS with increasing time (mean ± SD, n = 3).
Figure S5. Synthetic scheme of Dexa-CB[6] conjugate.
8
+ TGF-β3 Control
+ Dexa
+ Dexa-CB[6]
Figure S6. In vivo formation of monoCB[6]/DAH-HA hydrogels encapsulating hMSCs. Equal volumes of monoCB[6]-HA mixed with hMSCs (2.0 wt%, 5 × 106 cells in 0.2 mL) and DAH-HA (2.0 wt%, 0.2 mL) in PBS were subcutaneously injected to the back of six-week old Balb/c nude mice (left), with free dexamethasone (middle) or Dexa-CB[6] (right). After 4 weeks post-injection, the mice were sacrificed to obtain photographs of the skin flaps showing the appearance of the tissue surrounding the treated site.
9
