Four-armed poly(ethylene glycol) (PEG) with amine end groups (MW 10k; PEG4A) was purchased from SunBio PEG Shop (Orinda, CA). Diacrylated poly(ethylene glycol) (PEGDA) of molecular weight 3400 was purchased from Glycosan (Salt Lake City, UT). 3-Mercaptopropionic acid ethyl ester was purchased from TCI America (Portland, OR). 2-ClTrt chloride resin (1.55 mmol/g), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), and 1-hydroxybenzotriazole (HOBt) were purchased from Peptide International (Louisville, KY). Protected amino acids were purchased from NovaBiochem (La Jolla, CA). Acetonitrile and MeOH were from Burdick and Jackson. Trifluoroacetic acid (TFA) and ether were from J. T. Baker. Succinic anhydride, N,N-dimethylformide (DMF), N,N-diisopropylethylamine (DIEA), piperidine, 4-dimethylaminopyridine (DMAP), ninhydrin, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), ethyl 3-mercaptopropionate (EMP), and 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Calcein AM was purchased from Invitrogen (Chicago, IL).
Synthesis of Ethyl 3-Mercaptopropionate-Succinic Acid (EMP-SA)
EMP (2.95 g, 22 mmol) was added under argon to a stirring solution of succinic anhydride (2.0 g, 20 mmol) and DMAP (122 mg, 1 mmol) in 25 mL of acetonitrile–pyridine (9:1). The reaction mixture was stirred at room temperature overnight. The solution was concentrated under reduced pressure and dried in vacuo. The residue was dissolved in 50 mL of EtOAc. The EtOAc solution was washed with 0.1 N HCl aqueous solution 30 mL (×3) and H2O (×3) and dried over anhydrous MgSO4. After filtration, the solution was concentrated to dryness under reduced pressure and in vacuo (). 1H NMR (CDCl3, 500 MHz) δ 10.23 (s, br), 4.14 (2H, q, J = 7.0 Hz, -CH2-CH3), 3.13 (2H, t, J = 7.0 Hz, −S-CH2-), 2.88 (2H, t, J = 7.0 Hz, -S-CO-CH2-), 2.69 (2H, t, J = 7.0 Hz, -S-CH2-CH2-), 2.62 (2H, t, J = 7.0 Hz, -CH2-COOH), 1.25 (3H, t, J = 7.0 Hz, −CH2-CH3).
Synthesis of Thioester Macromonomer 1
The synthetic approach is shown in . A solution of EMPSA (0.234 g, 1 mmol) in DCM (2 mL) was added into a vial containing PEG4A (1 g, 0.4 mmol of amine) and BOP (0.221 g, 1 mmol), followed by addition of DIEA (0.348 mL, 2 mmol). The mixture was vortexed for 5 min and rocked for 2 h. The reaction was monitored by silica gel TLC (solvent system DCM–MeOH–HOAc = 100:3:1). The spots on the TLC plate were visualized by spray of 1% ninhydrin solution in ethanol containing 3% HOAc followed by heating at 105 °C. The purification of the product was performed by dilution with MeOH to a final volume of 50 mL. The solution was shaken thoroughly and frozen at −20 °C. The precipitate was collected by centrifugation (−9 °C, 6000 rpm, 20 min) and decanting the solvent. The purification cycle of dissolution in MeOH at room temperature, freezing at −20 °C, centrifugation at −9 °C, and decanting the MeOH was repeated four times, followed by precipitation with diethyl ether, and drying in vacuo. The ninhydrin test of the purified product gave a yellowish solution. 1H NMR (CDCl3, 500 MHz) δ 4.15 (2H, q, J = 7.0 Hz, -CH2-CH3), 3.79−3.41 (m, -O-CH2-CH2-O-), 3.12 (2H, t, J = 7.0 Hz, -S-CH2-), 2.92 (2H, t, J = 7.0 Hz, -S-CO-CH2-), 2.61 (2H, t, J = 7.0 Hz, -S-CH2-CH2-), 2.52 (2H, t, J = 7.0 Hz, -CH2-COOH), 1.26 (3H, t, J = 7.0 Hz, -CH2-CH3).
Protected Dipeptide Fragment Synthesis
Fully protected dipeptides Boc-Cys(Trt)-Gly-OH, Boc-Cys(Trt)-Glu(OtBu)-OH, Boc-Cys(Trt)-Asp(OtBu)-OH, Boc-Cys(Trt)-Trp(Boc)-OH, and Boc-Cys(Trt)-Arg(Pbf)-OH were synthesized manually as protected peptide fragments by Fmoc strategy on a 2-chlorotrityl chloride resin (Scheme S1
). A typical procedure of solid-phase synthesis is described below, performed with 2-chlorotrityl chloride resin (1.0 g, 1.55 mmol/g). The solution of Fmoc-amino acid-OH (1 mmol) dissolved in DCM (10 mL) and DIEA (1 mL) was added to a reaction vessel containing resin and rocked for 30 min, followed by washing with DMF three times. The resin was treated with DCM–MeOH–DIEA (8:1:1) for 20 min and washed with DMF three times. Fmoc was removed by treatment with 20% piperidine in DMF for 20 min and washed with DMF four times. The ninhydrin test of the resin gave a positive result. Boc-Cys(Trt)-OH (0.92 g, 2 mmol) and BOP (0.88 g, 2 mmol) were dissolved in DCM (8 mL), followed by addition of DIEA (522 μ
L, 3 mmol). After 10 min, the solution was added to the resin and rocked for 2 h, followed by washing with DMF and MeOH, three times each. The resin was dried in vacuo and the ninhydrin test of the resin gave a yellowish color. Then, protected peptide fragments were obtained by treatment of the resin with 1% TFA in dichloromethane (DCM), and the cleaved peptide sequences were confirmed by MALDI TOF-MS analysis. The analysis of the products by silica gel TLC (solvent system DCM–MeOH–HOAc = 100:3:1) gave a single spot on the plate.
Synthesis of N-Terminal Cysteine Macromonomers 2, 3a–e
The synthetic approach used for macromonomers 3a–e is shown in . The method used for macromonomer 2 was similar except for the use of Boc-Cys(Trt)-OH instead of dipeptide. The solution of Boc-Cys(Trt)-OH or protected dipeptide Boc-Cys(Trt)-AA-OH (0.25 mmol) in DCM (2 mL) was added into a vial containing PEG4A (0.5 g, 0.2 mmol of amino group) and BOP (0.11 g, 0.25 mmol), followed by addition of DIEA (44 μL, 0.25 mmol). The mixture was vortexed for 5 min, subsequently rocked for 2 h, and concentrated under a N2 flow. The residue was dissolved in 50 mL of MeOH and frozen at −20 °C. The precipitate was collected by centrifugation (−9 °C, 6000 rpm, 20 min) and decanting the solvent. The purification cycle of dissolution in MeOH at room temperature, freezing at −20 °C, centrifugation at −9 °C, and decanting the MeOH was repeated four times, followed by precipitation with diethyl ether, and drying in vacuo. The analysis of the products by silica gel TLC (solvent system DCM–MeOH–HOAc = 100:3:1) gave a single spot on the origin and no spot from Boc-Cys(Trt)-OH or protected dipeptides. The ninhydrin test of the purified product gave a yellowish solution. Proton NMR (CDCl3, 500 MHz) spectra of the protected cysteine-PEG4A conjugates confirmed their structures.
Synthesis of N-Terminal Cysteine Macromonomers 3a–e
Protected cysteine-PEG4A conjugates were then treated with 30 mL of TFA containing TIS (1 mL) and EDT (1 mL) at room temperature for 2 h and concentrated under reduced pressure, respectively. The residue was dissolved in 50 mL of MeOH and frozen at −20 °C. The precipitate was collected by centrifugation (−9 °C, 6000 rpm, 20 min) and decanting the solvent. The purification cycle of dissolution in MeOH at room temperature, freezing at −20 °C, centrifugation at −9 °C, and decanting the MeOH was repeated four times, followed by precipitation with diethyl ether, and drying in vacuo to generate the conjugates 2 and 3a–e TFA salt. Ninhydrin test gave a dark blue color, indicating that the Boc protection group was removed. Finally, the conjugates 2 and 3a–e TFA salt were dissolved in 0.1 M NH4HCO3 aqueous solution (25 mL), bubbled with argon for 20 min, frozen at −20 °C, and lyophilized to produce the salt-free conjugates 2 and 3a–e.
Gel formation was accomplished by mixing equimolar amounts of thioester macromonomer 1
in pure water (solution A) with one of the N
-terminal cysteine macromonomers 2
in buffer solution (buffer concentration, ×2; solution B) at 23 or 37 °C. N
-Terminal cysteine macromonomers were used in either TFA salt form (Table S1
) or salt-free form (Table S2
). The molar ratio of thioester macromonomer (1
) to N
-terminal cysteine macromonomer (2
) was generally 1:1 unless specifically noted, and the final polymer concentration ranged from 2–20% (w/v). No exogenous free thiol or reducing agent was added during the NCL reaction.
Screening of Gel Formation Time and Rheological Characterization
Rapid screening of gel formation time was performed by visual inspection as follows: to a stirring solution A (250 μ
L) in a test tube (100 × 13 mm) with a stirring bar (10 × 3 mm; 200 rpm) was added solution B (250 μ
L) and a stopwatch was started. Gel formation time was recorded when the stirring bar stopped rotating as a result of gel formation (Tables S1 and S2
). More detailed hydrogel characterization was performed using dynamic rheology in which gel formation occurred in situ. All oscillatory rheological experiments were performed with a Paar Physica MCR300 Rheometer with a Peltier temperature control device maintained at 20 °C using either a stainless steel cone/plate fixture (50 mm diameter, 1° cone) or a stainless steel parallel plate fixture (25 mm diameter, 1 mm gap). Two methods were used.
Method 1. A total of 500 μ
L each of solutions A and B were added to a vial. After vortexing, 590 μ
L of the mixture was immediately loaded onto the thermostatted rheometer plate, and a 50 mm/1° cone was positioned to confine the solution in a 0.05 mm gap at the center between the cone and the plate. After moisturized Kimwipes paper was applied to surround the cone/plate fixture for evaporation control, data were collected every 20 s over 140 min. The measurements of the storage modulus and loss modulus were taken in the oscillatory mode at 1 Hz frequency and 1% strain during cross-linking. At the conclusion of the gelation experiment, a frequency sweep experiment was performed from 0.01 to 10 Hz with 19 data points at 1% strain. Finally, a strain sweep experiment was performed with strain from 1 to 100% at 1 Hz frequency. Data obtained using this method are shown in and S1–S4
Oscillatory rheology of a mixture of 20% macromonomer 1 and 20% macromonomer 3b in 100 mM NH4HCO3, pH 8.3. Storage (G′) modulus vs time is shown (rheology method 1).
Method 2. Solutions A and B were mixed as described above, 500 μ
L of the mixture was immediately loaded onto the thermostatted rheometer plate (20 °C), and a parallel plate (25 mm in diameter) was positioned to confine the solution within a 1 mm gap. After moisturized Kimwipes paper was applied to surround the parallel plate fixture for evaporation control, data were collected every 20–30 s for up to 300 min. The measurements of the storage modulus and loss modulus were taken at 20 °C in the oscillatory mode at 1 Hz frequency and 1% strain during measurement. In selected experiments, frequency and strain sweeps were performed after the storage modulus reached a stable plateau value. Frequency sweeps were performed from 0.01 to 10 Hz at 1% strain. Strain sweeps were performed from 1 to 100% strain at 1 Hz frequency. Data obtained using this method are shown in , , and S5–S8
Oscillatory rheology of 10% macromonomer 2 in 100 mM NH4HCO3, pH 8.3. Storage (G′) and loss (G″) modulus vs time are shown (rheology method 2).
Oscillatory rheology of a mixture of 10% macromonomer 1 and 10% macromonomer 2 in 100 mM NH4HCO3, pH 8.3. Storage (G′) and loss (G″) modulus vs time are shown (rheology method 2).
Synthesis of Cell Adhesive Peptides
Peptides maleimide-GRGDSPG-NH2 and Ac-CGRGDSPG-NH2 were synthesized using standard solid phase peptide synthesis protocols on Rink amide resin (Anaspec, San Diego, CA) at 0.1 mmol scale. Each coupling step was carried out by mixing 3 equiv of Fmoc-protected amino acids, PyBop, and N-methyl morphiline with the resin beads for 4 h on a rocker. Upon completion of coupling indicated by Kaiser's test, the resin beads were washed thoroughly with DMF and then 20% piperidine in DMF was used to deprotect Fmoc group to expose the amine groups on the beads for the next coupling. After the last amino acid was conjugated, either maleimide-OSU ester (2 equiv) in DMF was used to attach the maleimide moieties, or acetyl anhydride (20 equiv) and triethylamine (5 equiv) in DMF was used to cap the N-terminal of resin-bound peptides. Cleavage of the peptides from the resin and deprotection of the amino acid side chains were accomplished by treating the resin with 95% (v/v) TFA, 2.5% H2O, and 2.5% TIS for 2 h at room temperature, after which the cleaved peptide solution was collected by filtration. Solvent was removed using a rotary evaporator; the product residues were dissolved in a minimal amount of TFA and precipitated with cold ether and by centrifugation at 4 °C. The product pellets were dissolved in deionized water, frozen, and lyophilized. Crude products were purified by preparative RP-HPLC and peptides were confirmed by MALDI-TOF MS.
Preparation of Peptide Functionalized NCL Hydrogel
Macromonomers 1 and 2 were dissolved at 10% (w/v) in H2O and 0.2 M NaHCO3 respectively. A total of 15 μL of each solution was pipetted into a 1 mL disposable syringe with the tip cut off. Gel was allowed to form for 15 min at 37 °C. Hydrogel disks (diameter = 5 mm, thickness = 1 mm) were removed from the syringe and placed into a well of a 96-well cell culture plate, washed with PBS, and then immersed in 100 μL of a solution of maleimide-GRGDSPG-NH2 in PBS (10 mM, pH 7.2) for 15 min. The gel disks were then washed thoroughly with PBS prior to seeding of hMSCs.
Human Mesenchymal Stem Cell Culture
Human mesenchymal stem cells (hMSC) were obtained from Lonza (Walkersville, MD) and used as received. In 75 cm2 tissue-culture treated flasks, 4 × 105 cells were seeded and cultured in hMSC basal media with mesenchymal cell growth supplements, l-glutamine, amphotericin-B, and gentamicin purchased from Lonza. Cells were split 1:5 approximately once a week and fed every 3 days until utilized, and cells that had been passaged twice were used for culture on hydrogel surfaces. Hydrogel disks formed by NCL were seeded with hMSCs at a density of 5000 cells/cm2 and cultured in hMSC growth media for 24 h. Cells were stained with Calcein AM (Invitrogen, Chicago, IL) and imaged by fluorescence microscopy to determine cell adhesion. Images are shown in grayscale and 80× original magnification. Cell adhesion on hydrogel surfaces was quantified using Metamorph Image Analysis software for eight fields per condition. Projected cell surface areas were measured in Metamorph to determine the percentage of hydrogel surface covered by cells.