Common use of Synthesis Clause in Contracts

Synthesis. (4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy) butanoic acid (AN) was prepared according to the procedure as reported.28,37 Briefly, 4-hydroxy-3-methoxyacetophenone was alkylated with ethyl 4-bromobutyate in DMF in the presence of K2CO3. Nitration was performed by treating with HNO3. The ketone was reduced with NaBH4 and followed by hydrolysis of the ethyl ester with 1M NaOH. Desired compound AN was obtained from the reaction with acryloyl chloride in the presence of triethyl amine. The product was purified by silica gel column chromatography. 1H NMR (400 MHz, CDCl3): δ 1.7 (3H, d, J = 6.4), 2.2 (2H, m), 2.6 (2H, t, J = 7.2), 3.9 (3H, s), 4.1 (2H, t, J = 6.4), 5.9 (1H, d, J = 1.2), 6.2 (1H, m), 6.4 (1H, d, J = 1.2), 6.5 (1H, q, J1 = 6.4, J2 = 6.4), 7.3 (1H, s), 7.6 (1H, s), 10.5 (1H, br); 13C NMR (100 MHz, CDCl3): δ 21.8, 23.8, 30.1, 56.1, 67.9, 68.4, 108.0, 108.8, 128.0, 131.3, 133.1, 139.5, 147.0, 153.9, 164.8, 178.8. Dex-AN was prepared by an esterification of the hydroxyl groups of dextran with AN using the procedure that we reported previously.4 0.5 g of AN was dissolved in 10 mL of DMSO, followed by the addition of DCC (0.5 g) and DPTS (0.07 g). To the mixture, dextran (0.7 g) solution in DMSO (10 mL) was added and stirred for 36 hours at room temperature. After filtration, the filtrate was put into a dialysis tube (MWCO 3500) and dialyzed against DMSO and water. After lyophilization, 0.4 g of the product was recovered. Dithiol functionalized poly(ethylene glycol) was prepared by following previously reported procedures.5,54 Macroscale hydrogels were obtained by mixing solutions of Dex-AN and DSPEG. Typical procedure was following: 150 µL Dex-AN (20 mg) and 50 µL DSPEG (12 mg) in phosphate buffered saline (PBS) solutions were mixed by vortexing (the ratio of acrylates to thiols was 1:1), and the resulted solution was incubated at 37 °C under dark. The gelation time was determined to be 60 min by the vial tilting method. (When there was no flow of the sample within 5 seconds, it was regarded as a gel.54) Sample for visco-elastic measurements was prepared by mixing two solutions of Dex-AN (30 mg in 150 µ L PBS) and DSPEG (18 mg in 150 µ L PBS). To probe viscoelastic properties of the gel, a small sample of the hydrogel was analyzed using TA Instruments AR-G2 rheometer with plate-plate geometry (plate of 40 mm in diameter and a 220 microns gap distance). Prior to the measurements, the strain-sweep tests were performed on the sample to determine the limits of the linear viscoelastic regime. Data acquisition started when steady state of the gel mechanical stress was reached, as indicated by normal forces. Frequency sweeps were done between 0.1 and 100 rad/s in the linear response regime at 37 °C. A handheld UV lamp (100 W, Black-Ray) at the wavelength of 365 nm was used for all the light irradiation with a fixed distance of 30 cm between the sample and the lamp. Scanning electron microscopy (SEM) was was conducted on a Nova NanoSEM (FEI) with an accelerating voltage of 10 kV and spot size of 3.5. Sample was coated with carbon before measurements. The FT-IR spectra were obtained using a ▇▇▇▇▇▇ ▇▇▇▇▇ Paragon 1000 FT-IR spectrometer. Samples for both FT-IR and SEM were prepared as follows: 150 µL Dex-AN (20 mg) and 50 µL DSPEG (12 mg) in water solutions were mixed by vortexing, and resulted solution was incubated at 37 °C under dark for 60 min and then kept under dark overnight at room temperature. The formed gel was divided into 2 portions, one was freeze dried immediately and the other was kept under UV irradiation for 72 hours before freeze drying. UV-vis spectra were recorded on a ▇▇▇▇ 3 Bio UV-vis spectrometer. The sample was prepared as follows: Dex-AN (1.2 mg) was dissolved in 600 µL PBS and then a solution of DSPEG (0.6 mg) in 600 µL PBS was added. The mixture was vortexed and incubated at 37 °C under dark for 60 min. The sample was then put under the UV irradiation. Dex-AN (5 mg) and DSPEG (3 mg) were dissolved into 50 µL PBS containing green fluorescent protein (GFP, 0.1 µg/µL) and vortexed, the resulted solution was incubated at 37 °C under dark overnight. The hydrogel (11 mg) was placed into a 1 cm quartz cuvette, centrifuged for 10 minutes to form a square at one of the bottom corners of the cuvette. After gentle washing with 1 mL PBS, 1 mL of fresh PBS was added. The released amount of GFP was monitored through the fluorescence from the solution part. Fluorescence measurements were performed using a luminescence spectrometer LS50B (▇▇▇▇▇▇ ▇▇▇▇▇). All spectra were obtained at room temperature. Each spectrum was measured with the excitation and emission slits of 5 nm. The excitation wavelength was 475 nm. The emission spectra were recorded every 10 mins while the cuvette was shaking at 50 rpm. After the experiment, the cuvette was vortexed vigorously to make a homogeneous solution and check the fluorescence intensity of 100 % release. An aliquot of the formed hydrogel was taken out and mounted to a poly(dimethylsiloxane) (PDMS) mold (created by using a metal master square pillars of 300 × 300 × 50 µm). The obtained hydrogel piece was then taken out from the mold to a Petri dish and 100 µL of PBS was added on top of the gel. The degradation process of the hydrogel under UV irradiation was monitored by a Leica MZ16FA stereo fluorescent microscope using bright field. Movies were recorded every 4 min with a pause of 1 min between each. The movie shown in the supporting information is in a combined form of three succeeding runs and 10 times faster than the real-time recording rate. 100 µL of GFP solution (0.1 µg/µL) was added to 50 µL Dex-AN (20 mg) PBS solution, and then 50 µL PBS solution of DSPEG (12 mg) were added and vortexed. After incubation at 37 °C under dark for 60 min, an aliquot of the formed hydrogel was taken out and mounted to the PDMS mold. The obtained hydrogel piece was then taken out from the mold to a Petri dish and 100 µL of PBS was added on top of the gel. The release profile was monitored with the Leica MZ16FA stereo fluorescent microscope by taking picture of the hydrogel every 3 min. Luminosity of the gel was determined as the mode value of the central part of the gel (200 × 200 pixels, whole gel piece was typically about 400 × 400 pixels) after subtracting the background value. In order to test whether, besides with 365 nm UV light, the hydrogel can also be degradated by two-photon excitation using NIR light, we use the following approach. A custom-build multifocal laser scanning two-photon microscope is used to illuminate a rectangular area of 25 × 25 µm in a hydrogel containing streptavidin-conjugated quantum dots (Qdot 565, Invitrogen). The wavelength of the laser was 780 nm. Since two-photon absorption depends on the square of the illumination intensity, only a thin slice of the hydrogel with thickness ~1 µm is expected to be degraded. A 3D scan though the sample showed that quantum dots were only moving a 1 µm thick slice (data not shown). Fluorescence images of the same area were acquired using a low power 532 nm solid state laser (Cobolt Samba) before and after the two-photon irradiation. To exclude the argument that local heating of the gel would cause the liberation of the quantum dots, we repeated the experiment with the laser set in CW mode. This way, the peak intensity is dramatically reduced to far below the two-photon absorption threshold, while the overall laser power remains the same.