Supporting Information

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1 Supporting Information Wavelength Selective Coupling and Decoupling of Polymer Chains via Reversible [2+2] Photocycloaddition of Styrylpyrene for Construction of Cytocompatible Photodynamic Hydrogels Vinh X. Truong*, Fanyi Li, Francesca Ercole, and John S. Forsythe*. Department of Materials Science and Engineering, Monash Institute of Medical Engineering, Monash University, Clayton, 3800 VIC, Australia CSIRO Manufacturing, Bayview Avenue, Clayton, VIC 3168, Australia. ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia. *corresponding authors: 1

2 1. General Considerations Materials. Solvents (CH2Cl2, diethyl ether, petroleum ether, acetone, tetrahydrofurane, methanol) were purchased from VWR in HPLC grade. 4-arm PEG20k-OH was purchased from Jen All other chemicals were purchased from Sigma-Aldrich and used as received. All synthesized compounds were stored in a fridge at 2-4 C and covered with aluminum foil. Glassware for synthesis was first treated in a base bath (KOH in isopropanol 2 M) overnight, rinsed with water, submerged in an acid bath (HCl 1 M) for 2-4 h, rinsed with reverse osmosis water and dried in an oven at 120 C for 6-12 h. Chromatography. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 alumina sheets (Merck) and visualized by UV light or potassium permanganate solution. Column chromatography was run on silica gel 60 ( mm, mesh ASTM, Merck). NMR spectra were recorded on a Bruker Avance III 400 MHz or 600 MHz with a 5 mm broadband auto-tunable probe with Z-gradients at 293 K. Chemical shifts are reported as δ in parts per million (ppm) and referenced to the chemical shift of the residual solvent resonances (CDCl3 δ = 7.26 ppm), couplings are shown as d: doublet, t: triplet, m: multiplet and bs: broad singlet. Polymer samples were prepared at a concentration of 20 mg ml -1. In most spectra traces of water appears as a broad singlet at around ppm. NMR spectra were processed using MestReNova software. Size exclusion chromatography (SEC) was performed using a Shimadzu modular system comprised of a SIL-20AD automatic injector, an RID-10A differential refractive-index detector and a mm guard column followed by three KF-805L columns (300 8 mm, bead size: 10 μm, pore size maximum: 5000 Å). N,N-Dimethylacetamide (DMAc, HPLC grade, 0.03% w/v LiBr) at 50 C was used for the analysis with a flow rate of 1 ml min -1. Reaction time course 2

3 samples dissolved in DMAc were filtered through 0.45 μm PTFE filters before injection. The GPC calibration was performed with narrow-polydispersity polystyrene standards ranging from 500 to g mol -1. Fluorescence spectra were obtained using a Shimadzu RF-5301 PC fluorescence spectrophotometer. 2.5 mg of (reaction time course) sample was diluted in 3 ml of 1:2 acetonitrile/h2o and then further diluted 1 in 10 (200 µl added to 1.8 ml of H2O). Fluorescence spectra were run using excitation wavelength 350 nm and emission scanned between 400 and 800 nm. 2. Synthesis Procedures 2.1 Synthesis of 1-bromopyrene (S1) Pyrene (4 g, 20 mmol) and HBr (3.5 ml, 65 mmol) were dissolved in a solution of MeOH/Et2O (20 ml/20 ml) and solution was cooled on an ice bath. H2O2 solution (2 ml) was added over 15 min, after which the solution was stirred at ambient temperature for 16 h. The solution was diluted with water (20 ml) and extracted with EtOAc (50 ml x 3). The combined organic phases were washed with NaOH 1 M solution (50 ml), water (50 ml), brine (ml), treated with MgSO4 and concentrated in vacuo to give product as yellow solid (yield: 5.43 g, 97%). 1 H NMR spectral data are in agreement with reported literature. 1 3

4 Figure S1. 1 H NMR spectrum of compound S1 (600 MHz, CDCl3; *: EtOAC). 2.2 Synthesis of (E)-4-(2-(pyren-1-yl)vinyl)benzoic acid (S2) 1-bromopyrene (2.81 g, 10 mmol), 4-vinyl benzoic acid (1.48 g, 10 mmol), Pd(OAc)2 (22.4 mg, 0.1 mmol), triphenylphosphine (52.5 mg, 0.2 mmol) and K2CO3 (1.38 g, 10 mmol) were added to a solution of DMF/NEt3 (40 ml, v/v = 3/1) and the mixture was stirred at 100 C for 16 h. The solution was allowed to cool to ambient temperature and diluted with saturated NH4Cl solution (200 ml). The solid was filtered and purified by column chromatography on silica gel 4

5 and running with CH2Cl2/MeOH (v/v = 1:0.1) to give product as yellow solid (yield: 2.01 g, 59%). This product contains some dimer due to light exposure during handling, and the percentage of the dimer is ca. 2% by integration of the chemical shifts in NMR spectrum. The dimer product was not observed in the NHS-ester product S3. 1 H NMR (δ, ppm): (bs), δ=8.81 (d, J=9.5 Hz), 8.55 (dd, J=13, 5.0 Hz), 8.32 (m, 4H), 8.19 (s, 2H), 8.09 (dd, J=8.0, 8.0 Hz), 8.00 (s, 4H), 7.65 ppm (d, J=16.5 Hz). HRMS: Cald for C25H16O2 (M + ): , found: Figure S2. 1 H NMR spectrum of compound S2 (400 MHz, d6-dmso; *: dimer product). 2.3 Synthesis of 2,5-dioxopyrrolidin-1-yl (E)-4-(2-(pyren-1-yl)vinyl)benzoate (S3) 5

6 S2 (696 mg, 2 mmol) was suspended in CH2Cl2 (10 ml), EDC.HCl (433 mg, 2.2 mmol) and NHS (248 mg, 2.2 mmol) were added. The solution became clear after 5 min of stirring at ambient temperature. The reaction was monitored by TLC until complete disappearance of the acid starting material (ca. 1 h). The solution was then washed with water (20 ml), brine (20 ml), dried (MgSO4), concentrated in vacuo and purified by column chromatography eluting with CH2Cl2/MeOH (v/v = 1/0.05) to give product as yellow solid (yield: 810 mg, 91%). 1 H NMR (δ, ppm): 8.87 (d, J=9.5 Hz), 8.72 (dd, J=13, 5.0 Hz), 8.38 (m, 4H), 8.16 (s), 8.09 (dd, J=8.0, 8.0 Hz), 8.12 (s), 7.76 (d, J=16.5 Hz), 2.94 (s). HRMS: Cald for C25H16O2: , found:

7 Figure S3. 1 H NMR spectrum of compound S3 (400 MHz, d6-dmso; *: CH2Cl2, #: methanol). 2.3 Synthesis of MeO-PEG-NH2 and 4-arm PEG-NH2 Methoxy poly(ethylene glycol) (4 g, 2 mmol) and NEt3 (0.6 g, 6 mmol) was dissolved in CH2Cl2 (20 ml) and the solution was cooled on an ice bath. Methanesulfonyl chloride (0.458 g, 4 mmol) was added dropwise over 30 min. The solution was stirred for 4 h and precipitated in diethyl ether (100 ml) to give product as white powder. The above product was dissolved in DMF (10 ml) and NaN3 (0.65 g, 10 mmol) was added. The solution was stirred at 70 C for 16 h and DMF was concentrated in vacuo. The solid was dissolved in CH2Cl2 (10 ml) and filtered. The filtrated was washed with water (20 ml), brine (20 ml), dried (MgSO4) and precipitated in diethyl ether to give product as white powder. The above product was dissolved in MeOH (20 ml) and triphenyl phosphine (2.1 g, 8 mmol) was added. The solution was heated at 80 C under refluxing conditions for 12 h. MeOH was concentrated in vacuo and water (20 ml) was added to the mixture. The solution was washed with Et2O (20 ml x 3) and extracted with CH2Cl2 (20 ml x 3). The combined CH2Cl2 phases were dried (MgSO4), concentrated in vacuo to ca. 5 ml and precipitated in Et2O solution (100 ml) to give product as white powder (total yield: 3.2 g, 80%). 1 H NMR (δ, ppm): 3.65 (bs), 3.47 (s), (2.88 t, J=5.2 Hz). 7

8 Figure S4. 1 H NMR spectrum of MeO-PEG-NH2 (400 MHz, CDCl3). 4-arm PEG-NH2 was prepared using similar synthetic procedure starting with 4-arm PEH-OH having molar mass of 20,000 g mol -1. 8

9 Figure S5. 1 H NMR spectrum of 4-arm PEG-NH2 (400 MHz, CDCl3). 2.4 Synthesis of MeO-PEG-styrylpyrene (polymer 1) MeO-PEG-NH2 (1 g, 0.5 mmol) and S3 (396 mg, 0.5 mmol) were dissolved in CH2Cl2 (5 ml) followed by addition of N,N-diisopropylethylamine (10 µl). The solution was stirred for 6 h at ambient temperature and precipitated twice in diethyl ether to give product as yellow solid (yield: 1.16 g, 86%). 1 H NMR (δ, ppm): 8.51 (d, J=9.3 Hz), 8.35 (d, J=8.4). 9

10 Figure S6. 1 H NMR spectrum of compound 1 (*: diethyl ether; 400 MHz, CDCl3). 4-arm PEG-styrylpyrene (polymer 3) was synthesized following similar procedure to give product as yellow solid (yield: 84%). 10

11 Figure S7. 1 H NMR spectrum of compound 3 (400 MHz, CDCl3). Normalised RI Response arm PEG-NH2 Polymer x10 4 4x10 4 6x10 4 8x Molecular Weight (g mol -1 ) Figure S8. SEC traces of 4-arm PEG-NH2 and styrylpyrene conjugated product (3). 11

12 3. Characterization Methods and Additional Data Light irradiation is undertaken using either a Mightex's WheeLED wavelength-switchable LED for λ = 365 nm/420 nm/445 nm (built in filter), or Omnicure S2000 curing lamp for λ = 340 nm using a hard coated OD 4 10nm bandpass filter from Edmund Optics, and λ = nm using filter provided with the Omnicure S2000 lamp. The intensity of light irradiance on the gel sample was tuned to the desired intensity using either a Cole Parmer Series 9811 UV light Radiometer (for 340 nm/365 nm wavelength) or a RM-12 radiometer (Opsytec) with a sensor VISBG nm (for nm/420 nm/445 nm wavelength). NMR studies: A solution (0.5 ml) of MeO-PEG-styrylpyrene in water at the concentration of c = 1 M was placed in a push cap vial (12x50 mm) equipped with a stirrer bar. Irradiation was induced using the Omnicure S2000 lamp with the light guide placing on top of the vial. The light intensity was adjusted to 20 mw cm -2 and the solution was stirred at the rate of 200 rpm. At predetermined time period, the irradiation and stirring were stopped and the solution was freeze-dried, dissolved in CDCl3 and subjected to NMR measurement. UV-vis spectra were recorded on a Cary60 spectrometer. A quartz cuvette with a transparent window above 220 nm was used and the recorded absorbance values were corrected for background and solvent absorbance. A solution (2.5 ml) of MeO-PEG-styrylpyrene at the concentration of c = 0.05 M was placed in a push cap vial (12x50 mm) equipped with a stirrer bar. Irradiation was induced using the Omnicure S2000 lamp with the light guide placing on top of the vial. The light intensity was adjusted to 20 mw cm -2 and the solution was stirred at the rate of 200 rpm. At predetermined time period, the irradiation and stirring were stopped and the solution was subjected to UV-Vis scan. 12

13 Figure S9. UV-Vis spectra of dimer product (2) under irradiation of light at λ = 340 nm, each spectrum was collected after 10 s interval. Arrows showing the directional increase/decrease of the absorption bands. Figure S10 a) Images of polymer solution (1), before and after being treated with irradiation at different wavelengths, under excitation of long wavelength UV lamp; b) fluorescent intensity of polymer solution (1) after being treated with visible light (λ = nm, I = 20 mw cm -2 ) at predetermined time period. 13

14 Figure S11. 1 H NMR spectra of polymer 2 under UV light irradiation at different time periods. Figure S12 SEC traces of polymer 2 under irradiation with UV light (340 nm) at different time periods. 14

15 Rheological experiments were carried out on an Anton Paar Physica rheometer with a plate-plate configuration. The lower plate is made of quartz and the upper plate is made of stainless steel with a diameter of 15 mm. A liquid light guild, which was connected to the light source was equipped below the quartz plate. In a typical experiment, a polymer solution (50 µl) was placed on the lower plate and the upper plate was lowered to a measurement gap of 0.2 mm. A layer of paraffin oil was applied on the edge of the stainless steel plate to prevent dehydration of hydrogel and the test was started by applying a 1% strain with the frequency of 0.1 Hz on the sample. In all rheological tests, an induction period of s was observed after light illumination and before a sharp increase in the storage modulus Figure S13. Rheology data of polymer solutions (6 wt%) under irradiation at λ = nm and different intensities: a) 5 mw cm -2 ; b) 10 mw cm -2 ; c) 15 mw cm -2 ; d) 20 mw cm -2. Red dashed lines indicate when the light was turned on. 15

16 16

17 Figure S14. Rheological data of polymer solution (6 wt%) under irradiation of light at a) 365 nm; b) 420 nm; and c) 445 nm. The intensity at all irradiation conditions was 20 mw cm -2 and the light was turned on immediately after the rheological measurements started. 17

18 Figure S15. Degradation profiles of hydrogels at different polymer concentrations under irradiation of UV light (340 nm, and I = 20 mw cm -2 ). Red dashed lines indicate when the light was turned on. Figure S16. Wavelength-gated reversible crosslinking of polymer solution at c = 3 mm. 18

19 4. Cell Culture hmscs (Lonza) were maintained in the low glucose DMEM with 10% fetal bovine serum (FBS) and 100 U ml -1 penicillin/streptomycin at 37 C with 5% CO2. In this study, cells were trypsinized and harvested at passage 6. The cells were then spun down and resuspended at a concentration of cells L -1. The styrlpyrene stock solution was freshly prepared in sterile DPBS at the concentration of 6 wt%. The stock solution and corresponding cell suspension were mixed well to achieve the final 5 wt% gel with cell encapsulation density of 2.5 million cells ml -1. Triplicate samples were loaded into a petri dish and subsequently cured under the visible light (λ = nm, I = 20 mw cm -2 ) for 10 min. The formed hydrogels have the thickness of approximately 2-3 mm. The samples were then washed twice with culture media and incubated at 37 C with 5% CO2. For UV light irradiation, the cell-laden hydrogels were cultured for 1 day and subjected to UV light irradiation (λ = 340 nm) at the intensity of 10 mw cm -2 for 10 min and the viability was assessed by live/dead staining after 1 day or another 2 days of culture. Live/Dead assay was performed on the hmsc-laden styrlpyrene gel at 24 hrs post encapsulation to determine the cell viability. The live/dead staining solution (Life technologies) was prepared according to the manufacturer s instructions. Images were obtained using a Nikon inverted confocal microscope and reconstructed through ImageJ. 19

20 Figure S17. Live/dead image of cell-laden hydrogels at different concentration after treatment with UV light at λ = 340 nm and I = 10 mw cm -2 for 10 min, compared to hmscs cultured on tissue culture plate (TCP), (Scale bar = 100 µm). Figure S18. Live/dead image of cell-laden hydrogels after 3 days under cell culture condition, cells in hydrogel with NO UV light treatment show ca 27% viability, whereas cells in hydrogel with UV light treatment after 3 days of culture show ca 45% viability (Scale bar = 100 µm).this may be due to softening of the hydrogels by UV light treatment, allowing cell movement and interaction. 20

21 Reference: 1. Kathayat, R. S.; Finney, N. S., Sulfoxides as Response Elements for Fluorescent Chemosensors. J. Am. Chem. Soc. 2013, 135, (34),