Photoregulated Hydrazone Based Hydrogel Formation for 3D Cell Encapsulation and Biochemical Patterning

Transcription

1 Supporting information for: Photoregulated Hydrazone Based Hydrogel Formation for 3D Cell Encapsulation and Biochemical Patterning Malar A. Azagarsamy a, Ian A. Marozas a, Sergio Spaans b and Kristi S. Anseth a * a Department of Chemical and Biological Engineering, the Howard Hughes Medical Institute and the BioFrontiers Institute, University of Colorado at Boulder, 596 UCB Boulder, CO 80303, USA. b Department of Biomedical Engineering, Eindhoven University of Technology, Post box 513, 5600 MB, Eindhoven, Netherlands. These authors contributed equally General: Material and Methods: 1 H-NMR spectra were performed on a 300 MHz Bruker NMR spectrometer, where the residual proton resonance of the solvent is used as an internal standerd for each molecule. Chemical shifts are shown in parts per million (ppm). The multiplicities of each peak are given in abbreviations such as: s, singlet; d, doublet; t, triplet; m, multiplet; bs, broad singlet. All chemicals used were purchased from commercial sources and used as such, unless otherwise mentioned.

2 Synthetic scheme for PEG-NB and PEG-HY:

3 Synthesis of compound 1a: O H NO 2 O O O 1a O Nitric acid (15 ml, 354 mmol) was poured in a 250 ml single-neck RB with a stir bar and a thermometer. The nitric acid was cooled down to below 5 C. Compound 1 (5g, 18.8 mmol) was then grinded to a fine powder with a mortar and pestel. The powder was added in small portions (~3 spatula scoops) to the nitric acid and allowed to dissolve before adding the next one (~5 min per portion). Once compound 1 has been dissolved, the reaction was removed from ice bath and heated with a water bath to 32 C while leaving the RB open. The temperature of the reaction was checked every 5 min. Once the reaction reached C, it was placed back on ice until the reaction has cooled down to below 20 C. When the temperature was below 20 C, it was returned to the 35 C water bath. These last steps were repeated until the total reaction time in the 35 C water bath was approximately 1h. The reaction mixture was added dropwise to a beaker on ice (500 ml) and was stirred overnight at 4 C. A yellow precipitate formed that was then filtered over a glass frit using a vacuum. The product was rinsed with deionized water and dried with air. The product was recrystallized in boiling absolute ethanol (500 ml, 78 C) in an Erlenmeyer flask and stored overnight at 4 C. The suspension was filtered over a glass frit and dried with air to yield the product 1a (761 mg, 13%). 1 H-NMR (300 MHz, CDCl 3 ) δ [ppm] 10.4

4 (s, 1H), 7.6 (s, 1H), 7.4 (s, 1H), (m, 4H), 4.0 (s, 3H), 2.55 (t, 2H), 2.2 (m, 2H), 1.25 (t, 3H). Synthesis of compound 1b: To a solution of ethanol (13 ml, 38 C) compound 1a (761 mg, 2.4 mmol) was dissolved under flow of argon. Once dissolved, sodium borohydrate (165 mg, 4.36 mmol) was added in small portions every 5 min and reacted for 3h. Reaction completion was confirmed with thin layer chromatography (TLC). The reaction mixture was added to deionized water in a large beaker (500 ml) and stored overnight at 4 C. The product was filtered through a glass frit filter and dried in a vacuum oven (37 C) for 30 min yielding the intermediate product (500 mg, 1.60 mmol). This product was then dissolved THF (6 ml) in a 250 ml RB flask while stirring it with a magnetic stir bar on ice. Sodium hydroxide (116 mg, 2.9 mmol) was then dissolved in H 2 O (6 ml) until completely dissolved. The solution of sodium hydroxide was then slowly added to the reaction mixture and reacted for 2 h. Product formation was then confirmed with TLC. The aqueous layer was extracted three times using ethyl acetate as an organic solvent and the resulting organic layer was dried over Na 2 SO 4 and evaporated to dryness. The crude product was then purified using silica gel column chromatography to yield the final product 1b (177 mg,

5 39%). 1 H-NMR (300 MHz, (CD 3 ) 2 SO). δ [ppm] (bs, 1H), 7.7 (s, 1H), 7.4 (s, 1H), 5.6 (bs, 1H), 4.1 (t, 2H), 3.9 (s, 3H), 2.4 (t, 2H), 2.95 (m, 2H). Synthesis of compound 2: To a solution of DMF (2ml), Et 3 N (140 µl, 1 mmol) and 4-arm PEG-amine (1 g, 0.1 mmol, 10 kda) was added until completely dissolved. Hereafter, product 1b (263 mg, 1 mmol) and DMAP (12.2 mg, 0.1 mmol) was added. Finally after adding DIC (156 µl, 1 mmol), the reaction was allowed to react overnight. The product was precipitated in cold diethyl ether, spun down and washed again with cold diethyl ether. This was repeated three times and dried under vacuum for 30 min. The product was then dissolved in deionized water and dialysis was performed with a cut-off of ~7 kda. Purified product was frozen and lyophilized to yield the final product 2 (926

6 mg, 90%). Product 2 was characterized by confirming hydrogel formation after mixing product 2 and 3a and exposing to collimated UV light. 1 H-NMR (300 MHz, (CD 3 ) 2 SO). δ [ppm] 7.66 (s, 1H), 7.4 (s, 1H), 5.6 (bs, 1H), 5.6 (bs, 1H), 4.83 (s, 2H), 4.04 (t, 2H), 3.92 (s, 3H), 3.50 (s, PEG), 2.26 (t, 2H), 1.96 (m, 2H). Synthesis of compound 3:

7 To a solution of DMF (4ml), Et 3 N (139 µl, 1 mmol) and 4-arm PEG-amine (1 g, 0.1 mmol, 10 kda) was added until completely dissolved. Hereafter, tri-boc-hydrazinoacetic acid (390 mg, 1 mmol) and DMAP (12.2 mg, 0.1 mmol) was added. Finally after adding DIC (156 µl, 1 mmol), the reaction was allowed to react overnight. The product was precipitated in cold diethyl ether, spun down and washed again with cold diethyl ether. This was repeated three times and dried under vacuum for 30 min. The product was then dissolved in deionized water and dialysis was performed with a cut-off of ~7 kda. Purified product was frozen and lyophilized to yield the product 3 (946 mg, 91%). Product 3 (946 mg, 0.09 mmol) was dissolved in 2% hydrazine in TFA:DCM (1:1, 10 ml) and reacted overnight. The product was precipitated in cold diethyl ether, spun down and washed again with cold diethyl ether. This was repeated three times and dried under vacuum for 30 min. The product was then dissolved in deionized water and dialysis was performed with a cut-off of ~7 kda. Purified product was frozen and lyophilized to yield the product 3a (400 mg, 44%). Product 3a was characterized by confirming hydrogel formation

8 after mixing product 2 and 3a and exposing to collimated UV light. 1 H-NMR (300 MHz, (CD 3 ) 2 SO). δ [ppm] 8.98 (bs, 2H), 8.26 (bs, 1H), 3.69 (t, 2H), 3.5 (s, PEG) Synthesis of NB-K(Dde)GRGDS: Solid-phase peptide synthesis was performed on a Protein Technologies Tribute SPPS using 2- (1-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as the coupling

9 agent. With this technique, the peptide K(Dde)-G-R(Pbf)-G-D(tBu)-S(tBu)-NH 2 was made on resin and the N-terminal amine of the peptide was used to couple product 1b (301 mg, 1.06 mmol) using DMAP (61 mg, 0.5 mmol) and DIC (194 µl, 1.25 mmol) in DMF (2ml). Finally, the peptide was cleaved from the resin by treating it with phenol (250 mg) in trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5). The peptide was precipitated in cold diethyl ether and washed three times with ether. The crude peptide was purified using reverse-phase high performance liquid chromatography (HPLC) with a linear gradient (10-95% acetonitrile in water). The product was then frozen and lyophilized to result in the peptide NB-K(Dde)GRGDS. The molecular weight of the peptide was confirmed with matrix-assisted laser desorptionionization time-of-flight mass spectrometry using α-cyano-4-hydroxycinnamic acid as the matrix: calculated ([M+H] + =1049); observed ([M+H] + =1049.8). See figure below.

10 Synthesis of NB-K(fluorescein)GRGDS:

11 After purifying NB-K(Dde)GRGDS (5 mg, 4.8 mmol), it was treated with 2% hydrazine monohydride in DMF (1 ml) and reacted for 20 min. The product was precipitated out using cold diethyl ether, washed using the same solvent and dried under vacuum. The product (4.25 mg, 4 μmol) was then dissolved in DMF (2 ml) followed by the addition of 5(6)-carboxyfluorescein succinimidyl ester (fluorescein) (5.68 mg, 12 μmol) and the mixture was allowed to react overnight. The product was then precipitated using cold diethyl ether and washed three times in ether. The product was dissolved in water, frozen and lyophilized to result in the peptide NB- K(fluorescein)GRGDS. The molecular weight of the peptide was confirmed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry using α-cyano-4-hydroxycinnamic acid as the matrix: calculated ([M+H] + =1245); observed ([M+H] + =1244.3). See figure below.

12 Kinetic Experiments. A solution of 4mM NB 1b and 12mM fumarate was prepared in ddmso for analysis by NMR. 500 μl of solution was transferred to a clean NMR tube and irradiated with intensities of light corresponding to hydrogel polymerization experiments (5, 10, 20 and 30 mw/cm 2 ) for up to 30 minutes of total exposure time. After each exposure interval, the sample was analyzed by 1 H-NMR (40 scans). The integration of the 1H aldehyde peak was normalized to the internal reference standard 2H fumarate peak at each time point and plotted as the natural logarithm of the ratio of the aldehyde integration at time t to the final aldehyde integration (see figure below), where the integration stopped changing significantly. One would expect that this

13 would result in a linear relationship where the slope is the pseudo-first order rate constant k. However, we observed a non-linear relationship that can be explained by the fact that the nitrosobenzaldehyde product of photocleavage is also an absorber of UV light, with a molar extinction coefficient similar to the NB alcohol 1b. Thus, as the reaction proceeds, the nitrosobenzaldehyde product decreases the effective light intensity throughout the sample for NB alcohol conversion and the reaction rate decreases. This observation was supported by a code written in Mathematica that was used to solve the PDEs describing light intensity and nitrosobenzaldehyde production as a function of time and sample depth and showed a non-first order production of nitrosobenzaldehyde. The solution of these PDEs demonstrated that the effective rate constant is a function of sample thickness, however, the NMR studies showed that the time to final conversion was faster for higher light intensities, as expected. ln(ald t /Ald f ) mW/cm 2 10mW/cm 2 20mW/cm Time (s) Rheology experiments. Rheology was performed on a TA Instruments DHR-3 rheometer with an 8mm flat plate. This was mounted with the Omnicure lamp to monitor gel formation during exposure of collimated UV light (365 nm).

14 Characterization of Hydrogel Properties. 10 wt% hydrogels were prepared by mixing stock solutions of 20 wt% PEG-NB and 20 wt% PEG-HY in phosphate buffered saline (PBS) to a final density of 5 wt% for each macromer. 10 µl of the precursor solution was placed on a TA Instruments DHR-3 rheometer with a 8mm flat plate at a gap height of 100 µm. Hydrogel gelation kinetics was characterized and evaluated in situ by measuring the storage and loss moduli (G and G ) during exposure of the prepolymer solution to 365 nm light (EXFO Omnicure high-pressure mercury arc lamp with internal 365nm band pass filter). A PBS bath was used during the measurement to prevent the hydrogel from dehydrating and the light source was kept on for the entirety of the experiment. Measurements were taken with an oscillatory shear strain of 1% (within the linear viscoelastic region) and a frequency of either 10 or 5 rad/s. There was no significant difference observed in the storage modulus (G ) of gels exposed to angular frequencies between 0.1 and 100 rad/s, thus both experimental results are comparable (see figure below) Hydrazone gel frequency sweep Storage Modulus (Pa) ω (rad/s)

15 The first set of results was based on varying the light intensity of the light source, while keeping other variables constant (10 rad/s, 1% strain, ph 6.0). Light intensities were based on previously used light intensities for cell experiments (i.e. 5, 10, 20 and 30 mw/cm 2 ). In Figure 2a, hydrogel evolution is shown as the storage modulus vs. the time of exposure. The second set of results was based on varying the ph of the buffer of the hydrogel precursors, while keeping other variables constant (5 rad/s, 1% strain, 20 mw/cm 2 ). PBS was used as a buffer and was acidified by HCl and alkalized by NaOH. The ph of the final hydrogel mixture was measured with ph-paper (±0.5 accuracy) and was varied between, i.e. ph 6.0, 7.5, and 8.5 (Figure 3c). In Figure 2c, hydrogel evolution is shown as the storage modulus vs time of exposure. An interesting property of the PEG-NB macromer is that it has the ability to homopolymerize to form a gel in aqueous conditions in under 10 minutes when irradiated with 365nm light at an intensity of 30mW/cm 2. However, the timescale of this gelation is almost three fold slower than the copolymerization of PEG-NB and PEG-HY, even at 10 wt% PEG-NB (see figure below). This result suggests that although there are competing reactions in this system, the condensation of the benzaldehyde and hydrazine functionalities predominates.

16 Storage Modulus (Pa) wt% PEG-NB 10 wt% PEG-NB 5 wt% PEG-NB + 5 wt% PEG-HY Time (s) Peptide Photopatterning. To demonstrate spatial and temporal control over biochemical patterning, hydrogels (0.5 off-stoichiometry) were first prepared with 10 wt% PEG-HY and 5 wt% PEG-NB and exposed to collimated UV light for 10 min. Prefabricated hydrogels were incubated in 10 mm NB-K(fluorescein)GRGDS in PBS, and then irradiated with collimated UV light (365 nm, 20 mw/cm 2, EXFO Omnicure Lamp) through a chrome on a quartz photomask (250 µm lines with 250 µm spacing) for 5 min. The patterned hydrogel was then washed in fresh PBS on an orbital rotator for 2h. This was repeated for another 2h. The patterned hydrogel was then imaged at 10x magnification through a water immersion lens on a Zeiss NLO confocal laser scanning microscopy. Cell Encapsulation and Live/Dead Assay. To test the cytocompatibility of this new hydrogel crosslinking chemistry, hmscs (isolated from human bone marrow) were encapsulated in 40 µl hydrogels, and then viability was assessed via Calcein AM (for Live) and ethidium bromide (for Dead) 24, 48 and 120 h post-encapsulation. hmscs were seeded at passage 2 at a density of 5 x

17 10 6 cells/ml, resuspended in solution of 5 wt % of PEG-Hydrazine and 5 wt % PEG-NB and 1 mm NB-K(Dde)GRGDS and exposed to collimated UV light (20 mw/cm 2 ) for 10 min. Hydrogels with hmscs were imaged at 10X magnification through a water immersion lens on a Zeiss NLO confocal laser scanning microscopy. Cell viability was quantified using a particle counting module in FIJI.