Hydrogel patterning by diffusion through the matrix and subsequent light-triggered chemical immobilization

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1 Supporting Information ydrogel patterning by diffusion through the matrix and subsequent light-triggered chemical immobilization Zheyi Yi, Yu Zhang, Sujit Kootala, Jöns ilborn, and Dmitri A. ssipov* Science for Life Laboratory, Department of Chemistry-Ångström Laboratory, Uppsala University, Uppsala, SE Uppsala, Sweden Corresponding Author Dmitri ssipov, General. Linkers used in preparation of modified hyaluronan (A) derivatives were synthesized according to the literature procedures. Their structures are given in Scheme S1. Particularly, 3,3'- Dithiobis(propionic hydrazide) 1 1 and disulfanediylbis(ethane-2,1-diyl) bis(2-(6- ((hydrazinecarbonyl)oxy)hexanoyl) hydrazinecarboxylate) 2 2 were used to attach thiol- and hydrazideterminated side chains to A backbone respectively. Tartaric acid dihydrazide 3 3 was used to modify A with aldehyde groups. A derivatives were synthesized according to Scheme S2 following the reported protocols. 2,4,5 Scheme S1. Structures of modifying linkers used in the study. Synthesis of 3-(acrylamido-1-hydroxypropane-1,1-diyl) bis(phosphonic acid) (BP-acrylamide). Pamidronate hydrochloride (543 mg, 2 mmol) was dissolved in 2 ml of 2 wt. % a. The solution was cooled to C and acryloyl chloride (.64 ml, 8 mmol) was added into 4 portions (162 µl for each portion) over 45 min while the reaction mixture was kept at C by an external ice-bath cooling. After third addition of acryloyl chloride, the p was adjusted with 2 wt. % a from 2 to 1. After the addition, the mixture was stirred for 1.5 h with the temperature raising to room temperature. The resulting mixture was extracted with ethyl acetate (2 ). The aqueous phase was evaporated and the residue was triturated with methanol. Insoluble in methanol crystals were collected by filtration and dried under vacuo to give 468 mg (81% yield) of BP-acrylamide as a white solid. 1 -MR (D 2 ): 6.21 and 6.12 (2, dd and d, C 2 =, J = 16.1 z, J = 9.9 z), 5.69 (1, d, C=, J = 1.3 z), 3.53 (2, t, C 2, J = 7.7 z), (2, m, C 2 C()(P 3 2 ) 2 ). 31 P-MR:

2 R 1 /R 2 A-hy Ac R 1 = (9%) R 2 = 5 2 (1%) DTT R 1 = (9%) 2 EDC, Bt Ac A 3 EDC, Bt ai 4 R 2 = A-al (1%) 1 & 2 EDC, Bt DTT R 1 /R 2 /R 3 A-hy-S Ac R 1 = (7%) R 2 = R 3 = S (2%) 5 2 (1%) Scheme S2. Synthesis of A derivatives utilized in the preparation of hydrogels. Synthesis of hydrazide and thiol dually modified A (A-hy-S). 4 mg of A (MW 15 kda) was dissolved in 5 ml of de-ionized water. Linker 1 (71.4 mg,.3 mmol) was added to the A solution. Linker 2 (92 mg,.15 mmol) was first dissolved in 4 ml of methanol and the obtained methanolic solution was added to the A solution. -hydroxybenzotriazole (Bt) (153 mg, 1 mmol) was separately dissolved in.5 ml of -methyl-2-pyrrolidone and added to the solution of A. The p of the resultant solution was adjusted to 4.5 after which the coupling reaction was initiated by addition of solid EDC (115 mg,.6 mmol) to the reaction mixture. The mixture was stirred overnight. The reaction solution was basified to 8.5 with 1M a and DTT (347 mg, 2.25 mmol, i.e. 5-fold molar excess relative to the applied reagents 1 and 2) was added to the solution to provide cleavage of disulfide bond of the coupled reagents. The mixture was stirred overnight, after which the solution was acidified to 3.5 with 1M Cl and transferred to a dialysis tube (M w cutoff = 35). After exhaustive dialysis against dilute Cl (p 3.5) containing.1 M acl, followed by dialysis against dilute Cl, p 3.5 two times, the solution was lyophilized to give 367 mg of A-hy-S (91% yield). The incorporation of hydrazide and thiol groups was verified by 1 -MR. Specifically, the peaks corresponding to the native A protons, such as acetamide protons at 1.9 ppm, 2', 3', 4', 5', and 6'-protons of A disaccharide unit at ppm, as well as anomeric 1'-protons at 4.4 ppm, were compared with newly appeared peaks corresponding to the peaks at 2.58 and 2.73 ppm corresponding to C 2 C 2 S methylene protons (see Figure S1 for designation). This indicated that 2% of the A disaccharide units were modified with hydrazideterminated side chains. The degree of incorporation of hydrazide (1%) groups in A-hy was verified by integration of the peaks at 4.9, 2.23, 1.57 and 1.3 ppm corresponding to α, β, γ, δ, and ε methylene protons of C 2 C 2 C 2 C 2 C 2 C 2 side chain (Figure S1). 2

3 anomeric protons sugar protons f1(ppm) Figure S1. 1 -MR spectrum of A-hy-S derivative. Synthesis of hydrazide-modified A (A-hy). The procedure was analogous to the synthesis of Ahy-S derivative except that one linker 2 was used in the coupling step for preparation of hydrazide modified A derivative. The degree of incorporation of hydrazide (1%) groups in A-hy was the same as for A-hy-S derivative. Synthesis of aldehyde-modified A (A-al). 4 mg A (MW 15 kda) was dissolved in 5 ml of de-ionized water. Dihydrazide linker 3 (27 mg,.15 mmol) was added to the A solution. - hydroxybenzotriazole (Bt) (153 mg, 1 mmol) was separately dissolved in 5 ml of 1:1 (v/v) mixture of acetonitrile-water and added to the solution of A. The p of the resultant solution was adjusted to 4.5 before the coupling reaction was initiated by addition of solid EDC (58 mg,.3 mmol) to the reaction mixture. The mixture was stirred overnight and then dialyzed (M w cutoff = 35) first against dilute Cl (p 3.5) containing.1 M acl, followed by dialysis against dilute Cl, p 3.5 two times. The dialyzed solution was lyophilized to give the intermediate A derivative modified with 1,2-diol groups (41 mg yield). The obtained 1,2-diol-modified A (41 mg) was dissolved in 5 ml of de-ionized water. 5.6 ml of.15 M aqueous solution of sodium periodate (214 mg, 1 mmol) was added to the above A solution to achieve 1:1 molar ratio between periodate and A disaccharide units. The mixture was stirred for 1 min at room temperature, after which ethylene glycol (.52 ml, i.e. 1 molar equivalents per amount of periodate used) was added to the mixture. The mixture was stirred in the dark overnight. The reaction solution was dialyzed against pure water two times (M w cutoff = 35) and finally lyophilized (361 mg or 9 % yield). The amount of aldehyde groups was obtained by reaction with tert-butyl carbazate (TBC) followed by reduction with ab 3 C. Briefly, A-aldehyde (~2 mg) was dissolved in 2 ml of water and to this solution was added the.5 M aqueous solution of TBC (1-fold excess per molar amount of sodium periodate that was used in the preparation of A-aldehyde derivative). The mixture was stirred for 1 h at room temperature after which.5 M aqueous solution of ab 3 C (equimolar amount to that of TBC) was added to the mixture. The mixture was allowed to react for 24 h at room 3

4 temperature. The TBC-modified A was recovered by dialysis in 35 MW cut off tubing against water twice. 1 MR of the obtained product was examined and the peak corresponding to the tert-butyl substituent ((C 3 ) 3 CC, δ = 1.38 ppm) was compared with the peak of A acetamide protons at 1.9 ppm. Degree of aldehyde functionalization in the obtained A-al derivative (~ 1 %) was calculated from the amount of reacted TBC reagent. A Direction of diffusion After UV fixation but before swelling in.17m acl After swelling in.17m acl B Figure S2. (a) yaluronic acid (A) hydrogel with a gradient concentration of bisphosphonate ligands covalently attached to the matrix. (b) Schematic presentation of sectioning of the gradient hydrogel for subsequent EDXS analysis. 4

5 A 3, Amount of CytC loaded, mg 2,5 2, 1,5 1,,5, Feeding amount of BPacrylamide, mg B Concentratio of CytC in the gel, mg/ml 3,5 3, 2,5 2, 1,5 1,,5, Feeding amount of BPacrylamide, mg Figure S3. Dependence of cyt c loading capacity of A matrix expressed as absolute amount (a) or concentration (b) of the loaded protein on the feeding amount of BP-acrylamide used in preparation of the hydrogels. 5

6 Image analysis of hydrogels prepared under different patterning conditions. ydrogels patterned under different conditions were photographed and the obtained TIF images were analyzed with Java image processing program ImageJ. Particularly, rectangular area covering maximally a hydrogel image was selected. Two-dimensional graphs of the intensities of pixels along the rectangular selections were obtained. X-axis represented the horizontal distance through the selection, while the Y-axis represented the vertically averaged pixel intensity. The images were geometrically positioned that the direction of diffusion was co-linear with the horizontal X-axis. 1 1 o BP-acrylamide 8 All conditions for thiol-ene addition reaction are met 8 Colour value 6 4 Colour value Distance, mm Distance, mm 8 o UV 8 o S Colour value 6 4 Colour value Distance, mm Distance, mm Figure S4. Image analyses of cyt c loaded hydrogels that were patterned under different conditions. Red curve corresponds to hydrogel in which all conditions of thiol-ene bisphosphonate immobilization were met. Black, blue and pink curves correspond to hydrogels in which BP-acrylamide, UV light, and thiol in the matrix were respectively excluded from the gradient hydrogel preparation. 6

7 Quantification of loaded and released cytochrome c. Calibration was performed in order to quantify cyt c liberated from hydrogels as a result of the hydrogels degradation or diffusion from the hydrogels in the course of release experiments..1 mg/ml,.5 mg/ml,.25 mg/ml, and.1 mg/ml solutions of cyt c in PBS were prepared by diluting the 1 mg/ml stock solution of the protein. UV-vis absorbance of the samples was measured at 41 nm. Linear fitting of the calibration data yielded the following equation for the determination of concentration: c = A/7.838 mg/ml..7.6 y = x R= Conc mg/ml Figure S5. Standard UV-Vis calibration curve for solutions of cyt c at different concentrations. 7

8 Cell survival, % Concentration of cyt c, mg/ml Figure S6. Calibration of MCF-7 cell viability exposed to different cyt c concentrations. 8

9 Calcium assay of mineralized gradient hydrogels. Calibration was performed in order to quantify Ca 2+ extracted from different sections of gradient hydrogels. For this purpose, 1.5 mm stock solution of CaCl 2 in.5m acetic acid was prepared. 5 µm, 25 µm, 1 µm, and 5 µm solutions of CaCl 2 in epes buffer containing Arsenazo III were prepared by diluting the 1.5 mm stock solution (2 µl, 1 µl, 4 µl, and 2 µl respectively) with.4 mm Arsenazo III / 1 mm epes (5.4 ml, 5.5 ml, 5.9 ml, and 5.9 ml respectively) followed by neutralization with 5M a (22 µl, 11 µl, 44 µl, and 22 µl respectively). The neutralized solutions were filled up to 6 ml with.4 mm Arsenazo III in 1 mm epes. UV-vis absorbance of the samples was measured at 65 nm. Linear fitting of the calibration data yielded the following equation for determination of concentration: c = (A )/ µm y = x R= Concentration microm Figure S7. Standard UV-Vis calibration curve for CaCl 2 solutions of different concentrations treated with calcium sensitive dye Arsenazo III. 9

10 Figure S8. Schematic presentation of sectioning of the cyt c loaded gradient hydrogel for subsequent study of enzymatic oxidation of TMB in the hydrogel section. Quantification of concentrations of TMB, 2 2, and cytochrome c in gradient loaded hydrogel. Calibration was performed in order to quantify amount of TMB loaded into gradient hydrogel for its subsequent in-hydrogel oxidation by hydrogen peroxide. For this purpose,.2 mg/ml stock solution of TMB in DMS- 2 mixture (1 : 4, v/v) was prepared µg/ml, 6.7 µg/ml, 3.3 µg/ml, and 1.7 µg/ml solutions of TMB were prepared by diluting the stock solution (2 µl, 1 µl, 5 µl, and 25 µl respectively) with DMS- 2 mixture (1 : 4, v/v) (2.8 ml, 2.9 ml, 2.95 ml, and ml respectively). UV-vis absorbance of the samples was measured at 288 nm. Linear fitting of the calibration data yielded the following equation for the determination of concentration: c = A/.1968 µg/ml: 1

11 .35.3 y = x R= TMB microgram/ml Figure S9. Standard UV-Vis calibration curve for solutions of TMB at different concentrations. After incubation of the middle section of the gradient hydrogel in TMB solution, 5 µl of the used feeding solution was diluted with 2.95 ml of DMS- 2 mixture (1 : 4, v/v) and UV spectrum was recorded. Basing on the obtained absorbance (A 288nm =.3282) and the dilution factor (3 µl / 5 µl = 6), the concentration of TMB left in the feeding solution after incubation was 3.22 µg/ml 6 =.193 mg/ml. Because volume of TMB solution after incubation was 4.57 ml, the amount of TMB left in the feeding solution was.193 mg/ml 4.57 ml =.883 mg. During incubation of gradient hydrogel in hydrogen peroxide solution, TMB could diffuse out of the hydrogel. After the oxidation reaction in the hydrogel, 3 ml aliquot of the hydrogen peroxide incubation solution (8.97 ml) was therefore also examined by UV-vis spectrometry. Basing on the obtained absorbance (A 288nm =.1441), the concentration of TMB left in the feeding solution after incubation was 1.54 µg/ml and the absolute amount of TMB was 1.54 µg/ml 8.97 ml = 13.8 µg. Because initial amount of TMB used for the hydrogel feeding was 1 mg, the amount of TMB loaded into the hydrogel was finally found as 1 mg.883 mg.14 mg.1 mg. Volume of the hydrogel section after TMB loading was.386 ml which allowed final quantification of TMB in the hydrogel during the oxidation reaction: [TMB] =.1 mg/.386 ml =.26 mg/ml. [ 2 2 ] = 2 mg / ( ) ml =.19 mg/ml As was found previously, loading of cyt c into gradient hydrogel was 34.4 %. Middle section of gradient hydrogel used for the oxidation reaction represented one forth part of the whole hydrogel by mass (basing on the masses of the section and the whole hydrogel, i.e mg and mg respectively. Thus, the concentration of cyt c in the middle section of the gradient hydrogel was as follows: [cyt c] = 5 mg.344 (165.9 mg/664.5 mg) /.386 ml = 1.1 mg/ml. 11

12 Enzyme kinetics of cytochrome c peroxidase activity in the presence of additives. Control oxidations of TMB with varying cyt c concentrations were performed first. Solution of.6 mg of TMB in 3 µl DMS was added to 2.7 ml of PBS containing either.6 mg or 3 mg of cyt c..6 µl of 2 2 was finally added to the TMB substrate and UV absorbance was measured at 65 nm and 45 nm continuously over 16 hours. Absorbance 5, 4,5 4, 3,5 3, 2,5 2, 1,5 1,,5 85 min cyt c + A-hy high cyt c + A-hy, Time, min Absorbance 3, 2,5 2, 1,5 1,,5 1 min 27.5 min cyt c + A-BP high cyt c + A-BP, Time, min Absorbance 2, 1,5 1,, min 85 min cyt c + A-hy cyt c + A-BP, Time, min Absorbance 1 min 85 min 4,5 4, 3,5 3, 2,5 2, 1,5 1,,5 high cyt c + A-hy high cyt c + A-BP, Time, min Figure S1. Time-dependent absorbance changes at 652 nm TMB using cytochrome c at.17 mg/ml or.5 mg/ml (high cyt c) concentrations in different reaction systems: cyt c + A-hy (green and purple curves) and cyt c + A-BP (blue and red curves). The time at which maximal amount of intermediate TMB semiquinone-imine cation electron free radical is produced corresponds to peak of absorbance at 652 nm (shown by dashed lines). 12

13 Table S1. Amount of phosphorous in weight percentage of all elements composition determined in different sections of the gradient hydrogel. Sections were numbered from the side of the hydrogel where diffusion of BP-acrylamide started. Section 1 Section 2 Section 3 Section 4 Spot ± ± ±.9.86 ±.9 Spot ± ± ±.9.53 ±.4 Spot ± ± ±.8.66 ±.6 Average 5.34 ± ± ±.9.68 ± ± ± ± ±.19 Table S2. Properties of homogeneous A hydrogels prepared with different feeding amounts of BPacrylamide. Feeding amount of cyt c, mg ydrogel volume before loading of cyt c (V before), mm 3 ydrogel volume after loading of cyt c (V after ), mm 3 Relative decrease of volume after loading, % a) Mass of cyt c loaded (m), mg Concentration of the loaded cyt C in hydrogel, mg/ml b) ± ± ± ± ± ± 59.4 ± ± ± ± 25.4 ± 16.7 ± ± ± 8.5 ±.77 ± ± ± ± ± ± ± ± ± ±.17 a) (V before V after ) / V before 1%; b) m / V after 1 REFERECES (1) Shu, X. Z.; Liu, Y.; Luo, Y.; Roberts, M. C.; Prestwich, G. D. Disulfide Cross-Linked yaluronan ydrogels, Biomacromolecules 22, 3, (2) ssipov, D. A.; Piskounova, S.; Varghese,. P.; ilborn, J. Functionalization of yaluronic Acid with Chemoselective Groups via a Disulfide-Based Protection Strategy for In Situ Formation of Mechanically Stable ydrogels, Biomacromolecules 21, 11, (3) Varcruysse, K. P.; Marecak, D. M.; Marecak, J. F.; Prestwich, G. D. Synthesis and In Vitro Degradation of ew Polyvalent ydrazide Cross-Linked ydrogels of yaluronic Acid, Bioconjugate Chem. 1997, 8,

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