SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATION Dynamic display of biomolecular patterns through an elastic creasing instability of stimuli-responsive hydrogels Jungwook Kim, Jinhwan Yoon, and Ryan C. Hayward * Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, USA Figure S1 Lateral resolution of biomolecular patterns. The normalized fluorescence intensity for each polyelectrolyte, PLL-g-PEG and PLL-g-PEG-X, projected along the depth direction, is measured laterally across a patterned stripe (reproduced above from the cross-sectional image in Fig. 2b). For PLL-g-PEG-X, the width of the boundary regions, defined as the distance between the lateral positions corresponding to 90 % and 10 % of the maximum projected intensity, is determined to be 2 μm. nature materials 1

2 supplementary information Table S1 Templating crease locations with underlying substrate patterns. Hydrogels attached to substrates with periodic raised lines, whose pitch (S) and width (W) are varied at constant initial gel thickness (thicker portion, H 0 : 160 m and thinner portion, h 0 : 70 m), show either one or two creases over each raised line, depending on the pattern geometry. For each S and W in Table S1, the upper and lower entries represent the number and location of creases, respectively. Whenever W is larger than 3d (d = 60 m in this case), two creases form, each a distance d from the corresponding edge of the substrate pattern. The numbers separated by semicolons represent: the distance from the left corner of the raised substrate feature to the left crease; the distance between the two creases; the distance from the right crease to the right corner. For images a and b, gel surfaces are stained with fluorescently tagged PLL in the unfolded state, and then imaged with LSCM in the creased state. The upper and lower images show the inplane and the cross-sectional views of creases, respectively, and white dashed and solid lines represent the positions of the substrate patterns. 2 nature MATERIALS

3 supplementary information Figure S2 Location of the maximum in-plane compression calculated from FEA. A series of FEA calculations to simulate the swelling of a hyperelastic gel attached to the substrate with a step pattern (as in Figure 3a) reveal that the distance of the maximum in-plane compressive strain from the pattern edge (d) increases linearly with the thickness of the thinner portion of the gel (h 0 ) at constant overall gel thickness, H 0 (here H 0 = 8, in arbitrary units). An equilibrium linear swelling ratio (i.e. the linear expansion in size of an unconstrained gel) of 1.4 was used for the calculations shown, corresponding to the degree of swelling at 23 o C for a hydrogel of identical composition to those in Figure 1 and 2. The experimentally-determined location of creases at 23 o C (red circle, d = 60 m) for surface-attached hydrogels with H 0 = 160 m and h 0 = 70 m (Table S1) coincides closely with the FEA predictions. The predicted value of d is negligibly influenced by the material parameters such as shear modulus or Flory interaction parameter between the gel and the solvent, though the values used for the calculations shown are G = 1 kpa, and = 0.2, respectively. nature materials 3

4 supplementary information Figure S3 Dynamic interactions between a scaffold and microscopic beads. Bright-field optical micrographs of streptavidin-coated beads adhered to a biotin-patterned scaffold at 37 o C (top) and 23 o C (bottom). The white arrow denotes the edge of the underlying substrate pattern. Figure S4 Polymeric network density of a creased hydrogel. LSCM was used to investigate spatial variations in polymer network density for a creased hydrogel attached to a topographicallypatterned substrate. The fluorescent monomer fluorescein o-acrylate (Sigma-Aldrich) was incorporated in the pre-gel solution (0.3 mol % of total monomer), and LSCM was used to measure the fluorescence intensity throughout the hydrogel in its swollen (creased) state, which is taken to be proportional to the local density of polymer within the gel (no deconvolution has been applied). It can be seen from the resulting density profile and the inset (cross-sectional image along crease from Fig. 4a, added to scale) that the functionalized surface areas (red, inset) and thus the captured objects (streptavidin-functionalized PS beads; yellow, inset) are surrounded by regions of locally enhanced polymer density. The location of the crease is indicated by a black dotted line. 4 nature MATERIALS

5 supplementary information Movie S1 Detachment of beads upon crease formation. At sufficiently low surface density of biotin (~ 2.5 μm) adherent beads are detached from the hydrogel surface as a crease forms, as evidenced by the resumption of Brownian motion. This movie is sped up by a factor of 30. Table S2 Relative enzymatic activities (arbitrary units) of scaffolds. For each scaffold, two consecutive cycles of unfolding (38 o C) and folding (26 o C) are performed with a given substrate. Since the enzymatic activity is largely recovered in the 2 nd cycle at 38 o C, we conclude that the observed reduction in activity at 26 o C predominantly reflects the sequestration of enzyme within creases, not the irreversible denaturation of enzyme upon folding. METHODS Synthesis of polylysine graft copolymers PLL-g-PEG and PLL-g-PEG-biotin: 10 mg of poly-l-lysine hydrobromide (PLL, M w = 150 ~ 300 kg mol -1, Sigma-Aldrich) and either 73 mg of methoxy succinimidyl carboxymethyl PEG (mpeg-nhs, M w = 5.2 kg mol -1, Laysan Bio) or 55 mg of mpeg-nhs with 18 mg of biotin N- hydroxy succinimidyl PEG (biotin-peg-nhs, M w = 5.2 kg mol -1, Jenkem Technology) were nature materials 5

6 supplementary information dissolved in 1.0 ml of anhydrous DMSO containing 20 L of triethylamine (TEA). In both cases, 1 out of every three lysine residues was grafted with PEG; in the case of PLL-g-PEG-biotin, 1/4 of the grafted PEG chains contained terminal biotins. The solution was stirred for 12 hr at room temperature, then either 0.57 mg of fluorescein isothiocyanate (FITC, Sigma-Aldrich) or 0.64 mg of tetramethylrhodamine isothiocyanate (TRITC, Sigma-Aldrich) was added with additional stirring for 4 hr. The solution was dialyzed (50 kda molecular weight cutoff, Spectra/Por) against phosphate buffered saline (PBS, 10 mm phosphate buffer in 138 mm aqueous NaCl, Sigma- Aldrich) for 1 day, then deionized water for 2 days, and finally lyophilized to yield fluorescein tagged PLL-g-PEG (orange color) or rhodamine tagged PLL-g-PEG-biotin (red color). The NHSamine coupling reaction was confirmed by 1 H-NMR (D 2 O, Bruker Avance 400) via the presence of a broad peak at PLL-g-PEG-RGD: 0.9 mg of poly-l-lysine hydrobromide and 6.1 mg of maleimide N- hydroxy succinimidyl PEG (MAL-PEG-NHS, M w = 3.6 kg mol -1, Jenkem Technology) were dissolved in 250 L of anhydrous DMSO. 432 L of DMSO containing 0.51 vol % TEA (0.9 molar equivalents compared to MAL-PEG-NHS) were added slowly (0.2 ml/hr) by syringe pump with vigorous stirring at room temperature; this slow addition maintained a low concentration of deprotonated lysine units such that the amine-nhs coupling overwhelmed the Michael reaction between amine and MAL, which otherwise led to gels of PLL crosslinked by the bifunctional PEG. Upon completing TEA addition, 1.0 mg of a cyclic peptide containing the Arg-Gly-Asp sequence (c(rgdfc), Peptides International) was dissolved in 300 L of anhydrous DMSO and added to the polymer solution, such that the Cys residue of the peptide coupled with MAL through the Michael reaction. Remaining unreacted MAL groups were quenched with an excess of 2-mercaptoethanol. After addition of 0.06 mg of TRITC and 4 hr of stirring, the solution was dialyzed and lyophilized as described above. Characterization of dynamic scaffolds Hydrogel scaffolds were imaged using either a Zeiss Axiovert 200 inverted optical microscope (10x, 20x, and 40x objectives) or a Zeiss LSM 510 META laser scanning confocal fluorescence microscope (LSCM) (10x and oil-immersion 40x objectives). For LSCM measurements, 6 nature MATERIALS

7 supplementary information an Argon laser (wavelength 488 nm) and a HeNe laser (wavelength 543 nm) were used to excite fluorescein (FITC, detection filter: nm) and tetramethyl rhodamine (TRITC, detection filter: 560 nm), respectively. The fluorescence intensity from labeled PLL graft copolymers deposited at and below the hydrogel surface was converted to a concentration of lysine residues using a calibration curve from labeled PLL solutions of known concentrations. The extent of vertical swelling of surface-attached hydrogels (h/h 0 ) as a function of temperature was measured by incorporating fluorescent polystyrene beads (2 x 10-6 wt%, average diameter 2 μm, Polysciences) in the pre-gel solution. Following polymerization, the gel was immersed in PBS within a temperature-controlled microscope stage, and the apparent size of fluorescent beads measured via epi-fluorescence microscopy. At a fixed vertical position of the microscope stage and objective, changes in the vertical positions of each bead due to swelling of the surface-attached hydrogel changed the defocusing of each bead and thus its size in the resulting image 1. The radii of seven fluorescent beads measured at each isothermal equilibrium swelling were averaged and compared to their initial values obtained prior to swelling, with uncertainties estimated from the standard deviation of these seven measurements. The degree of swelling was determined from a calibration curve of the defocusing induced by a known translation of the microscope objective. Lipase immobilization on scaffolds Lipase from Candida Rugosa (EC , Sigma-Aldrich) was biotinylated using biotin- PEG-NHS. 15 mg of lipase were dissolved in 1 ml of 0.1 M PBS (ph 8.0, NaCl 0.14 M), and the solution was centrifuged to remove insoluble residue. 27 mg of biotin-peg-nhs and 0.4 mg of TRITC were dissolved in 50 L of anhydrous DMSO, and the solution was added drop-wise to the lipase solution. This solution was stirred overnight and dialyzed against PBS at 4 o C. Biotinylation of the enzyme was confirmed by SEC-HPLC (Waters Corporation). Biotinylated lipase was next immobilized on streptavidin-functionalized polystyrene beads (average diameter: 2 m, Polysciences) as described by the manufacturer s protocol, and immobilization of lipase on the beads was confirmed by the fluorescence emission of the beads (detection filter: 620 ± 30 nm) due to presence of TRITC-labels on lipase. To fabricate lipase-patterned scaffolds, biotin-patterned nature materials 7

8 supplementary information scaffolds were incubated with avidin (from egg white, Invitrogen) solution (1 mg / 4 ml of PBS) at 37 o C for 5 minutes, washed thoroughly with PBS, incubated with a solution containing the biotinlipase-streptavidin functionalized polystyrene beads at 37 o C for 30 minutes, and finally washed with PBS. Cell culture on scaffolds LLCPK porcine epithelial cells in a 1:1 mixture of Opti-MEM (Invitrogen) and Ham s F10 (Sigma-Aldrich) media supplied with 7.5 % of serum were seeded on the RGD patterned scaffold and cultured in a humidified 5% CO 2 atmosphere incubator at 37 o C. Prior to cell culture, the scaffold was rinsed with sterile PBS several times and placed in a plastic Petri dish. After incubation for 1 d, cells adhered selectively on the RGD-functionalized regions of the hydrogel surface. The cells and scaffold surface were imaged via optical microscopy while the temperature was varied between 37 o C and 23 o C using a temperature-controlled microscope stage. To assess viability of cells following encapsulation within creases, a solution of trypan blue (0.1 wt% in PBS, Aldrich) was used to selectively stain dead cells. After incubating cells on the scaffolds at 37 o C for 1 d, we performed (i) five cycles of folding and unfolding, i.e., 23 o C for 20 min then 37 o C for 10 min, or (ii) continuous encapsulation within folds for 60 min. For each cycle, the number of adherent cells was counted; all cells observed maintained adhesion throughout. The scaffolds were then rinsed with sterile PBS and exposed to trypan blue solution for 3 min which stains dead cells dark blue. Greater than 99% of cells were found to be viable following either encapsulation procedure. References 1 Wu, M., Roberts, J. W. & Buckley, M. Three-dimensional fluorescent particle tracking at micron-scale using a single camera. Exp. Fluids 38, (2005). 8 nature MATERIALS