Core–shell patterning of synthetic hydrogels via interfacial bioorthogonal chemistry for spatial control of stem cell behavior

A new technique is described for the patterning of cell-guidance cues in synthetic extracellular matrices.


Synthesis of tetrazine functionalized hyaluronic Acid (HA-Tz)
Hyaluronic acid ( (0.60 mmol, 88%) HA-Tz as a pink fluffy solid. The product was stored at -20 °C prior to use.

Synthesis of PEG-TCO
To a round bottom flask was added the MeO-PEG 12 -amine (67 mg, 98% purity, 0.116mmol), CH 2 Cl 2 (2 mL) and Et 3 N (65µL, 0.464mmol). After the addition of sTCO 4-nitrophenyl carbonate (55 mg, 0.175 mmol), the solution was stirred at room temperature overnight, at which point the reaction was analyzed for completion by UPLC-MS analysis. The solvent was evaporated under reduced pressure and the residue was passed through a short column of deactivated C 2 silica gel first eluting with CH 2 Cl 2 to remove the excess carbonate and then 10% MeOH in CH 2 Cl 2 to elute fractions containing the product. The solvent was concentrated under reduced pressure and the yellow crude product was purified by reverse phase chromatography using a Biotageon C 18 silica gel using a gradient of 10% to 90% MeOH in neutral water to give the product (

Synthesis of RGD-TCO
Prior to TCO conjugation, RGD peptide with a sequence of GKGYGRGDSPG was synthesized following standard solid phase peptide synthesis protocol. The cleaved product, with C-amidated and N-acetylated, was allowed to react with nitrophenyl carbonate-derived sTCO in anhydrous DMF to install TCO through the lysine amine. The product was purified by HPLC and analyzed by ESI-MS, as reported in our previous publication. 3

Synthesis of PEG-dTCO
To a round bottom flask was added the MeO-PEG 12 -amine (58 mg, 98% purity, 0.102mmol), CH 2 Cl 2 (2 mL) and Et 3 N (56µL, 0.400mmol). Next, dTCO 4-nitrophenyl carbonate (53mg, 0.152mmol, 91:9 ratio of 2 diastereomers) was added and the solution was stirred at room temperature overnight. When the reaction was complete, as judged by UPLC-MS analysis, the solvent was evaporated under reduced pressure and the residue was passed through a short C 2 deactivated silica gel column first eluting with CH 2 Cl 2 to remove the excess carbonate and then 10% MeOH in CH 2 Cl 2 to get fractions containing the product. The solvent was concentrated under reduced pressure and the yellow crude product was purified by reverse phase chromatography on C 18 silica gel using a gradient of 10% to 90% MeOH in neutral water to give the product ( 2. Analytical methods.

Percent tetrazine incorporation in HA-Tz.
The percent tetrazine incorporation in HA-Tz was determined collectively by UV-vis and 1 H NMR analyses. UV-vis quantification was based on the tetrazine absorption at  max 267 nm, employing Beer-Lambert law. Using an aqueous solution of Tz-hydrazide at a concentrations from 4.7 mM to 0.47 mM as the standard ( Figure   S1A-B), the molar extinction coefficient of the tetrazine moiety (ε Tz ) was determined as 2.3 × 10 4 L Mol -1 cm -1 . Taking into consideration the change of the molecular weight for HA disaccharide repeats after tetrazine incorporation, the degree of tetrazine incorporation was calculated as 18.6% ( Figure S1C). By 1 H NMR ( Figure S11), tetrazine incorporation in HA was calculated as 18.0%, analyzed by comparing the integration between the aromatic protons (7.4-8.4 ppm) to the anomeric protons of HA. As expected, some EDC-activated carboxyl groups in HA were transformed to N-acylurea. 5 Figure S1. UV-Vis spectra of aqueous solutions of Tz-hydrazide (A, 4.7 mM to 0.47 mM) and HA-Tz (C, 0.27mM). The extinction coefficient was determined from the standard curve with a linear regression (B). A UV cuvette with a pathlength of 1 cm was used.

Analysis of reaction kinetics.
The reaction was run under pseudo-first order conditions and monitored by UV-Vis spectroscopy at 267 nm using an Applied Photophysics SX.18MV-R stopped-flow dual mixing spectrometer.
The K obs was determined by fitting a non-linear curve of ln(A/A 0 ) vs time, where A 0 and A was absorbance at time 0 and t, respectively. The kinetic runs were measured in triplicate, and the average K obs was 13.51 ± 0.01 s -1 and 2.130 ± 0.003 s -1 for PEG-TCO and PEG-dTCO, respectively. The second order rate constant (k 2 ) was calculated to be 6.70 × 10 4 M -1 s -1 and 9.94 x 10 3 M -1 s -1 for PEG-TCO and PEG-dTCO, respectively.

Mechanical Properties.
Hydrogel microspheres were tested under compression using a micro-materials tester, with a parallel plate (Video S1). The upper platen was positioned ~150 µm above the hydrogel sphere. Following hydration by a single drop of PBS the platen was driven toward the hydrogel microsphere at 45 µm/s to a target depth of 700 µm. Due to the known load cell compliance (0.0341 mN/µm) and surface offset (~150 µm), the compression rate and deformation of the sample were typically ~40 µm/s and ~500 µm, respectively. Preliminary experiments demonstrated that the compression response was relatively insensitive (± 0.7 kPa or ± 4.7%) to compression rates between 5 and 400 µm/s. From Figure S3, it can be seen that as the glass flat approaches the hydrogel microsphere, the force remains at zero until a critical point at which the formation of a fluid meniscus pulls the cantilever beam of the load cell. We define the point of initial contact as the point during which the meniscus is first formed. As the glass platen continues downward the force increases nonlinearly with the compression of the sphere in accordance with Hertz's theory for the contact between spherical elastic bodies. A Hertzian analysis of the loading portion of the test was used to quantify the compressive modulus of each gel. Hertz's solution to the deformation of an elastic sphere against a rigid flat is: The model determines the contact modulus (E c ) of the sphere based on the measured variables: force (F), radius of the sphere (R), and deformation (δ). The analysis assumes the glass is rigid relative to the hydrogel microspheres and has infinite curvature, i.e.
flat. Young's modulus (E) can be calculated from the contact modulus through the relationship: , which requires prior knowledge of the Poisson's ratio (ν). Here we assume ν = 0.5. Note that the R of each sphere was measured using calipers prior to compression testing and was paired with its indentation profile for model fitting. It is worth noting that between each indentation, the sample was removed and placed back in PBS while the surfaces were wiped free of any liquid. Figure S3. Representative data set demonstrating the full approach and retraction phases. Note that the formation and breaking of the fluid meniscus do not occur at the same position.
Furthermore, note the apparent hysteresis between the approach and retraction curve. It is thought that the majority of the hysteresis seen here is due to the meniscus forces.   Modulus was altered by tuning the relative concentration of mono-functional capper, PEG-TCO.

Gel degradation and cell morphology
As-synthesized, fully swollen hydrogel spheres were introduced to glass cylinders of known weights and the initial gel mass was recorded. Hank's balanced salt solution (HBSS,pH 7.4,200 μL) containing 100 U/mL collagenase type IV was introduced to the cylinders and enzymatic degradation was monitored every 30 min for up to 4 h. At each time point, the buffer was aspirated and the combined weight of the glass cylinder and the gel was measured. Three repeats for each condition were included and the results were reported as the average gel mass remaining as a function of incubation time.

Immunostaining for collagen I.
After 7 days of culture, constructs were stained for F-actin using Alexa Fluor 568 phalloidin, with the nuclei counter stained by DAPI, following our previous protocols. 6 Samples were incubated with primary anti-Collagen I antibody (Abcam) at a 1:100 dilution in 1x PBS containing 3% BSA for 2 h at room temperature. Samples were then treated with Alexa Fluor 488-conjugated secondary antibody at a 1:200 dilution in the same buffer for 2 h at room temperature. Stained samples were imaged using a Zeiss 710 NLO confocal microscope with a 40X objective. Figure S10. Characterization of cellular expression of collagen I by immunostaining and confocal imaging (40X). Cells were cultured for 7 days in homogeneous hydrogels prepared using either PEG-bisTCO (Gel A) or GIW-bisTCO and RGD-TCO (Gel B). DAPI, Phalloidin and Collagen I were stained blue, red and green, respectively. Scale bar: 50 μm.              Figure S34. HRMS spectrum of PEG-dTCO.