SUPPLEMENTARY INFORMATION - Nature Research · Figure S7 Post-patterning peptide diffusion from...

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nmat2473

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Supplementary Information

Figure S1 Statistical gelation model for step-growth networks (Flory and Stockmayer)To verify that hydrogel network formation occurs via the proposed step growth mechanism, a series of stoichiometrically-imbalanced gel polymerizations were monitored via real time rheology. Measurements for tan δ, ultimate elastic moduli, and the time required for gelation (estimated by the crossover point) were obtained for 13.5 wt% gels in PBS at room temperature with compositions shown in the table below. Here, tan δ is defined as the loss modulus divided by the storage modulus (tan δ = G’’/G’); therefore, a value less than unity indicates a solid material, while values greater than one are indicative of a liquid.

According to the Flory-Stockmeyer equation for statistical gelation of step growth networks, the conversion required for gelation (ρc) can be predicted as a function of the stoichiometry ratio of reactants r where r ≤ 1, fazide and fyne are the degree of functionality for the PEG azide and cyclooctyne peptide monomers, respectively1. For our system, we have fazide= 4 and fyne = 2.

𝜌𝜌𝜌𝜌𝑐𝑐𝑐𝑐 =1

�𝑟𝑟𝑟𝑟(𝑓𝑓𝑓𝑓𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 − 1)�𝑓𝑓𝑓𝑓𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑎𝑎𝑎𝑎 − 1�=

1√3𝑟𝑟𝑟𝑟

By definition, conversion in any reactive system cannot exceed unity. As such, for a gel to form, ρc must be less than or equal to 1. It follows that gels will form in this system only when r ≥ 0.33. Our data is consistent with this step-growth prediction in that gels are unable to form in systems where r = 0.3.

Azide:acetylene ratio

r ρc Time to gel point (sec)

tan δ Final G' (kPa)

3.3 : 1.0 0.3 1.1 No gelation 8.1 ± 3 0 ± 0.1 2.0 : 1.0 0.5 0.82 511 ± 40 0.74 ± 0.1 2.9 ± 0.4 1.0 : 1.0 1.0 0.58 290 ± 30 0.28 ± 0.1 12 ± 0.6 1.0 : 2.0 0.5 0.82 573 ± 50 0.81 ± 0.1 2.3 ± 0.5 1.0 : 3.3 0.3 1.1 No gelation 9.8 ± 3 0 ± 0.1

1. Odian, G.G. Principles of polymerization, xxiv, 812 p. (Wiley-Interscience, Hoboken, N.J., 2004).

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SUPPLEMENTARY INFORMATION doi: 10.1038/nmat2473

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Figure S2 Kinetics of network formation from dynamic MAS 1H NMR

In these experiments, 1H NMR spectra were acquired at 400.15 MHz using a Varian Inova-400 NMR spectrometer system equipped with a Varian gHX-Nano Probe for MAS operation. Spectra were acquired with sample spinning speeds of 3.0-3.5KHz at the magic angle roughly every 10 minutes (left) and integral values for the characteristic peaks were exponentially fit to give a second-order rate constant for the reaction (right). The characteristic peak has completely disappeared 1 hour into the polymerization, indicating that all alkyne functionalities have fully reacted at this point.



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Figure S2 Kinetics of network formation from dynamic MAS 1H NMR

In these experiments, 1H NMR spectra were acquired at 400.15 MHz using a Varian Inova-400 NMR spectrometer system equipped with a Varian gHX-Nano Probe for MAS operation. Spectra were acquired with sample spinning speeds of 3.0-3.5KHz at the magic angle roughly every 10 minutes (left) and integral values for the characteristic peaks were exponentially fit to give a second-order rate constant for the reaction (right). The characteristic peak has completely disappeared 1 hour into the polymerization, indicating that all alkyne functionalities have fully reacted at this point.

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Figure S3 MALDI-TOF Spectra of Ac-KRRK(alloc)GGPQGILGQRRK-NH2



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Figure S4 MALDI-TOF Spectra of Ac-K(DIFO3)RRK(alloc)GGPQGILGQRRK(DIFO3)-NH2



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Figure S4 MALDI-TOF Spectra of Ac-K(DIFO3)RRK(alloc)GGPQGILGQRRK(DIFO3)-NH2

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Figure S5 MALDI-TOF spectra of FAM-KGWLGPAK(FAM)GKC-NH2



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Figure S6 Viability of 3T3s and hMSCs within click-based hydrogels over 1 week of cultureTo verify viability of cells following encapsulation and subsequent material patterning, 3T3s and human mesenchymal stem cells (hMSCs) were stained and imaged using confocal microscopy on days 1, 2, and 7 (live cells are shown in green, while dead cells are red). 24 hour post encapsulation, a portion of the gels were bulk modified with RGDSC via the thiol-ene reaction. The images indicate a predominately viable cell population for each cell type (>95% viability, no statistic significance between any two conditions). While we did not explicitly assay for proliferation, it does appear that the 3T3s are proliferating in the presence of the patterned RGDSC. Images represent 200 µm confocal projections. Scale bar = 100 µm.



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Figure S6 Viability of 3T3s and hMSCs within click-based hydrogels over 1 week of cultureTo verify viability of cells following encapsulation and subsequent material patterning, 3T3s and human mesenchymal stem cells (hMSCs) were stained and imaged using confocal microscopy on days 1, 2, and 7 (live cells are shown in green, while dead cells are red). 24 hour post encapsulation, a portion of the gels were bulk modified with RGDSC via the thiol-ene reaction. The images indicate a predominately viable cell population for each cell type (>95% viability, no statistic significance between any two conditions). While we did not explicitly assay for proliferation, it does appear that the 3T3s are proliferating in the presence of the patterned RGDSC. Images represent 200 µm confocal projections. Scale bar = 100 µm.

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Figure S7 Post-patterning peptide diffusion from click-based hydrogelsFluorescently-labeled peptide was swollen into the hydrogel network and allowed to equilibrate for 2 hours. Confocal microscopy was used to determine peptide concentration profiles throughout the gel with time. The gel was placed in fresh media on an orbital shaker at room temperature in between time points to facilitate transport. The peptide was found to be completely released within two hours of treatment.

The diffusion of peptide from the click-based hydrogel was calculated using solutions to Fick’s second law of diffusion for a planar sheet with a uniform initial condition (C = C0, -l < z < l, t = 0), and sink boundary conditions (C = Cl = 0, z = ±l, t ≤ 0):

𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕 = 𝐷𝐷𝐷𝐷

𝜕𝜕𝜕𝜕2𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝑎𝑎𝑎𝑎2

Using separation of variables, an exact analytical solution is achieved1:

𝜕𝜕𝜕𝜕 − 𝜕𝜕𝜕𝜕0

𝜕𝜕𝜕𝜕𝑙𝑙𝑙𝑙 − 𝜕𝜕𝜕𝜕0= 1 −

4𝜋𝜋𝜋𝜋�

(−1)𝑦𝑦𝑦𝑦

2𝑦𝑦𝑦𝑦 + 1

∞

𝑦𝑦𝑦𝑦=0

exp �−𝐷𝐷𝐷𝐷(2𝑦𝑦𝑦𝑦 + 1)2 𝜋𝜋𝜋𝜋2𝜕𝜕𝜕𝜕

4𝑙𝑙𝑙𝑙2 � cos �(2𝑦𝑦𝑦𝑦 + 1)𝜋𝜋𝜋𝜋𝑎𝑎𝑎𝑎

2𝑙𝑙𝑙𝑙 �

A visual fit of experimentally obtained data for a 500 µm thick slab gel gives a diffusion coefficient on the order of 10-6 cm2/s, which is comparable to that of small molecules in water (10-5 cm2/s). For gels that are of reasonable thickness for 3D cell culture (~200 microns to 1 mm), this gives characteristic diffusion time scales on the order of a few minutes to a few hours.

1. Crank, J. The mathematics of diffusion, viii, 414 p. (Clarendon Press, Oxford, [Eng], 1975).



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Figure S8 Stereolithographic patterning within click-based hydrogelScale bars = 200 µm (left) and 100 µm (mid).



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Figure S8 Stereolithographic patterning within click-based hydrogelScale bars = 200 µm (left) and 100 µm (mid).

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Figure S9 Additional illustration of 2-photon patterning within click-based hydrogelFalse coloring was used to enhance reader visualization. Scale bar = 50 µm.



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Figure S10 Patterning of multiple peptides within a single gelAF488-AhxRGDSC-NH2 was first swollen into a gel at 3 mg/mL in 0.05 wt% I2959 in clear media for one hour. 365 nm light was exposed to the gel through a photomask at 10 mW/cm2 for 10 min. The gel was then placed in media for two hours to swell out any unreacted material. The process was repeated again for AF546-AhxPHSRNC-NH2 and then AF405-AhxKRTGQYKLC-NH2, the full process taking ~10 hours. The pattern was subsequently imaged using confocal microscopy. Images represent a single optical slice within the hydrogel. AF546-AhxPHSRNC-NH2 is shown in red on the top left, AF405-AhxKRTGQYKLC-NH2 in blue on the top right, and AF488-AhxRGDSC-NH2 in green on the bottom left. The overlay of all peptides is shown in the bottom right. Scale bar = 100 µm.



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Figure S10 Patterning of multiple peptides within a single gelAF488-AhxRGDSC-NH2 was first swollen into a gel at 3 mg/mL in 0.05 wt% I2959 in clear media for one hour. 365 nm light was exposed to the gel through a photomask at 10 mW/cm2 for 10 min. The gel was then placed in media for two hours to swell out any unreacted material. The process was repeated again for AF546-AhxPHSRNC-NH2 and then AF405-AhxKRTGQYKLC-NH2, the full process taking ~10 hours. The pattern was subsequently imaged using confocal microscopy. Images represent a single optical slice within the hydrogel. AF546-AhxPHSRNC-NH2 is shown in red on the top left, AF405-AhxKRTGQYKLC-NH2 in blue on the top right, and AF488-AhxRGDSC-NH2 in green on the bottom left. The overlay of all peptides is shown in the bottom right. Scale bar = 100 µm.

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Figure S11 Synthesis and 1H NMR of four-arm PEG Tetraazide (MW~10000)

Synthesis of PEG-tetraazide: 5 grams of 4-arm poly(ethylene glycol) (Mn ~ 10,000 Da) (Jenkem) was dried overnight and dissolved in a 4:1 mixture of dichloromethane:pyridine under argon at 0 °C. Methanesulfonyl chloride (1.15 g, Sigma) dissolved in minimal DCM was added dropwise over 10 minutes in a five-fold molar excess to hydroxyl groups and allowed to react overnight. The solution was concentrated, washed with sodium bicarbonate (Fisher), dried over MgSO4

(Fisher), and precipitated in diethyl ether (Fisher). 1H NMR (DMSO-d6): 4.30 (8H, m, 4 x MsOCH2), 3.67 (8H, m, 4 x MsOCH2 CH2), 3.47-3.57 (m, [CH2CH2O]n), 3.18 (12H, s, 4 x CH3SOO-). The activated mesylate was co-dissolved with 5 molar excess sodium azide (650 mg, Fluka) in 10 mL anhydrous DMF and stirred overnight under argon at 80 °C. The precipitate was removed via Celite filtration and subsequently concentrated. The product was dissolved in distilled water and dialyzed for two days and lyophilized to give the desired product. 1H NMR (CDCl3): 3.50-3.77 (m, CH2[CH2CH2O]nCH2), 3.39 (8H, m, 4 x CH2N3).

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