The RGD-containing peptide, Ser–Asp–Gly–Arg–Gly–Gly–Lys(PEG3-TAMRA)–Gly–Cys was synthesized by AnaSpec. A polyethylene glycol ×3 (PEG3) spacer linked the peptide’s lysine to the TAMRA dye. Succinimidyl ester–functionalized PA gels were prepared with Acryloyl-X SE as described above. Gels were next functionalized with amines by incubation overnight with 200 mM glutamine solutions in 50 mM Hepes buffer (pH 8) at 4°C. Functional MAL groups were introduced through MAL-dPEG2-NHS (N-hydroxysuccinimide) ester (Quanta Biodesign) coupling. PA soft gels (E = 140 Pa) were incubated in 2 mM MAL-dPEG2-NHS ester dissolved in 1:1 dimethyl sulfoxide (DMSO):PBS solution, and PA stiff gels (E = 22,000 Pa) were incubated in 0.5 mM MAL-dPEG2-NHS ester dissolved in 1:1 DMSO:PBS solution, both at room temperature for 2 hours. Last, gels were coupled with 10 μM TAMRA-RGD by incubation at room temperature for 2 hours on a shaking platform. Between each chemical treatment, the gels were rinsed with PBS. Silicone gels were functionalized by treatment with 0.95% 3-aminopropyltriethoxysilane in methanol for 5 to 10 min, followed by rinsing with methanol and water and then baking the gels at 60°C for 1 hour. The amine-functionalized gels were coupled with 5 mM MAL-dPEG2-NHS ester in 1:1 DMSO:PBS solution and then 10 μM TAMRA-RGD, as described for PA gels.

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