Synthesis and Characterization of RGD- and PEG-modified Alginate

Sodium alginate rich in guluronic acid blocks and with a high molecular weight (280 kDa, LF20/40) was purchased from FMC Biopolymer, and was prepared as has been described previously^{3 (link)}. Briefly, high molecular weight alginate was irradiated by a 3 or 8 Mrad Cobalt source to produce lower molecular weight alginates. RGD-alginate was prepared by coupling the oligopeptide GGGGRGDSP (Peptides International) to the alginate using carbodiimide chemistry at concentrations such that 2 or 20 RGD peptides were coupled to 1 alginate chain on average for high molecular weight alginate (peptide molar concentrations in low molecular weight alginates were kept the same according to high molecular weight alginate for each degree of substitution, respectively). For FRET experiments, either GGGGRGDASSK(carboxyfluorescein)Y or GGGGRGDASSK(Carboxytetramethylrhodamine)Y were used instead of standard RGD peptide sequence, and were coupled at a concentration of 2 peptides per alginate chain on average for high molecular weight alginate (peptide molar concentrations in low molecular weight alginates were kept the same according to high molecular weight alginate). The coupling efficiency using this procedure was previously characterized using ¹²⁵I labeled RGD peptides^{3 (link)}. These correspond to densities of 150 μM and 1500 μM RGD in a 2% wt/vol alginate gel. Alginate was dialyzed against deionized water for 2–3 days (molecular weight cutoff of 3.5 kDa), treated with activated charcoal, sterile filtered, lyophilized, and then reconstituted in serum free DMEM (Life Technologies).
Polyethylene glycol (PEG)-alginate was prepared by coupling PEG-amine (5 kDa, Laysan Bio) to the low molecular weight alginate (35 kDa) using carbodiimide chemistry with a similar procedure to the RGD coupling^{3 (link)}. In brief, 295 mg of PEG-amine was mixed with 50 mL of 10 mg/mL alginate in 0.1 M MES (2-(N-morpholino)ethanesulfonic acid, Sigma-Aldrich) buffer at pH 6.5. Then 242 mg of EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, Sigma-Aldrich) and 137 mg of Sulfo-NHS (N-hydroxysulfosuccinimide, Thermo Fisher Scientific) were added into the solution. The reaction was carried out for 20 hours under constant stirring. The product was dialyzed against deionized water for 3 days (molecular weight cutoff of 10 kDa), filtered with activated charcoal, sterile filtered, and lyophilized. The structure of the PEG-alginate was confirmed with nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). Based on the change of molecular weight of alginate before and after PEG coupling (from 35 kDa to 45 kDa), an average of 2 PEG molecules were coupled to 1 alginate chain. This number was confirmed by ¹H NMR spectroscopy (Supplementary Fig. S14).