5 aminofluorescein

RGDS-Alginate was synthesised using carbodiimide chemistry based on previous publications (Rowley et al., 1999) . Briefly, a 1% solution of alginate in 100 mM MES, pH6.8 was activated using 10 mM Sulfo-NHS (Sigma Aldrich) and 9 mM EDC (Alfa Aesar). Then, RGDS peptide (Alfa Aesar, Heysham, UK) was added (0.01% w/v or 1% of alginate mass) and the reaction stirred for 1 h. The solution was then dialysed against Milli-Q water using a 3.5 kDa MW membrane cassette (Thermo Fisher, Loughborough, UK) for 3 days with 8 water changes. After dialysis, RGDS-Alginate was precipitated by adding ethanol to 70% volume and washed several times with more ethanol. The modified alginate was then further purified by treating it with chloroform and activated charcoal. The solid was then freeze dried. The same reaction was performed for fluorescein alginate by adding 10 mM 5-aminofluorescein (Sigma Aldrich) in place of the RGDS peptide. Purity of alginate, G/M ratio and conjugation efficiency were assessed using 1H-NMR in a Bruker AVIII400 with water suppression; high power 1H pulse of 10 μs at -1.83 dB; number of scans 256 with a relaxation delay of 1 s and acquisition time of 1 s. Line broadening 1 Hz.