Synthesis of Maleimide-Conjugated Chondroitin Sulfate

To incorporate the negatively charged chondroitin sulfate to the cell surface, maleimide-conjugated chondroitin sulfate (MCS) was synthesized using the modified version of a previously reported method [28 (link)]. Briefly, 2 g of chondroitin sulfate sodium salt (CS, cat# C0335, Tokyo Chemical Industry, Tokyo, Japan) was dissolved in 40 ml of PBS with vigorous magnetic stirring. Next, 286 mg of N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (EDC, cat# 39391, Sigma-Aldrich, St. Louis, MO, USA) and 328 mg of N-hydroxysulfosuccinimide sodium salt (NHS, cat# 56485, Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 10 ml of PBS. After the CS was completely dissolved, the mixture containing EDC and NHS was added to the CS solution and stirred for 20 min at room temperature. Then, 250 mg of N-(2-aminoethyl)maleimide trifluoroacetate salt (maleimide, cat# 56951-1G-F, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 ml of PBS, added to the mixture, and incubated for 5 h with gentle stirring at room temperature. Afterward, the solution was dialyzed with a dialysis membrane (cat#132676, Spectrum Laboratories, CA, USA) against distilled water for 4 days (distilled water was renewed every 12 h) at room temperature and freeze-dried until future use.