Water soluble poly(ethylene oxide)-graft-methotrexate (PEO-g-MTX) conjugates with a robust amide linkage via the amine or carboxylic acid groups of MTX were designed,prepared and investigated for in vitro anti-tumor effects.MTX was conjugated to multi-functional PEO containing multiple pendant carboxylic acid (PEO-g-COOH)or amine groups (PEO-g-NH2) via the carbodiimide chemistry,which afforded PEO-g-MTX conjugates with an amide bond to the aminopteridine ring or carboxylic acid groups of MTX (denoted as PEO-g-MTX(COOH) and PEO-g-MTX(NH2),respectively).Dynamic light scattering (DLS) revealed that all PEO-g-MTX conjugates,with MTX contents varying from 4.8 to 19.6 wt％,existed as unimers in phosphate buffer (PB,pH 7.4,20 mmol·L-1).Interestingly,MTT assays showed that PEO-g-MTX(COOH) exhibited potent anti-tumor activity in HeLa,A549,KB and NIH3T3 cells with cytotoxicity profiles comparable to that of free MTX.In contrast,PEO-g-MTX(NH2)revealed diminishing cytostatic effect with IC50 (half maximal inhibitory concentration) ten to hundred times higher than that of PEO-g-MTX(COOH).Moreover,PEO-g-MTX(COOH) conjugates allowed facile conjugation with targeting ligands.Notably,folate-decorated PEO-g-MTX(COOH) macromolecular drugs showed apparent targetability to folate receptor-overexpressing KB cells with an IC50 over 12-fold lower than non-targeting PEO-g-MTX-(COOH) control and about 2-fold lower than free MTX under otherwise the same conditions.
