• functional poly(ethylene oxide);
  • methotrexate;
  • macromolecular drug;
  • folate;
  • drug delivery


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.