Doxorubicin-loaded, charge reversible, folate modified HPMA copolymer conjugates for active cancer cell targeting.

Although folate exhibits many advantages over other targeting ligands, it has one major defect: poor water solubility. Once it was conjugated to hydrophilic drug carrier such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, the hydrophobic folate may be buried inside the random polymer coil and not exposed to be accessible to its receptor on the cell surface, thus losing its active targeting ability. To address this folate dilemma, the positive charge was introduced in the present study. The obtained cationic folate-functionalized HPMA copolymers exhibited a synergistic enhancing effect on cellular uptake by folate receptor (FR) positive Hela cells via electrostatic absorptive endocytosis and folate receptor-mediated endocytosis, with the involvement of multiple internalization pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and energy-dependent endocytosis. As demonstrated in binding efficiency study, the FR antibody bound to 71.2% of tested cells in the competition with neutral folate modified HPMA copolymers, while the FR antibody-bounded cells decreased to only 34.0% in competition with cationic folate modified HPMA copolymers, indicating that the positively charge could probably amplify the binding efficiency of folate to its receptor due to close proximity of the conjugates to the cell surface by the electronic adhesion. In addition, the cell uptake study on FR negative A549 cells also confirmed the specific role of folate as targeting ligand. Then, to avoid non-specific binding by positive charge in the circulation, the charge shielding/deshielding approach was further employed. With selective hydrolysis of the charge shielding groups 2,3-dimethylmaleic anhydride (DMA) at tumor extracellular pH 6.8, the conjugates underwent a quick charge-reversible process with more than 80% DMA cleavage within 2 h and endocytosed into the endo/lysosomes much more rapidly than at physiological pH 7.4. And then the drug release was triggered by the cleavage of hydrazone spacer at another level of pH 5 in endo/lysosomal compartment. Furthermore, the anticancer activity results showed that Dox-loaded, charge-switchable, folate modified HPMA copolymer conjugates could indeed lead to enhanced cytotoxicity, stronger apoptosis and greater tumor spheroid inhibition towards Hela cells, indicating the great potential feasibility of this multiple responsive drug delivery system.