Comparative studies of polyethylenimine-doxorubicin conjugates with pH-sensitive and pH-insensitive linkers

Doxorubicin (DOX) was conjugated to polyethylenimine (PEI) in several synthetic ways and created PEIsuccinic anhydride (SUC)DOX, PEIN-succinimidyl 3-maleimidopropionate (SMP)DOX and PEI3-maleimidopropionic acid hydrazide (MPH)DOX conjugates using pH-insensitive linkage (amide bond) or pH-sensitive linkage (hydrazone bond). The effects of the conjugates on anticancer therapy against human breast cancer cells were investigated for in vitro release under different pH, cellular uptake, intracellular localization and antiproliferative activity. DOX release from PEIMPHDOX formed via hydrazone bonds occurred in an acid-triggered manner and increased with decreasing pH. But PEISUCDOX and PEISMPDOX formed via amide bonds were quite stable and released negligible DOX under both neutral and acidic conditions. The results from flow cytometry and confocal laser scanning microscopy analysis showed that conjugates uptakes were PEISUCDOX > PEISMPDOX > PEIMPHDOX, which was consistent with the primary amine content and more efficient than free DOX due to the intense nonspecific interaction between the positively charged conjugates and the negatively charged cell surface proteoglycans. PEIMPHDOX showed higher antiproliferative activity than that of PEISUCDOX and PEISMPDOX. The antiproliferative activity of PEIMPHDOX was achieved through the endocytosis, nuclear entry of released DOX and interaction with the DNA. However, PEISUCDOX and PEISMPDOX showed more endocytosis but less nuclear entry leading to lower antiproliferative activity. According to the measurements, we determined that enhanced endocytosis and pH-sensitive drug release were the critical processes in determining anticancer therapy of drugpolymer conjugate. The PEIMPHDOX conjugate could be sifted to use as a prodrug for cancer therapy and also as a novel delivery carrier to codelivery DOX and gene in further research. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.