Polyethylenimine-conjugated Hydroxyethyl Cellulose for Doxorubicin/Bcl-2 siRNA Co-delivery Systems

Abstract

Hydroxyethyl cellulose (HEC), widely known for its biocompatibility and water solubility, is a potential material for pharmaceutical use, especially in delivery systems. Here, we synthesized polyethylenimine (PEI)-conjugated hydroxyethyl cellulose (HECP2k) and evaluated HECP2k as a novel gene/drug carrier. HECP2k was synthesized by reductive amination with PEI and oxidized HEC. Using 1H NMR, the molar amounts of primary amine were calculated to 2.7, 5.2, and 7.0 mmole/g for HECP2k 1X, 5X, and 10X, respectively. Also, by primary amine quantification, the molar amounts of primary amine were double-checked to 1.48, 2.13, and 3.86 mmole/g for HECP2k 1X, 5X, and 10X. The weight average molecular weights were measured as 13.92, 9.38, and 7.27 kDa for HECP2k 1X, 5X, and 10X, respectively. pDNA condensing ability of HECP2k increased as the PEI conjugation ratio increased. HECP2k/pDNA polyplexes retained the size of 100–300 nm while their zeta-potential was about 30 mV. Although HECP2k 1X showed a bit of cytotoxicity, HECP2k 5X and 10X showed above 90% of relative cell viability. In the transfection test, HECP2k 10X/pDNA polyplex with the optimized weight ratio exhibited 35–127 times enhanced efficiency than the positive control in serum-free condition, and 9-19 times enhanced efficiency in serum condition. HECP2k 10X was optimized to the experimental group (HECP2k) for the following experiments. Flow cytometry analysis and Confocal laser scanning microscopy (CLSM) images verified the superior cellular uptake and intracellular trafficking behavior of HECP2k. In addition, HECP2k had excellent serum stability compared to PEI25k. The drug loading content and the drug loading efficiency of HECP2k@Dox were 13.83% and 41.49%, respectively. The Z-average sizes and zeta-potentials of HECP2k/siRNA and HECP2k@Dox/siRNA were 153.7 nm, 14.67 mV, and 241.3 nm, 16.97 mV, respectively. In contrast to HeLa cells, HECP2k@Dox/siRNA nanocomplexes caused synergistically enhanced antitumor effect to HepG2 cells. HeLa cells were almost killed even at low concentration treatment of doxorubicin, but HepG2 cells showed above 60% of viability even at high concentration of Dox treated. However, when treated with HEPC2k@Dox/siRNA, the cell viability decreased by less than 50% in HepG2 cells. To estimate the synergistic effect of Bcl-2 siRNA/Dox co delivery, a median effect analysis was conducted. By Annexin V staining analysis confirmed that HECP2k@Dox/siRNA caused cell death by inducing apoptosis. Consequently, HECP2k can be considered a promising Bcl-2 siRNA/Dox co delivery carrier due to its high serum stability, great gene transfection efficiency, and improved antitumor effects.