SYNTHESIS AND CHARACTERIZATION OF A CROSSLINKING POLYETHYLENIMINE AS SMART GENE CARRIER AND EFFECTS OF PEGYLATION DEGREE

Abstract

A smart biodegradable cationic polymer (CBA-PEI) based on the disulfide bond-containing cross-linker cystamine bisacrylamide (CBA) and low molecular weight branched polyethylenimine (1800-Da, PEI1800) was successfully synthesized by Michael addition reaction in our recent study. Furthermore, a series of copolymers (CBA-PEI-PEG) with different PEGylation degree were obtained by the mPEG-SPA (5000-Da) reacting with CBA-PEI at various weight ratios directly. The molecular structures of the resulting polymers CBA-PEI and CBA-PEI-PEG were evaluated by nuclear magnetic resonance spectroscopy ((1)H-NMR) and capillary viscosity measurements, all of which had successfully verified formation of the copolymers. The polymer/DNA complexes based on CBA-PEI and CBA-PEI-PEG were measured by dynamic light scattering and gel retardation assay. The results showed that the particle size and zeta potential of complexes were reduced with increasing amount of PEG grafting, even no particle formation. The particle size of CBA-PEI/DNA complexes was in range of 103.1 to 129.1 nm, and the zeta potential was in range of 14.2 to 24.3 mV above the 2:1 weight ratio. In the same measure condition, the particle size of CBA-PEI-PEG complexes was reduced to a range of 32.2 to 55 nm, and the zeta potential was in range of 9.3 to 13.8 mV at the 2:1 weight ratio. In the gel retardation assay, CBA-PEI showed a strong DNA-binding capability. Plasmid DNA was completely retarded at the 0.1:1 weight ratio. By contrast, CBA-PEI-PEG which consists of PEG/CBA-PEI (W/W, 38.9%) or PEG/CBA-PEI (W/W, 95.8%) displayed a poor retardation effect even at the high weight ratio. PEG-grafted showed a shield effect in polymer/DNA complexes. However, it showed the reduced DNA binding ability if the cationic polymers were modified with too much PEGs. Moreover, the CBA-PEI and CBA-PEI-PEG were evaluated in CHO and KB cells for their cytotoxicity and transfection efficiency. MTT assay revealed that both CBA-PEI and CBA-PEI-PEG had very lower cytotoxicity in CHO cell lines compared with PE125k. Although CBA-PEI showed 4 times higher transfection efficiencies compared with PE125k, the transfection efficiency of CBA-PEI-PEG was reduced obviously with increasing amount of PEG grafting.