Protein moiety in oligochitosan modified vector regulates internalization mechanism and gene delivery: Polyplex characterization, intracellular trafficking and transfection

Oligochitosan-modified proteins have gained attention as efficient non-viral vectors for gene delivery. However, little information exists if protein moieties can serve as an important role for internalization and endosome escape ability of the genetic material. To explore this issue, we designed two cationic oligochitosan-modified vectors that consist of different proteins, namely a hydrophobic plant protein (zein) and a hydrophilic animal protein (ovalbumin (OVA)) to deliver pDNA to epithelial cell line CHO-K1 and HEK 293 T. These cationic vectors were systematically characterized by molecular weight, infrared (IR) structural analysis, transmission electron microscopy (TEM) morphology, and surface charge. A remarkable impact of protein moieties was observed on physiochemical properties of the developed vectors. Oligochitosan-modified zein containing hydrophobic protein exhibited high buffering capacity and excellent DNA binding ability compared to the oligochitosan-modified OVA. The data on transfection in the presence of endocytic inhibitors indicated that the caveolae-mediated pathway (CvME) played a key role in the internalization of the zein-based polyplex. However, the OVA-based polyplex was internalized in CHO-K1 cells via CvME and in HEK 293 T cells via the lipid-mediated pathway. Moreover, oligochitosan-modified zein exhibited lower cytotoxicity, greater lysosomal escape ability, better plasmid stability, and better transfection efficiency than the oligochitosan-modified OVA. This study offers a facile procedure for the synthesis of cationic vectors and elucidates the relationship that exists between protein moieties and transfection activity, thus providing an alternative, non-viral platform for the gene delivery.