Arginine and Histidine-modified Layered Double Hydroxides Facilitate Transgene Expression in Cancer Cells in Vitro

Abstract

Layered double hydroxides (LDHs) have interesting properties and structures that enable them to carry nucleic acids, such as deoxyribonucleic acid (DNA). This study synthesized LDHs using the co-precipitation method and functionalized with the amino acids arginine (Arg) and histidine (His) to promote proton-sponge activity for enhanced transgene expression. The LDHs were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). The interaction of the LDHs with the reporter gene plasmid DNA (<em>pCMV-Luc DNA</em>) was determined using agarose gel electrophoresis. Cytotoxicity and transgene expression was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) and luciferase reporter gene assay in the human embryonic kidney (HEK293), colorectal carcinoma (Caco-2) and hepatocellular carcinoma (HepG2) cells. The DNA: LDH complexes were relatively non-cytotoxic to all cells, and the highest transgene expression was achieved in the HEK293 cells exhibiting the most significant degree of transfection, followed by the Caco-2 cells. The His-LDH complexes displayed more than a two-fold increase in transfection than the Arg-LDHs, especially in the HEK293 cells at the optimal binding ratio. The non-functionalized LDHs demonstrated high transfection, which exceeded that of the His-LDH and Arg-LDH by 20% and 30%, respectively, in the Caco-2 cells. Little difference was noted in the HepG2 cells, which presented with the lowest transfection. These LDHs have demonstrated the potential to bind, protect, and efficiently deliver pDNA <em>in vitro</em>.