Improved biocompatibility of dendrimer-based gene delivery by histidine-modified nuclear localization signals

Polyamidoamine (PAMAM) dendrimers have been explored as an alternative to polyethylenimine (PEI) as a gene delivery carrier because of their relatively low cytotoxicity and excellent biocompatibility. The transfection efficiency of PAMAM dendrimers can be improved by the addition of nuclear localization signal (NLS), a positively charged peptide sequence recognized by cargo proteins in the cytoplasm for nuclear transport. However, increased positive charges from NLS can cause damage to the cytoplasmic and mitochondrial membranes and lead to reactive oxygen species (ROS)-induced cytotoxicity. This negative effect of NLS can be negated without a significant reduction in transfection efficiency by adding histidine, an essential amino acid known as a natural antioxidant, to NLS. However, little is known about the exact mechanism by which histidine reduces cytotoxicity of NLS-modified dendrimers. In this study, we selected cystamine core PAMAM dendrimer generation 2 (cPG2) and conjugated it with NLS derived from Merkel cell polyomavirus large T antigen and histidine (n = 0-3) to improve transfection efficiency and reduce cytoxicity. NLS-modified cPG2 derivatives showed similar or higher transfection efficiency than PEI 25 kDa in NIH3T3 and human mesenchymal stem cells (hMSC). The cytotoxicity of NLS-modified cPG2 derivatives was substantially lower than PEI 25 kDa and was further reduced as the number of histidine in NLS increased. To understand the mechanism of cytoprotective effect of histidine-conjugated NLS, we examined ROS scavenging, hydroxyl radical generation and mitochondrial membrane potential as a function of the number of histidine in NLS. As the number of hisidine increased, cPG2 scavenged ROS more effectively as evidenced by the hydroxyl radical antioxidant capacity (HORAC) assay. This was consistent with the reduced intracellular hydroxyl radical concentration measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) assay in NIH3T3. Finally, fluorescence imaging with JC-1 confirmed that the mitochondrial membranes of NIH 3T3 were well-protected during the transfection when NLS contained histidine. These experimental results confirm the hypothesis that histidine residues scavenge ROS that is generated during the transfection process, preventing the excessive damage to mitochondrial membranes, leading to reduced cytotoxicity.