Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

Kehai Liu^1,2,*, Xiaoyu Wang^1,*, Wei Fan¹, Qing Zhu², Jingya Yang², Jing Gao³, Shen Gao<sup>1

1</sup>Department of Pharmaceutics, Shanghai Hospital, Second Military Medical University, ²Department of Biopharmaceutics, School of Food Science and Technology, Shanghai Ocean University, ³Department of Pharmaceutics, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China

*The first two authors contributed equally to this work

Background: To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed.
Methods: First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in avß3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro.
Results: The vector showed controlled degradation, strong targeting specificity to avß3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/µg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 µg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa.
Conclusion: P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy.

Keywords: nonviral gene vector, polyethylenimine, P123, αvß3, cell-penetrating peptides