Co-assembled Ca2+ Alginate-Sulfate Nanoparticles for Intracellular Plasmid DNA Delivery

Matan Goldshtein; Stav Shamir; Ekaterina Vinogradov; Alon Monsonego; Smadar Cohen

doi:10.1016/j.omtn.2019.03.006

Co-assembled Ca²⁺ Alginate-Sulfate Nanoparticles for Intracellular Plasmid DNA Delivery

Mol Ther Nucleic Acids. 2019 Jun 7:16:378-390. doi: 10.1016/j.omtn.2019.03.006. Epub 2019 Mar 28.

Authors

Matan Goldshtein¹, Stav Shamir¹, Ekaterina Vinogradov², Alon Monsonego³, Smadar Cohen⁴

Affiliations

¹ Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
² Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, The National Institute of Biotechnology in the Negev, and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
³ Shraga Segal Department of Microbiology, Immunology, and Genetics, Faculty of Health Sciences, The National Institute of Biotechnology in the Negev, and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel; Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel.
⁴ Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel; Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel; The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel. Electronic address: scohen@bgu.ac.il.

Abstract

Successful gene therapy requires the development of suitable carriers for the selective and efficient delivery of genes to specific target cells, with minimal toxicity. In this work, we present a non-viral vector for gene delivery composed of biocompatible materials, CaCl₂, plasmid DNA and the semi-synthetic anionic biopolymer alginate sulfate (AlgS), which spontaneously co-assembled to form nanoparticles (NPs). The NPs were characterized with a slightly anionic surface charge (Zeta potential [ζ] = -14 mV), an average size of 270 nm, and their suspension was stable for several days with no aggregation. X-ray photoelectron spectroscopy (XPS) validated their ternary composition, and it elucidated the molecular interactions among Ca²⁺, the plasmid DNA, and the AlgS. Efficient cellular uptake (>80%), associated with potent GFP gene expression (22%-35%), was observed across multiple cell types: primary rat neonatal cardiac fibroblasts, human breast cancer cell line, and human hepatocellular carcinoma cells. The uptake mechanism of the NPs was studied using imaging flow cytometry and shown to be via active, clathrin-mediated endocytosis, as chemical inhibition of this pathway significantly reduced EGFP expression. The NPs were cytocompatible and did not activate the T lymphocytes in human peripheral blood mononuclear cells. Proof of concept for the efficacy of these NPs as a carrier in cancer gene therapy was demonstrated for Diphtheria Toxin Fragment A (DT-A), resulting in abrogation of protein synthesis and cell death in the human breast cancer cell line. Collectively, our results show that the developed AlgS-Ca²⁺-plasmid DNA (pDNA) NPs may be used as an effective non-viral carrier for pDNA.

Keywords: alginate sulfate; calcium; gene therapy; plasmid DNA; polymeric nanoparticles.