Cell-Division-Independent Rapid Expression of DNA Delivered with α-Synuclein–Gold Nanoparticle Conjugates

Gene delivery is a primary technology employed in diverse areas of biomedical science, from gene therapy to gene editing, cancer treatment, and stem cell research. Here, we introduce a gene delivery system utilizing an intrinsically disordered protein of α-synuclein (αS) demonstrated to interact with lipid membranes by transforming its original random structure to an α-helix. Since the helix bundle formation is a signature of cell-penetrating peptides for membrane translocation, a multitude of αS(Y136C)s replacing tyrosine at the C-terminus with cysteine were covalently attached onto gold nanoparticles (AuNPs) in a specific orientation with the helix-forming basic N-termini exposed outward. The resulting αS(Y136C)-AuNP conjugates were found to exhibit a rapid gene expression without causing cytotoxicity when the gene of the enhanced green fluorescent protein (EGFP) was delivered with the conjugates into the cells. Based on inhibition studies toward endocytosis and mitosis, the αS(Y136C)-AuNP/DNA complex was demonstrated to take both endosomal and non-endosomal intracellular transport pathways. The DNA translocation into the nucleus was independent of cell division. This nondisruptive and rapid DNA transfection by αS(Y136C)-AuNPs allowed a successful delivery of granzyme A gene leading to cellular pyroptosis. Modifications of αS(Y136C)-AuNP/DNA complex, such as antibody immobilization and replacement of DNA with biological suprastructures including RNA, protein, and nonbiological fusion materials, would allow the intracellular delivery system to be applied in diverse areas of future biotechnology.