Gene expression manipulation is pivotal in therapeutic approaches for various diseases. Non-viral delivery systems present a safer alternative to viral vectors, with reduced immunogenicity and toxicity. However, their effectiveness in promoting endosomal escape, a crucial step in gene transfer, remains limited. To address this drawback, we developed a reducible conjugate combining the StIW111C mutant of Sticholysin I, a pore-forming protein, with a polylysine peptide. This conjugate aims to enhance plasmid DNA (pDNA) release from endosomes, thereby improving gene expression. A 16-polylysine peptide was attached to StIW111C via a disulfide bridge to block its membrane-binding site, enabling controlled modulation of pore-forming activity in response to a reductive environment. This modification also enhances the conjugate's positive charge, facilitating binding to nucleic acids. Formation of positively charged nanometric complexes was achieved by mixing pDNA with the bio-responsive StIW111C conjugate and polylysine peptide. In vitro assays showed the conjugate could permeabilize endosomes, but reporter gene expression was limited, likely due to the largest complexes or aggregates that reduced conjugate entry and blocked nucleic acid release. CryoTEM imaging revealed the presence of small aggregate fraction, highlighting the need for further redesign to prevent aggregation and optimize endosomal release of non-viral systems for enhanced gene expression.