Efficient nucleic acid delivery into target cells remains a critical challenge in gene therapy. Due to its advantages in biocompatibility and safety, recent research has increasingly focused on non-viral gene delivery. . A series of copolymers-synthesized by integrating thermally sensitive poly(N-isopropylacrylamide) (PNIPAm), hydrophilic poly(ethylene glycol) (PEG) grafts, and a polycationic poly(L-lysine) (PLL) block of varying lengths ((PNIPAm)--(PEG)--(PLL), z = 10-65)-were investigated. Plasmid DNA complexation with the copolymers was achieved through temperature-modulated methods. The resulting polyplexes were characterized by evaluating complex strength, particle size, zeta potential, plasmid DNA loading capacity, resistance to anionic stress, stability in serum, and lysosomal membrane destabilization assay. The copolymers' potential for plasmid DNA delivery was assessed through cytotoxicity and transfection studies in cancer cell lines. Across all complexation methods, the copolymers effectively condensed plasmid DNA into stable polyplexes. Particle sizes (60-90 nm) ranged with no apparent correlation to copolymer type, complexation method, or N/P ratio, whereas zeta potentials (+10-+20 mV) and resistance to polyanionic stress were dependent on the PLL length and N/P ratio. Cytotoxicity analysis revealed a direct correlation between PLL chain length and cell viability, with all copolymers demonstrating minimal cytotoxicity at concentrations required for efficient transfection. PNL-20 ((PNIPAm)--(PEG)--(PLL)) exhibited the highest transfection efficiency among the tested formulations while maintaining low cytotoxicity. The study highlights the promising potential of (PNIPAm)--(PEG)--(PLL) copolymers for effective plasmid DNA delivery to cancer cells. It reveals the importance of attaining the right balance between polyplex tightness and plasmid release to achieve improved biocompatibility and transfection efficiency.