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Designed for gene therapy of chronic diseases, HBP–DEAPA 60 is a non-toxic biodegradable amine modified hyperbranched polyester. This candidate was chosen from a series of hyperbranched polymers for further characterization as it showed the best transfection efficiency and fastest degradation rate. HBP–DEAPA 60/DNA complexes were investigated with regard to stability, uptake and formation to gain a better insight into HBP–DEAPA 60/DNA complex properties. We investigated HBP–DEAPA 60/DNA complex uptake into A 549 cells by FACS and CLSM. Their stability was investigated by a heparin displacement assay as well as by DNAse I assay. Morphology was shown by AFM. HBP–DEAPA 60/DNA complex formation was further characterized in terms of thermodynamic parameters. We studied the conformation of DNA in nano-complexes via circular dichroism (CD) spectroscopy for different NP ratios. Thermodynamic studies showed that binding enthalpies were endothermic; the nano-complex formation was entropically driven. Although PEI/DNA and HBP–DEAPA 60/DNA complexes showed similar behavior with regard to uptake, heparin stability, DNA helicality and their entropically driven complex formation they differ in their binding constant Ka and in their ability to protect the DNA from DNAse. Concerning Ka and DNAse stability, HBP–DEAPA/DNA complexes should be further optimized. This shows that different characterization studies are necessary to fully characterize polyplex stability and properties.