AbstractPrimary ovine fibroblast (POF) is the preferred cell line in biotechnology fields such as cloning, cancer treatment, rare diseases treatment, and gene therapy. It is difficult to achieve high transfection efficiency due to its notable resistance. The aim of this study is to develop and evaluate a stable and biocompatible nanosuspensions based on Zn/Al–NO3 layered double hydroxides (nLDHs) as inorganic gene carrier to be used for the transfection on POF cell lines, for the first time. For this purpose, the synthesis optimization, structural and physicochemical characterizations, in vitro studies including gene binding capacity, cytotoxicity, and transfection efficiency of the nLDHs were realized and evaluated their transfection efficiencies on POF and standard cell lines HEK293T. The stable nanosuspensions produced at different Zn/Al mole ratios (nLDH2:1 and nLDH3:1) with favorable characteristics were fabricated using two step-synthesis method by varying the reaction parameters. nLDH2:1 and nLDH3:1 with particle sizes smaller than 150 nm, narrow size distributions, and zeta potential of ∼70 mV have the acceptable characteristics required for transfection in terms of physicochemical properties, stability, cytotoxicity, and gene binding capacity. The highest transfection efficiencies of nLDH2:1 and nLDH3:1 at nLDH:pEGFN1 (w/w) ratio 50:1 for HEK293T cell lines were obtained as 75%±5.8% and 70%±7.1%, respectively. The transfection efficiency of nLDH2:1 (51.5%) for POF cell lines was obtained very close to that of lipofectamine (52.7%) which is a widely used commercial transfection agent. This study proved that nLDH2:1 and nLDH3:1 could be suggested as effective and safe inorganic gene carriers to be used in nanobiotechnology. [ABSTRACT FROM AUTHOR]