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Synthesis and application of layered nanomaterials are known as emerging field in nanotechnology. Inorganic layered nanomaterials with nanometer scale, high aspect ratios, and large-surface area generate various scientific and technological interests in several potential areas of application such as separation technology, medical sciences, chromatography drug delivery, and catalysis. In this research, naproxen was opted as a unique model of the drug to intercalate into three-dimensional layered double hydroxide (LDH) nanostructures by calcination–reconstruction method. The designed-naproxen nanostructures were synthesized and used as novel drug nanocarriers. The synthesized nanomaterial characteristics were confirmed by FTIR, XRD, and SEM. The results of antibacterial activity indicated that homemade nanostructure can inhibit bacterial growth. Furthermore, MTT assay analysis showed that synthesized-nanostructure in physiological concentration has not cytotoxicity on C2C12 myoblast cells in vitro. Subsequent to intercalation with naproxen according to calcination–reconstruction method, the basal spacing of LDH increased to 2.62 nm from primary 0.77 nm in diameter, which confirms successful intercalation of naproxen into inserted layer of LDH by bridging interaction. Also, in-vitro drug release tests in PBS (pH 7.4) showed constant release profile for naproxen. Consequently, due to the facile and low-cost fabrication of three-dimensional LDH nanostructure, it can be considered as a potential alternative for traditional and high cost drug delivery. [ABSTRACT FROM AUTHOR]