It is very desirable to design multifunctional nanocomposites for theranostic applications via flexible strategies. The synthesis of one new multifunctional polycationic Au nanorod (NR)‐coated Fe3O4 nanosphere (NS) hierarchical nanocomposite (Au@pDM/Fe3O4) based on the ternary assemblies of negatively charged Fe3O4 cores (Fe3O4‐PDA), polycation‐modified Au nanorods (Au NR‐pDM), and polycations is proposed. For such nanocomposites, the combined near‐infrared absorbance properties of Fe3O4‐PDA and Au NR‐pDM are applied to photoacoustic imaging and photothermal therapy. Besides, Fe3O4 and Au NR components allow the nanocomposites to serve as MRI and CT contrast agents. The prepared positively charged Au@pDM/Fe3O4 also can complex plasmid DNA into pDNA/Au@pDM/Fe3O4 and efficiently mediated gene therapy. The multifunctional applications of pDNA/Au@pDM/Fe3O4 nanocomposites in trimodal imaging and combined photothermal/gene therapy are demonstrated using a xenografted rat glioma nude mouse model. The present study demonstrates that the proper assembly of different inorganic nanoparticles and polycations is an effective strategy to construct new multifunctional theranostic systems.

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