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An increasing number of non-viral vectors are being developed for the use of gene delivery nowadays, among which cationic polymers and lipoplexes receive most attention. Most of these researches are focused on how to increase the transfection efficiency of non-viral vectors as well as the reduction of toxicity. In this review, we go over new strategies to reduce the toxicity of cationic polyplexes such as poly(ethylene-imine) and the construction of highly effective gene transfer vector lipoplexes. In addition, since transformation of gene expression system from two-dimensional (2D) substrate to 3D scaffold triggers far better transfection efficiency, the non-viral vectors applied in 3D transfection system have also been reviewed.