Introduction:Electrotransfection (ET) is a non-viral approach widely used for delivery of naked nucleic acids. Its efficiency can be increasedin vitroby treatment of cells with various small molecule enhancers. However, these enhancers often fail to improve ETin vivo, presumably due to rapid clearance in tissues after local injection, reducing their cellular uptake. To this end, we propose to develop a new type of ET enhancers, which we term nanoenhancer, that diffuse slowly in tissues and are poorly absorbed by blood and lymph microvessels.Methods:Two nanoenhancers were synthesized with alginate (Alg) and chitosan (Chi) with or without poly (ethylene imine) (PEI). They were used to treat cellsin vitroor mouse muscle in the hind legin vivoprior to ET of plasmid DNA coding reporter genes. At 24 hours post ET, the efficiency of ET was quantified, and compared with that in the untreated controls. Changes in lysosomal size and acidity post nanoenhancer treatment were measured with fluorescence microscopy techniques.Results and discussion:We observed that the pretreatment of cells with the nanoenhancers could enhance the ET efficiency and cell viability in both C2C12 and HCT116 cellsin vitro, and the nanoenhancer pretreatment had similar effects on the ET efficiencyin vivo. Mechanisms of the enhancement were related to transient inactivation of lysosomal functions triggered by the nanoenhancer treatment. The concept of nanoenhancer will lead to development of new enhancers that can be used to improve ET efficiencyin vivo, highlighting its potential in clinical applications.