Nonmesoporous Janus silica nanobowls (NBs) are unique in that they possess two different nonporous surfaces per particle for loading biological molecules and can thus be designed with multifunctional properties. Although silica NBs have been successfully employed for both targeted therapeutic and diagnostic applications, their ability to deliver DNA has not yet been fully explored. The purpose of this study was to design and develop an in vitro transfection agent that would exploit the distinct characteristics of the silica NB. First, we determined that the NB surface can be linked to either supercoiled cDNA plasmids or vectorless, linear cDNA constructs. Additionally, the linearized cDNA can be functionalized and chemisorbed on NBs to obtain a controlled release. Second, the successful transfection of cells studied was dependent on lipid coating of the NB (LNBs). Although both NBs and LNBs were capable of undergoing endocytosis, NBs appeared to remain within vesicles as shown by transmission electron microscopy (TEM). Third, fluorescence microscopy and Western blotting assays revealed that transfection of four different cell lines and acutely isolated rat sensory neurons with LNBs loaded with either linear or supercoiled cDNA constructs coding for the fluorescent protein, clover and tdTomato, resulted in protein expression. Fourth, two separate opioid receptor-ion channel signaling pathways were functionally reconstituted in HEK cells transfected with LNBs loaded with three separate cDNA constructs. Overall, these results lay the foundation for the use and further development of LNBs as in vitro transfection agents.