UC San Diego

Non-mesoporous Janus silica nanobowls (NBs) are unique in that they possess two different non-porous 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. In this work, we have demonstrated that such silica NBs can be used for in vitro (cell lines and primary neurons) and ex vivo transfections (dorsal root ganglia, DRG). We have shown that the NB system can be effectively used for supercoiled as well as linearized DNA loading and delivery in cells with dose-modulated protein expression efficiencies in a cell line dependent manner. NBs were shown to successfully internalize in vitro (cell lines and neurons), ex vivo (rat DRG) and in vivo (rat DRG and salivary gland) with high cell viability. This work also demonstrates the use of lipid encapsulation on the silica NBs to overcome vesicular entrapment following endocytosis, which is a well-known bottleneck to non-viral nano delivery systems.

After the characterization and optimization of the lipid encapsulated silica NB system (LNB), four different gene delivery applications show its versatility. First, controlled release of linearized DNA was demonstrated with NBs in the presence of reducing agents and their relative protein expression efficiencies were compared with other constructs i.e. linearized and supercoiled. Second, the LNB system was used to transfect neurons and glia in the DRG of rats in vitro or ex vivo. Thirdly, the LNB system was used to co-deliver three different constructs simultaneously to functionally reconstruct the coupling of two membrane proteins relevant in opioid-dose response in the nervous system. G-protein coupled opioid receptors and G-protein coupled inversely rectifying K+ channels (GIRKs) were functionally co-expressed by LNB transfection in HEK cell line and demonstrated as an in vitro model for testing potent opioids like fentanyl and oxycodone with a high throughput membrane potential assay. Finally, the LNB system was further modified with SPIONS and explored as a dexamethasone/silencing RNA co-delivery platform relevant in therapeutic interventions in cancer and infectious diseases like Covid19.

Overall, these results lay the foundation for the use and further development of silica NBs as non-viral transfection agents.