Nanowire cell penetration for drug delivery [electronic resource]

Introduction of biomolecules across the cell membrane with high efficiency is a challenging yet critical technique in biological and medical research. Vertically aligned nanowire (NW) arrays have been recently reported to offer new opportunities to access a cells' interior by directly breaching the cell membrane. This nanoscale physical membrane penetration circumvents conventional biochemical pathways to deliver materials into cell, and may avoid accompanying degradation routes such as endocytosis. In spite of the early success, the microscopic understanding of how and when the nanowires penetrate cell membranes is still lacking, and the degree to which actual membrane penetration occurs is controversial. In order to advance this field, in this work efforts were devoted to understand the NW cell penetration mechanism, release the structure restriction of NWs from planar substrate, and enhance the NW cell penetration efficiency. First, to elucidate the possible penetration mechanisms, a continuum elastic cell mechanics model is presented to address how penetration occurs, and explore the characteristics that affect penetration. Cell Penetration process is investigated through two mechanisms based on experimental observation, namely through "impaling" as cells land onto a bed of nanowires, and through "adhesion mediated" penetration, which occurs as cells spread on the substrate and generate adhesion force. Our results reveal that cell penetration is likely to occur only for a limited time window during cell adhesion. The penetration effects of NW geometry (radius, height and spacing) and cell properties (membrane stiffness, cell adhesion, and membrane strength) are systematically evaluated. These results provide a guide to designing nanowires for applications in cell membrane penetration. Second, NW cell penetrations are restricted to in vitro applications because the NWs are attached to a planar 2D substrate. To overcome this structure limitation, we propose suspended nanoparticles covered with NWs, namely "spiky particles", to deliver biomolecules by utilizing NWs' cell membrane penetration capability together with the natural occurring of particle engulfment by cellular phagocytosis. The NWs may assist particles penetrating cell membrane during particles engulfment, allowing direct releasing of bound biomolecules into cytosol. To explore this design, we developed suspended nanoparticles with ZnO NWs grown on them, and demonstrated the spiky particles fabrications, cell-particle interfaces, spiky particles cytotoxicity, and spiky particle-mediated DNA transfection, and used a reverse transfection approach to independently test the transfection capability of the particles' NWs. Third, notable drawbacks of solid NW-based delivery are limited dose and temporal control. In addition, NW cell penetration efficiency is low, and were unable to achieve efficient DNA plasmid transfection. A nano-electroporation platform was developed to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield co-transfection and sequential transfection.

Keywords: Materials Science and Engineering.
Advisor: Melosh Nicholas A. primary advisor. Thesis advisor
Cui Yi
Heilshorn Sarah advisor. Thesis advisor