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 In Vitro Single Cell Injection
In this project we apply nanoscale tools to address challenges in cell biology and therapeutic development. We leverage a core technology from our Probe-Based Nanomanufacturing efforts, the Nanofountain Probe (NFP), to study nanomaterial-mediated drug delivery, cellular pathways, gene expression, and toxicity.
Using the NFP, we have demonstrated two distinct modes of delivery for single cell interrogation:
In the first mode of delivery, bioagents (e.g., drugs, functional nanoparticles, or biomolecules) are patterned directly on glass substrates using the NFP [1]. Cells are then cultured on the patterned substrates and their response to a given dose observed. The sub-100-nanometer patterning resolution of the NFP enables extremely precise spatial control over dosing. For example, each dot feature contains approximately 10-24 grams of drug. This technique is currently being employed in dosing studies to determine, for example, the minimum required chemotherapy dose to achieve a desired efficacy, and has broader applications in nanomanufacturing of implantable drug delivery devices [2]. In the second mode of delivery, we take advantage of the unique tip geometry of the NFP to inject nanomaterials directly into live cells (Figure 1) with minimal invasiveness [1,2]. This enables unique studies of nanoparticle-mediated delivery, as well as cellular pathways and toxicity. Whereas typical in vitro studies are limited to cell populations, these broadly-applicable tools enable multifaceted interrogation at a truly single cell level. As an initial demonstration, fluorescently-labeled diamond nanoparticles were injected directly into live cells (Figure 2) and their diffusion coefficient within the cells characterized [1]. This ability was demonstrated on multiple cancerous and wild type cell lines. The technique is now being applied for cellular pathway, toxicity, and rescue investigations, as well as intercellular particle mobility studies. For example, by targeting and injecting an individual cell and observing how its neighbors are affected, we can investigate cellular pathways or the intercellular signaling as a result of the injected agents. Research and development of therapeutics can also be greatly aided by tools capable of reliably transfecting individual cells in a culture. In a typical “rescue” experiment, a diseased state is first induced in vitro. The efficacy of a given therapeutic can then be tested based on its ability to “rescue” cells back to their normal state. Here the NFP can be used to target and inject individual cells in the culture with a given therapeutic. The effectiveness of this therapeutic and/or the ability of the rescued cell to rescue its neighbors can then be assessed by observing the proliferation (or lack thereof) of rescued cells. Similarly, the NFP can be used to induce a diseased state in a culture of primarily wild type cells.
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