Cell squeezing : a vector-free microfluidic platform for intracellular delivery of macromolecules
| Autor: | Sharei, Armon R. (Armon Reza) |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: | |
| Druh dokumentu: | Diplomová práce |
| Popis: | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. "June 2016." Cataloged from PDF version of thesis. Includes bibliographical references (p. 158-165). Intracellular delivery of material is a long-standing challenge for both therapeutic and research applications. Existing technologies rely on a variety of mechanisms to facilitate delivery. Vector-based methods, such as polymeric nanoparticles and liposomes, form complexes with the target material and subsequently facilitate its uptake by the cell of interest, often through endocytosis. Although effective in some applications, these methods have had difficulty translating to patient-derived primary cells, especially stem cells and immune cells. Moreover, these vectors are often limited in the range of target materials they can deliver and leave much material trapped in endocytotic vesicles. Physical methods, such as electroporation and sonoporation, have been able to address some of the challenges with vector-based methods by providing a platform for physical disruption of the cell membrane. By eliminating the need for vector materials and circumventing the endocytotic pathway, these methods have shown an advantage in some applications, especially those involving primary cells that are recalcitrant to vector-based methods. However, both electroporation and sonoporation suffer from high cell toxicity and have had limited success in delivering materials such as proteins and nanomaterials. Electroporation in particular has been shown to damage certain target materials, such as quantum dots. Microinjection, an alternative method in which cells are punctured by a microneedle, can address a variety of target materials and cell types however its low throughput has hindered its adoption for most applications. There is thus a need for more effective intracellular delivery methods. This dissertation describes a microfluidic approach to intracellular delivery that seeks to embody the advantages of a physical method, while mitigating issues related to toxicity and damage to the target material. In our method, the cells of interest are prepared in suspension with the target delivery material and flown through a parallel network of microfluidic channels. Each channel contains a constriction point where the cells are rapidly deformed, or squeezed, as they pass through. This process induces temporary disruption of the cell membrane thereby enabling diffusive transport of material from the surrounding buffer into the cell cytosol. These disruptions persist for less than 5min before membrane integrity is fully restored. This method has thus far been demonstrated in over 15 cell types and has been able to deliver a variety of functional materials including, DNA, RNA, proteins, quantum dots, carbon nanotubes, and small molecules. Our cell squeezing technology has further illustrated its enabling potential in a number of applications detailed herein. Quantum dots are a promising alternative to organic fluorescent dyes due to their superior spectral properties and stability. These nanoparticles have much potential as imaging agents in vitro and in vivo. Delivery of undamaged quantum dots to the cell cytoplasm has been a challenge with existing techniques. Vector-based methods have resulted in aggregation and endosomal sequestration of quantum dots while electroporation can damage the semi-conducting particles and aggregate delivered dots in the cytosol. In our work, we demonstrated efficient cytosolic delivery of quantum dots without inducing aggregation, trapping material in endosomes, or significant loss of cell viability. Moreover, we have shown that individual quantum dots delivered by this approach are detectable in the cell cytosol, thus illustrating the potential of this technique for single molecule tracking studies. These results indicate that our method could potentially be implemented as a robust platform for quantum dot based imaging in a variety of applications. The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has much potential in its ability to address existing challenges in regenerative medicine by providing a patient-specific source of pluripotent stem cells to generate new tissue. The mechanism of this reprogramming process, however, is still poorly understood and existing technologies suffer from chronically low reprogramming efficiencies by Armon R. Sharei. Ph.D. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|