Xuan Liu

Date of Award

Spring 5-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Science and Nanotechnology

First Advisor

Shengnian Wang

Abstract

Emerging as an effective option, immunotherapy is gradually being recognized by the cancer treatment community. However, due to its wide use of viral vectors for gene delivery, many limitations and safety concerns impede its adoption in clinical practice. This promotes the emergence of many non-viral gene delivery methods including the use of inorganic or organic nanoparticles to carry and deliver genetic probes as complexes through chemical mechanisms and the use of mechanical or electrical forces to physically breakdown delivery barriers of the cell membrane to allow quick probe internalization. Compared to the former (chemical methods), the latter (physical methods) shows advantages in fast and efficient delivery performance, among which electroporation has attracted the most attention in the past two decades. Electroporation designates the use of short, high-voltage pulses to make the subjected cell membrane permeable. It has many advantages like simple operation, quick delivery, non-restrictions on gene or cell types, and low cost. However, compared to the viral delivery method, electroporation still faces great challenges in terms of transfection efficiency. To solve these problems, we developed a carbon nanotube (CNT) based micropillar array electrode (MAE) structure and nanosecond electroporation pulse schemes to improve the overall transfection performance of electroporation technology. The new carbon nanotube micropillar array electrodes are used to replace the traditional aluminum plate electrodes. The superior conductivity of CNTs on the micropillar surface makes the new electrode as good as their traditional counterparts in terms of conductance while its inert surface avoids undesired electrochemical reactions and the release of Al3+ ions in the cell buffered solution which are believed to be toxic to cells. More important, the epoxy base of the micropillars helps partially embed those CNTs and fix them permanently on the electrode surface while the exposed portion of CNTs serves as separated nanoelectrodes on the micropillar surface to focus the electric pulse strength. This allows cells of different sizes to receive uniform treatment during electroporation, resulting in significant improvement in the transfection efficiency of genetic probes. Besides the CNT nanoelectrodes, we also applied nanosecond pulses to further improve the transgene expression of electroporation by deeper penetration than the traditional/standard millisecond electrical pulses. Under those nanosecond electrical pulses, not only does the cell membrane becomes more fragile but also the cell nuclear membrane becomes permeable. This allows genetic probes to enter the cell nucleus more quickly to start the transcription process eventually speeding up the overall transfection kinetics.

In this project, leukemia cells and carcinoma cells were used as the representative suspension and adherent cell models respectively to demonstrate these two new electroporation technologies. Plasmids encoding pMaxGFP and small interfering RNA that precisely silence GFP translation were used as large and small genetic probe representatives to test their delivery effectiveness. We found that carbon nanotube micropillar array electrodes can achieve three times or more transfection efficiency than the commercial electroporation counterparts without sacrificing cell viability. Combining nanosecond and millisecond pulse scheme, the needed transgene expression time was shortened by nearly 20 hours, demonstrating with GFP plasmids. These new electroporation technologies were further applied to treat model blood cells by mixing primary blood cells with K562 cells at various ratios. The results show that our nano electroporation system could double the transfection efficiency compared to the commercial electroporation system for both plasmid and therapeutic microRNA, miR29b. The targeted protein level of miR-29b is further downregulated 20-25% from what is achieved in commercial electroporation systems. Its success might further promote the wide adoption of electroporation technology in cell immunotherapy.

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