The Nanoinjector Lance Array has been developed to inject foreign material into thousands of cells at once using electrophoresis to attract and repel particles to and from the electrically-charged lances. A mathematical computer model simulating the motion of attracted or repelled proteins informs the design of the nanoinjection lance array system. The model is validated by accurately predicting protein velocity in electrophoresis experiments. A complete analysis of parameters is conducted via simulations and specific research questions regarding the counter electrode of the nanoinjector lance array system are explored using the model. A novel technique for fabricating lance arrays from collapsed carbon nanotube forests is explored and detailed. Experiments are conducted using the Nanoinjector Lance Array, attempting to inject three different kinds of protein molecules into a culture of HeLa cells. The experimental results are encouraging and suggest possibilities for future success. Other recommendations are made for future research regarding the model, carbon nanotube fabrication, and experimental testing.

The ability to deliver sequences of DNA and other molecular loads across the membrane of a cell and into its nucleus is an area of interest in the medical community. One of its many applications is that of gene therapy. In contrast to other forms of treatment, gene therapy seeks to treat diseases at the cellular level. The success of these treatments depends on the technologies for cell transfection that are available. Physical methods are sometimes able to overcome poor efficiencies of chemical methods and the safety concerns of viral methods, but are usually impractical due to the limited number of cells that are able to be transfected at a time, isolation, and immobilization of the cells. Nanoinjection is capable of using millions of small lances in an array to inject hundreds of thousands of cells simultaneously with relatively high efficiencies and viabilities. The solid nature of the lances also allows them to be smaller than their hollow-needle counterparts, which results in higher cell viability. Propidium Iodide (PI), a dye whose fluorescence increases greatly when bound to nucleic acids, was used as an injection molecule for testing the efficacy of the nanoinjection process on HeLa 229 cancer cells in a portion of the experiments, with a GFP plasmid of DNA being used in the rest. After injection, flow cytometry was used to detect the concentration of PI or the expression of the GFP in the injected cells. Since PI cannot normally penetrate the membrane of living cells, those found with high concentrations of PI were either successfully injected or dead, which can be determined by the flow cytometry. Investigation of the parameters that affect the efficiency of the nanoinjection process will help improve it for further research. Some of these parameters that were investigated include the force of injection, the material used for the lances (silicon versus carbon nanotubes), and the injection speed of the lance arrays. An injection device capable of small changes in deflection was designed to ensure accurate increments in force for testing, as well as a pulsed current control injection system. Results for injections of varying forces indicate a slow rise in PI uptake from 0 to 1.8 Newtons where it reaches a maximum uptake of 4.11 when normalized to the PI uptake of the positive controls. The PI uptake then remains relatively level as the force continues to increase, averaging an uptake of approximately 3.1. The slow rise is likely due to more of the cells being punctured as the force increases until most have been punctured and the PI uptake levels off. The viability of the injected cells was close to that of the controls with no clear trend. A comparison of lance arrays made from silicon and carbon nanotubes using DNA as the molecular load shows little difference between materials. Different injection speeds tested show that only 1-5% of the cells in the injection process are lost for speeds in the range of 0.08-0.16 mm/sec, whereas 49-69% of the cells are lost using speeds between 0.6-3 mm/sec.

Fundamentally altering cellular function at a genetic level is a major area of interest in the biologic sciences and the medical community. By engineering transfectable constructs that can be inserted to dysfunctional cellular systems, scientists can mitigate aberrant genetic behavior to produce proper molecular function. While viral vectors have been a mainstay in the past, there are many limitations, particularly related to safety, that have changed the focus of genome editing to incorporate alternative methods for gene delivery. Lance Array Nanoinjection (LAN), a second-generation microfabricated transfection biotechnology, is one of these alternative technologies. LAN works by utilizing both simultaneous electrostatic interaction with molecular loads and physical lancing of hundreds of thousands of target cell membranes. The purpose of this work is to demonstrate LAN in the context of in vitro transfection of immortalized culture cells and primary cells. As part of that exploration, three distinct areas of investigation are considered, which include: characterizing environmental factors that impact LAN transfection, demonstrating LAN genetic modification of immortalized HeLa 229 culture cells using an indicator marker, and lastly, investigating the effects of LAN on human primary, neonatal fibroblasts.