Date of Award

2015

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering (PhD)

Administrative Home Department

Department of Biomedical Engineering

Advisor 1

Megan C. Frost

Committee Member 1

Jeremy Goldman

Committee Member 2

Bruce P. Lee

Committee Member 3

Xiaoqing Tang

Abstract

Nitric oxide (NO) is recognized as the most important small signaling molecule in the human body. An imbalance of NO is closely associated with many serious diseases such as neurological disorders, cardiovascular diseases, chronic inflammations and cancers. Herein two chemiluminescence-based devices (a real-time NO measurement device and a controllable NO delivery device) were developed to facilitate the NO quantitative study and obtain information for NO related drug design.

The first device used for real-time measuring NO(g) flux from living cells was developed and validated. The principle was to use a two-chamber design, with a cell culture chamber and a gaseous sampling chamber separated by a selective PDMS-based (polydimethylsiloxane-based) membrane. The membrane was polydopamine coated to improve the cell adhesion and growth. The signal response of the devices was validated by using the controlled NO releasing polymer SNAP-PDMS (S-Nitroso-N-acetyl-D-penicillamine covalently linked to PDMS). The real-time NO generation profiles of the macrophage cell-line RAW264.7 stimulated by lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) were successfully measured. Data also shows that the change of NO generation by different exogenous factors can also be tracked in real-time by the device. The maximum NO flux characterized by the device varies from approximately 2.5 to 9 pmol/106cell/s under 100 ng/ml LPS and 10 ng/ml IFN-γ stimulation depending on different culture conditions, indicating the current NO reporting method by using one single NO flux value is not sufficient to represent the dynamic character of NO in biological samples. By using the similar principle and device characterization processes, an innovative cross-membrane delivery system was developed to successfully deliver NO to the cultured cells in a dose and temporally controlled manner. Different levels and durations of NO were delivered to the smooth muscle cell line MOVAS cultured in the device to validate the controllability of the NO delivery. The device characterized an NO flux of 1.0x10-10 mol/cm2/min as a threshold level, above which NO may cause MOVAS death, and the effect of this lethal level was also duration and cell-type dependent. To further show the utilization of the two devices, the NO measurement device was used to systematically study different NO donors' potencies. It is suggested that measuring the real-time NO profiles of specific NO donors in specific biological conditions can help us avoid generating confusing data and understand the potencies of NO releasing drugs.

Current methods for NO quantitative experiment lack the means to understanding the time-related aspects of NO in biological environment and controlling the actual NO level and duration that cells experience. The two innovative devices solved these problems in cell culture models, achieving quantitatively studying the behavior of NO in the biological conditions and potentially assisting the design of the NO releasing drugs/materials.