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Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Michael J. Maroney
Michael J. Knapp
Alejandro P. Heuck
Chemistry | Inorganic Chemistry
Iron is an essential element for the body and plays important roles in many metabolic processes. A transient labile iron pool (LIP) has been proposed to play key roles in cellular iron trafficking and metabolism. However, free iron ions (Fe3+ and Fe2+) in this pool are toxic and damaging to cells due to their involvement in the production of oxygen radicals. The damages may lead to aging and various diseases including stroke, cancer, and several neurological disorders like Parkinson’s disease, Alzheimer’s disease and atherosclerosis. Determination of free iron ions in cells may contribute to a better understanding of iron’s function and transport pathways under physiological and pathological conditions. Early methods to identify a labile and transient iron pool needed to disrupt cells. Over the past two decades, fluorescent sensors have emerged to visualize metal ions in living cells without any damage. To visualize intracellular iron ions, specific fluorescent sensors are needed. Even though fluorescent sensors have been applied into cells for two decades, only a few Fe3+-selective sensors are capable of cellular imaging with limited success.
Chapter 1 introduces the biological background of iron as well as recently developed fluorescent sensors for iron detection.
In chapter 2, a new rhodamine-based sensor, RPE, was synthesized and characterized by 1H NMR, 13C NMR, and MS. The sensor responds to Fe3+ via coordination induced fluorescent activation (CIFA) mechanism and gives a distinct rapid and reversible fluorescence response upon the alteration of intracellular Fe3+ levels with little interference from other biologically relevant metal ions. RPE can readily detect endogenous chelatable Fe3+ via a confocal microscope in live human SH-SY5Y cells at subcellular resolution in real time, with two labile Fe3+ pools being successfully located in mitochondria and endosomes/lysosomes in both untreated and Fe3+-loaded human SH-SY5Y cells for the first time. RPE is thus a promising tool for probing the cell biology of Fe3+.
Sensors, such as RPE, which emit lights in visible range, suffer from some significant drawbacks, including intrinsic signal caused by auto-fluorescence, high light scattering, and poor light penetration when they are applied to biological tissues. To overcome these limitations, near infrared sensors can be used because they emit at longer wavelength so they display low autofluorescence background, deeper penetration to tissues and cause less damage to biological samples. In chapter 3, a heptacyanine based sensor, IRPE, was synthesized and characterized by 1H NMR, 13C NMR, and MS. IRPE showed selective response to Fe3+ and binds it in a 1:1 stoichiometry with an apparent binding constant 2.0×105 M−1 in ACN/HEPES (1/1 v/v) solution. The sensor displays a change in color and fluorescence upon the alteration of Fe3+ levels in solution with a reversible response and little interference with other biological relevant metal ions. IRPE is a good Fe3+-selective sensor but, cell studies showed that it was not capable of detecting free iron ions in cells.
To get cell permeable and Fe3+-selective near infrared sensors, it was decided to change the strategy for near-infrared sensor design. In chapter 4, a Changsha based sensor, NIRh-Ac, was developed. The sensor gives a distinct rapid and reversible fluorescence response upon the alteration of intracellular Fe3+ levels with little interference from other biologically relevant metal ions. Confocal experiments showed that NIRh-Ac could readily detect exogenous chelatable Fe3+ in live human SH-SY5Y cells and live fibroblast cell (ws1) at subcellular resolution, with the chelatable Fe3+ pools located in mitochondria and endosomes/lysosomes for SH-SY5 cells and in mitochondria for ws1 cells. Kinetic experiments with the sensor provided a visual imaging of Fe3+ transport pathway in human fibroblast cells in real time, i.e., from endosomes to lysosomes and finally to mitochondria via a direct “Lyso-mito docking” mechanism, bypassing the cytosol. Studies using zebrafish clearly demonstrated the capability of the NIRh-Ac sensor in imaging Fe3+ in live animals.
In chapter 5, another Changsha-based near infrared sensor, NRPA, was developed to detect endogenous Fe3+ ions in cells. It is a highly sensitive, highly selective, and reversible turn-on near infrared fluorescent sensor for Fe3+. The sensor gives a rapid and reversible fluorescence response upon the alteration of intracellular Fe3+ levels with little interference from other biologically relevant metal ions. Confocal imaging studies demonstrate that NRPA can readily detect endogenous free Fe3+ in live human SH-SY5Y cells and live fibroblast cell (ws1) at subcellular resolution, with the chelatable Fe3+ pools located in mitochondria and endosomes/lysosomes in SH-SY5 cells while in mitochondria only in ws1 cells. It was concluded that different cell lines store/handle iron in different ways. The ability of NRPA to detect endogenous free Fe3+ ions in zebrafish was also demonstrated and free Fe3+ ions were found located in liver/gall bladder of 6-days-old zebrafish.
In chapter 6, highly sensitive, highly selective, and reversible turn-on near infrared Changsha-based fluorescent Fe3+-sensors, NRPK and NRP were described. The initial goal was to synthesize Fe(II)-selective fluorescence sensor; however, NRPK turned out to be a sensor for Fe (III). Both NRP and NRPK coordinate Fe3+ with O/N/N binding motif with 2:1 ratio. The NRPK appears not to use the carbonyl group linked to the pyridine ring for coordination. Cell imaging experiments with NRPK showed that it could readily detect endogenous free Fe3+ in live bovine aortic endothelial cells (BAEC) and human SH-SY5Y cells at subcellular resolution, with free iron (III) ions located in mitochondria and endosomes/lysosomes for both BAEC and SH-SY5 cells. Finally, NRPK demonstrated the ability to detect exchangeable free iron(III) in zebrafish in vivo.
Finally, a novel sensor was developed to quantify the concentration of Fe3+ in the LIP and to confirm that the images observed by the sensors such as RPE, NIRh-Ac, NRPA and NRPK reflect the true locations of cellular chelatable Fe3+, not instead the locations of the sensors themselves. A ratiometric near infrared sensor, CR-PK, was presented in chapter 7.The CR-PK sensor shows NIR and visible emission in its spirolactam ring-open and closed forms, respectively. The reaction of CR-PK with Fe3+ leads to the ring opening of the spirocyclic moiety of CR-PK, causing a large red shift ~222 nm of the absorption band. Cell imaging experiments with CR-PK revealed that CR-PK is evenly distributed in the cells except the nuclei region; however, chelatable labile Fe3+ ions are located in certain organelles in live bovine aortic endothelial cells (BAEC), fibroblast (ws1) cells and human neuroblastoma cell (SH-SY5Y). The ratiometric sensor CR-PK enables the direct determination of endogenous labile Fe3+ concentration in the cells for the first time, with a value of~0.6 µM determined (0.43 ± 0.23 µM by Method 1 and 0.8 ± 0.28 µM by Method 2 ) for ws1 cells, ~1.78 µM determined (2.18 ± 0.35 µM by Method 1 and 1.38 ± 0.47 µM by Method 2 ) for BAEC cells, and ~3.05 µM determined (3.2 ± 0.63 µM by Method 1 and 3.1 ± 0.53 µM by Method 2 ) for SH-SY5Y cells.
The various highly selective Fe3+ sensors developed in this work offer novel tools for molecular imaging of Fe3+ in live cells and live animals. The sensors, covering wavelength in the visible and near infrared regions with affinity to Fe3+ in micro to nanomolar levels, are ideal to image Fe3+ at subcellular resolution in real time, with the chelatable Fe3+ pools identified in various cell lines and live animals for the first time. Moreover, the ratiometric sensor enabled the determination of Fe3+ concentration in live cells for the first time. The Fe3+ sensors will contribute to a better understanding of the cell biology of iron and its related pathology and medical applications.
Aydin, Ziya, "Developing Fluorescent Sensors for the Bioimaging of Chelatable Iron(III) Ions" (2015). Doctoral Dissertations. 281.