Graduate Thesis Or Dissertation
 

Reversible Covalent Immobilization of Desferrioxamine B (DFOB) on Aminated Polystyrene Beads for Citrate-Bound Fe(III) Chelation

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/v405sg90v

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  • The immobilization of chelators to solid supports has recently gained renewed interest due to the increasing biomedical and environmental applications made possible by these molecules’ ability to bind Fe(III) and other metal ions. The uses of immobilized chelators have expanded to include the treatment of metal overload in serum, the detection of metal ions and living bacteria, the reduction of tissue degradation and inflammation, and the separation of high-valence metal ions, among other applications. In this work, desferrioxamine B (DFOB) was reversibly and covalently immobilized to aminated 1 μm polystyrene beads for the chelation of Fe(III)-citrate complex. This iron complex was chosen given its relevancy in blood plasma, natural waters, plants and pathogenic bacteria. After linking Pyridyldithiol-Activated DFOB to the polymeric support via cleavable disulfide bonds, citrate-bound Fe(III) prepared in HEPES at a pH of 7.4 was successfully complexed by the immobilized chelator. It was subsequently shown that the active DFOB species were mostly covalently bound to the beads and not adsorbed on the polymeric surface. Additionally, the polystyrene support was effectively regenerated via cleaving the disulfide linkage between the resulting DFOB-Fe(III) complex and the surface, followed by recoupling to fresh DFOB. This ability to regenerate the support allows for its multiple use and can prove economically advantageous for small and large-scale operations. The kinetics and equilibrium studies for the adsorption of citrate-bound Fe(III) on DFOB-activated beads have also been performed under a constant chelator loading on the beads. Fitting the pseudo first-order, pseudo second-order and Elovich kinetics models to the experimental data did not lead to a firm conclusion regarding the most suitable model due to the similar calculated values of the correlation coefficients R². Moreover, modelling the Langmuir and Freundlich adsorption isotherms yielded relatively close correlation coefficients R² values of 0.992 and 0.953, respectively. However, since the Langmuir physics closely matches our understanding of the iron chelation by DFOB system, we expected that the Langmuir model offers a better description of the adsorption data, with a calculated Langmuir constant KL = 12.81 (L/mg-(Fe(III)) and a maximum iron coverage qmax = 0.206 (mg-(Fe(III))/g-adsorbent). Additionally, relating the pseudo first and pseudo second-order models via the Langmuir kinetics equation using the previously obtained KL and qmax values showed a dependency of the adsorption kinetics on the initial Fe(III) concentration; with a pseudo first-order model expected to most accurately describe the adsorption kinetics for initial Fe(III) concentrations lower than 22 μM or higher than 68 μM, while a pseudo second-order model was expected to offer the best fit to the data for values approaching 35 μM. Moreover, comparing the kinetics profiles of immobilized and free DFOB, it was found that approximately 180 minutes were required to reach constant concentration values of adsorbed Fe(III) with immobilized DFOB, compared to 30 minutes with the chelator in solution. These results showed that the immobilization of DFOB significantly reduced the rate of citrate-bound Fe(III) adsorption, which can be attributed to a possible hindrance of the iron’s citrate-DFOB ligand exchange mechanism. The causes of this obstruction can be related to a reduced diffusion of dimeric iron citrate complexes to the immobilized DFOB and/or a slower mechanism of metal ion complexation by the chelator’s hydroxamates groups. The effects of serum protein on the activity of immobilized DFOB were also investigated. Washing DFOB-activated beads with bovine serum albumin in phosphate-buffered saline (BSA-PBS) at its serum concentration of 40 mg/mL, and a total exposure time of 30 minutes, reduced the amount of chelated iron by more than 50% compared to washing with PBS. Additionally, Fe(III)-citrate solutions prepared in BSA (40 mg/mL) and 25% v/v equine plasma diluted with HEPES buffer had approximately 80% and 70% less iron chelation levels compared to protein-free Fe(III)-citrate, respectively. These results illustrate the negative effects of protein surface adhesion on the activity of DFOB. This can be possibly ascribed to a reduced access of Fe(III)-citrate molecules to the immobilized chelator caused by the presence of the larger surface proteins and/or their negative effects on the ability of the DFOB’s hydroxamates to complex the metal ions. Given the versatility of DFOB in chelating different metal ions, coupled with the polystyrene’s suitability for mass-production and support generation, it is hoped that this study can contribute in diffusing affordable and small-scale microfluidics and biosensors technologies, suited for different biomedical and environmental applications. Provided that the required protein-repulsing surface modifications are performed, this system can potentially offer a safer chelation treatment for metal overload patients. Other immediate applications can include the simple detection of metals and bacteria in physiological and environmental fluids and the separation of high-valence metals, along other potential applications.
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  • Ongoing Research
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  • 2018-11-02 to 2019-06-03

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