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Engineered binding sites for indoxyl sulfate and p-cresol sulfate
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- Author / Creator
- Li, Shuhui
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The removal of uremic toxins is of great importance in preventing the progression of chronic kidney disease. The investigation of the interaction of uremic toxins with serum and dialysis materials will contribute to the advancement of techniques in uremic toxins removal. This study investigates the host-guest system of uremic toxins with human serum albumin and polymer-coated particles. First, the driving force of uremic toxins binding with human serum albumin is investigated and determined. Second, a series of novel polymer-coated magnetic nanoparticles are created and tested for toxin removal ability and hemocompatibility.
Although the removal of uremic toxins has been researched for decades and well progress has been achieved on water-soluble small molecules removal. Protein-bound uremic toxins are retained in the blood causing detrimental complications. Herein, through an in-depth thermodynamic investigation using isothermal titration calorimetry, the driving force for protein-bound uremic toxins binding to human serum albumin was identified unveiling the contribution of hydrophobic interaction and electrostatic interaction to the binding behavior. Furthermore, STD NMR provided solid evidence that identified the binding site of indoxyl sulfate and p-cresol sulfate on human serum albumin, that IS binds to Sudlow’s site I and site II whereas PCS only partially binds to site II. The human serum albumin secondary and tertiary conformation shifting upon addition of uremic toxins is reflected through the tryptophan fluorescence changing and the spatial formation determined by fluorescence spectrometry and circular dichroism, respectively.
Secondly, the novel poly(β-Cyclodextrin-co-2-(methacryloyloxy)ethyl phosphorylcholine) coated magnetic nanoparticle is created to remove uremic toxins. Instead of investigating the two iconic protein-bound uremic toxins, indoxyl sulfate and p-cresol sulfate, a profile of uremic toxins has been researched. Native β-cyclodextrin was decorated with the alkane group and copolymerized with 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) through the route of atomic transfer radiation polymerization on the surface of initiator fixed magnetic nanoparticle. The study shows that surface chemistry and incubation time have an impact on the adsorbed toxinome profile, even with "low-fouling" polymers. The findings suggest a dynamic interaction between toxins and surfaces that is not solely driven by solution concentration, which could help in the design of adsorbent films for clearing uremic toxins.
Following the previous work, we investigated the biocompatibility of β-cyclodextrin-co-2-(methacryloyloxy)ethyl phosphorylcholine grafted magnetic nanoparticles (p(PMβCD-co-MPC) MNP). The impact of bare magnetic nanoparticles and coated particles on the structure of human serum albumin, α-Lactalbumin, and Lysozyme proteins was studied using circular dichroism and fluorescence spectroscopy. The study also examined the stability of plasma and uremic plasma in the presence of particles using a plasma clotting assay. Immunoblotting was utilized to study the adsorption of plasma proteins to different types of MNPs using UTX-treated and non-UTX-treated plasma. The results indicate that p(PMβCD-co-MPC) coated MNPs have better biocompatibility than bare MNPs, and that particle D (PMβCD: MPC=1:3) exhibited the strongest inhibition of clotting formation in the UTX-treated group. The findings provide important insights into the biocompatibility of p(PMβCD-co-MPC) MNPs and their potential application in biomaterials.
In conclusion, this study presents valuable insights into the host-guest system of uremic toxins with human serum albumin and p(PMβCD-co-MPC) MNPs. The investigation of the driving force for protein-bound uremic toxins binding with human serum albumin and the identification of their binding sites are significant contributions to the field. Additionally, the creation and testing of novel polymer-coated magnetic nanoparticles for toxin removal and hemocompatibility showed intriguing results. The study highlights the importance of surface chemistry and incubation time in the adsorption of uremic toxins and the biocompatibility of the coated nanoparticles. The findings provide a foundation for the design of adsorbent films and potential biomaterials for clearing uremic toxins. -
- Subjects / Keywords
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- Graduation date
- Fall 2023
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.