Honors Theses
Date of Award
Spring 5-2-2021
Document Type
Undergraduate Thesis
Department
Chemistry and Biochemistry
First Advisor
Susan Pedigo
Second Advisor
Davita Watkins
Third Advisor
Eden Tanner
Relational Format
Dissertation/Thesis
Abstract
Osteoarthritis and other debilitating diseases cause chronic pain that is difficult to manage. Nonsteroidal Anti-inflammatory Drugs (NSAIDs) are the current standard of treatment for many long-term pain management, but there are potential systemic effects when using NSAIDs long-term. Therefore, it isnecessary to have an alternative option for NSAID delivery that doesn’t include these systemic effects. Hydrogels can be that alternative option. Hydrogels’ distinct characteristics allow them to be viable in the body, degrade under unique conditions, and be modified through many different chemistries. Additionally, hydrogels can be engineered and their properties optimized for the use as a drug depot inside the body. Covalently attaching drugs to hydrogels with a hydrolysable linkage could allow for drugs to be present at the site of inflammation and only released during times of need, which eliminates the problematic systemic effects of taking drugs orally.
The purpose of this study is to extend previous work on a genetically-engineered, protein-based hydrogel to explore different modification mechanisms of the polymers. In this study, we will examine two different methods of modification on CCLP3 and CCLP3 BisCys (BC), two engineered polymers with sites of modification. Each of these polymers have either one or two cysteines, which are the modification sites of interest. The methods of modification will use two different reagents, BODIPY FL L-Cystine and BODIPY FL Maleimide, forming disulfides and thioethers, respectively.
The modification of CCLP3 and CCLP3 BC with both BODIPY FL L-Cystine and BODIPY FL Maleimide met with both successes and unfortunate setbacks. First, methods were established for performing the protein chemistry with small volumes of relatively low protein concentrations involving multiple dialysis and sample transferring steps. Each modification chemistry involved special considerations of pH and ratio of dye to proteins. Control and modified samples were assayed by SDS- PAGE, UV-vis spectroscopy and Mass Spectrometry. Each method yielded critical data, and not all analyses agreed.
Spectroscopic studies were plagued by an inability to trust the extinction coefficient for the fluorophore. There was evidence of modification, but the extent of the modification is still unknown. The reported extinction coefficients for both BODIPY FL L-Cystine and BODIPY FL Maleimide were in methanol and seem to be inaccurate in determining the actual level of modification in aqueous buffers. Furthermore, there appears to be significant formation of disulfide linkages in the control samples, regardless of the reducing conditions induced in the experiment. There also appears to be some proteolysis of the samples resulting in fragments and mixed dimers of fragments and monomers.
Despite the setbacks, there was still significant evidence towards modification of the polymers. The most compelling evidence was from LC-MS studies that showed a large percentage of CCLP3 and CCLP3 BC was modified by BODIPY FL Maleimide unlike the modification results calculated using UV-Vis. Since this measurement provides the ratio of modified to unmodified polymers directly, we have confidence in these values. Additionally, when calculating the potential amount of drug that can be attached using the lower values of modification, it is still a significant increase than the levels of drug in the body by taking a whole standard dose. This value can only be increased by creating an accurate method of determining the actual modification of CCLP3 and CCLP3 BC, thus creating a hydrogel that could be sufficient and effective enough to replace the current standard of care for Osteoarthritis.
Recommended Citation
Smith, Emilie, "Covalent Modification Of Genetically Engineered Polymers With BODIPY Fluorophores" (2021). Honors Theses. 1773.
https://egrove.olemiss.edu/hon_thesis/1773
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