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Item Designing virus-like nanoparticles as T 1-enhanced MRI contrast agents(Montana State University - Bozeman, College of Letters & Science, 2014) Qazi, Shefah Alma; Chairperson, Graduate Committee: Trevor Douglas; Lars O. Liepold, Md Joynal Abedin, Ben Johnson, Peter Prevelige, Joseph A. Frank and Trevor Douglas were co-authors of the article, 'P22 viral capsids as nanocomposite high-relaxivity MRI contrast agents' in the journal 'Molecular pharmaceutics' which is contained within this thesis.; Masaki Uchida, Robert Usselman, Riley Shearer, Ethan Edwards and Trevor Douglas were co-authors of the article, 'Manganese (III) porphyrins complexed with P22 virus-like particles as T1-enhanced contrast agents for magnetic resonance imaging (MRI)' in the journal 'Journal of bioinorganic chemistry' which is contained within this thesis.; Masaki Uchida, Hisanori Kosuge, Michael V. McConnell, and Trevor Douglas were co-authors of the article, 'Expression and biophysical characterization of RGD targeting peptide on surface of P22 via C-terminus extension of DEC and P22 coat protein' which is contained within this thesis.The field of nanotechnology is a rapidly growing field. In the past few decades, nanoparticles have been utilized for use in biomedical applications with a huge impact in enhancing diagnostic techniques. Protein cages and virus-like particles are biological examples of nanoparticles. They are highly symmetric, well-defined architectures made from multiple protein subunits and can be genetically or chemically engineered to impart desired new functionalities and have been used for design of nanomaterials for improving current diagnostic techniques, as discussed in this thesis. One of the main techniques for diagnosis used today is magnetic resonance imaging (MRI) as it provides good spatial resolution of soft tissues without using harmful ionizing radiation. However, due to poor sensitivity of this technique, contrast agents are often utilized by clinicians to aid in diagnosis of diseased tissues. The main MRI contrast agents used in T1-enhanced imaging are small Gd-containing molecules. Due to the toxicity of free Gd ions, these agents are administered in a tightly chelated form. Even in this form, high doses increase the risk of toxicity. Thus, it is important to reduce overall dosage of these contrast agents. In this thesis, we discuss design principles for virus-like particle based MRI contrast agents as next generation diagnostics which can overcome the above mentioned barriers. Conjugating clinically approved contrast agents to nano-sized virus-like particles changes the intrinsic properties of the contrast agent, directly impacting and increasing MRI contrast. Modifying the interior surface of these cage-like containers to grow functionalizable polymers provides multiple sites for conjugation of small molecule contrast agents, resulting in high payload of these agents. Modifying the exterior surface of these cage-like containers to present targeting ligands and enable them to localize at desired tissues of interest. All three of these design considerations contribute to higher contrast, significantly lower clinical dose requirements, and allow for safe administration of Gd (III) ions for enhanced imaging. As gadolinium-based contrast agents are directly linked with nephrogenic systemic fibrosis, a rare but deadly disease that causes hardening of tissues and organs, an alternate low-risk metal-complex, Mn (III) porphyrins, has also been explored for bioconjugation to virus-like particles.Item Exploring the potential of protein cages as MRI contrast agents with an emphasis on protein cage characterization by mass spectrometry techniques(Montana State University - Bozeman, College of Letters & Science, 2009) Liepold, Lars Otto; Chairperson, Graduate Committee: Trevor Douglas; Mark J. Young (co-chair)Described here is the development of a protein cages as efficient and potentially relevant MRI contrast agents. Three approaches are outlined to fuse high affinity Gd³+ chelating moieties to the surfaces of protein cages. In the first approach, a metal binding peptide has been genetically engineered into the subunit of Cowpea chlorotic mottle virus (CCMV) and to the small heat shock protein cage from Methanococcus jannaschii (HSP). The genetic fusion resulted in a 200x binding enhancement of Gd³+ to CCMV in comparison with wild type CCMV and metal binding functionality was added to the HSP protein cage. In a second approach DOTA-Gd was attached to CCMV by reactions with endogenous lysine residues on the surface of the viral capsids and resulted in r1 = 2,806 at 61 MHz for the 28nm diameter particle. Directed by the results of earlier generations of protein cage based contrast agents a next generation MRI contrast agent was designed. In this work a DTPA-Gd containing polymer was grown in the interior of HSP resulting in T1 particle relaxivities of 4,200mM-¹ sec-¹ for the 12nm particle. Relaxivity parameters were determined and this analysis suggests that the rotational correlation time of the Gd³+ chelate has been optimized while the exchange life time of Gd³+-bound water is slower than optimal. This synthetic approach holds much promise for the development of future generations of contrast agents. Throughout the evolution of the protein cage based contrast agents there has also been and evolution of our ability to characterize these cages with mass spectrometric techniques. Specifically refined methodologies are presented for QTof characterization of protein cage at the level of amino acids, protein subunits, protein complexes and their cellular expression. Furthermore, correct charge state assignment is crucial to assigning an accurate mass to supramolecular complexes such as protein cages analyzed by electrospray mass spectrometry. Conventional charge state assignment techniques fall short of reliably and unambiguously predicting the correct charge state for many supramolecular complexes. We provide an explanation of the shortcomings of the conventional techniques and have developed a robust charge state assignment method that is applicable to all spectra.