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Item High-density targeting of a viral multifunctional nanoplatform to a pathogenic, biofilm-forming bacterium(2007-04) Suci, Peter A.; Berglund, Deborah L.; Liepold, Lars Otto; Brumfield, Susan; Pitts, Betsey; Davison, William M.; Oltrogge, Luke; Hoyt, Kathryn Olivia; Codd, Sarah L.; Stewart, Philip S.; Young, Mark J.; Douglas, TrevorNanomedicine directed at diagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. Here, we targeted a spherical, icosahedral viral nanoplatform to a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding mediated through specific protein-ligand interactions exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto cells. The fluorescence-imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections.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.