13C PEG as an In Vivo Macromolecular Probe for MRI and Biophysical Studies of Detergent-Peptide and Detergent-Detergent Interactions using NMR

In the first part of this thesis, a new platform technology is demonstrated for the direct in vivo observation and quantification of macromolecular constituents, such as proteins and drug delivery systems, using 1H MRI. To overcome sensitivity challenges, a 28 kDa non-immunogenic tag is employed, consisting of 13C-enriched polyethylene glycol (PEG), whose spectroscopically equivalent 1H nuclei provide a signal amplification of 2500. Passing the 1H PEG signal through the directly coupled 13C nuclei enables the removal of background water and fat signal. Imaging capabilities are demonstrated by monitoring the real-time clearance of PEG and pegylated albumin in the hind leg of a mouse. Improvements to the initial imaging methodology are subsequently proposed, which increase the sensitivity and extend the imaging capabilities to abdominal and cranial regions. Ex vivo detection of pegylated constituents is also possible using NMR spectroscopy. Using blood as a sample biological fluid, clearance profiles of both unlabeled and 13C labelled PEG, as well as a pegylated protein were determined in rats. The second part of this thesis describes biophysical interactions of detergents with either peptides or other detergent molecules. Firstly, the effects induced by a single polar substitution on the structure and topology of model α-helical transmembrane segments were investigated. Upon substitution of a central isoleucine for an asparagine, diffusion NMR measurements revealed that the asparagine-containing peptide bound ~ 20% less detergent. Secondly, NMR was used to investigate the micellization process. A rapid method to determine thermodynamic and volumetric parameters, which relies on a single subtle change in the 1H chemical shift, was developed. The thermodynamic quantities were then related to spectroscopic studies of topology and micelle structure.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2015

Author dc:creator

• Alvares, Rohan

Advisors dc:contributor.advisor

• Prosser, Robert S
• Macdonald, Peter M

• HMQC
• molecular imaging
• MRI
• PEG
• poly(ethylene glycol)
• quantitative

Handle dc:identifier.uri

http://hdl.handle.net/1807/77748

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/77748