Date of Award

12-16-2015

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Jenny J. Yang

Second Advisor

Peng George Wang

Third Advisor

Siming Wang

Abstract

Protein based contrast agents (ProCAs) developed by the Yang lab exhibit unique capabilities in enabling magnetic resonance imaging (MRI) with significantly improved sensitivity and targeting capabilities by utilizing biomarkers which can target various carcinomas in animals. Further clinical in vivo human applications require modifications of these designed contrast agents to further improve organ and tissue biodistribution, biomarker and cell targeting capabilities, and reduction of immunogenicity. The aim of this thesis is to develop a novel protein modification on ProCA by glycosylation to improve liver distribution by targeting liver receptor, asialoglycoprotein receptor (ASGPR). Rat and humanized first generation and humanized third generation ProCA were expressed and purified using either glutathione s-transferase (GST) tagged or taggless methods. Rat ProCA1, rProCA1, was then used to optimize glycan modification with glycosylation achieved at the highest level using a 100:1 molar ratio and three lysine residues. Similar to non-modified rProCA1 and PEGylated rProCA1, metal binding affinity of gadolinium for glycan modified rProCA1, Glyco-rProCA1, was found to be 9.49 x 10-12 M, and relaxivity was found to be greater than clinically available contrast agents with 34.08 and 42.67 mM-1s-1 for r1 and r2 respectively. Glycosylation of rProCA1 has significantly increased human serum stability, and we have achieved significant liver MRI enhancement via tail vein injection due to high ASGPR expression in the liver altering biodistribution of glycan modified ProCA, and we have also imaged uptake in the secretory glands. These biodistribution changes were noted by immunohistochemistry (IHC) staining which was found to stain liver sinusoid with spaces in between. The distribution to the liver was further confirmed via inductively coupled plasma optical emission spectrometry (ICP-OES) which shows Glyco-rProCA1 has significant uptake of gadolinium in the liver tissue. This study represents the first achievement of in vivo liver imaging by glycosylation using a lactose targeting moiety covalently bonded to protein contrast agents for MRI showing promise for future more specific targeting or whole body imaging capabilities.

DOI

https://doi.org/10.57709/7924068

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