Antisense oligodeoxynucleotides (asODNs) show strong binding and high selectivity and can be constructed to recognize specific cellular targets such as gene regulated mRNA. Radiolabelled asODNs have the potential to image gene expression through mRNA targeting and could be a valuable tool in the early assessment of outcome to cancer treatment. We have explored the potential of in vivo imaging of p21 gene expression, using fluorine-18 labelled asODNs ([18F]asODNs) and in vitro techniques, recognizing the relationship between the expression of this gene and resistance of cancer cells to radiation therapy. Radiolabelling of fully phosphorothioated, 20-mer ODNs was performed using the [18F]-labelled prosthetic group, 4-N-[18F]fluorobenzyl-2-bromoacetamide ([18F]FBBA). [18F]FBBA was first synthesized in an automated synthesis unit, resulting in a modest radiochemical yield. Methods to improve the yield were investigated using a metal catalyst-assisted borohydride exchange resin. Alkylation of [18F]FBBA to ODN resulted in radiochemical yields of 40%. Cellular uptake and retention studies were performed in human carcinoma cells expressing p21+/+ (HCT116) and the p21 knock-out cell line, 80S4, using both [18F]-labelled antisense and random sequence ODNs. Nonradioactive FBBA-labelled ODNs were used to evaluate the antisense effectiveness and distribution of the FBBA-modified ODNs. In vitro studies demonstrated that FBBA did not interfere with the antisense effect of ODNs against p21 mRNA; however, the probes required a transfection agent to observe an antisense effect. Cell fractionation studies with [18F]ODNs revealed increasing accumulation of liposome-transfected [18F]asODN in the cytoplasm of HCT116 cells over time. A biocompatible spermine-grafted block copolymer (SP) was subsequently evaluated as a potential vector to improve the delivery of [18F]asODN into cells. SP was shown to direct [F]-labelled ODNs to the cytoplasm, whereas naked [F]ODNs remained sequestered in vesicles, and liposome-transfected [F]ODNs localized mostly in the nucleus. Selective uptake and retention of [18F]asODN was observed in p21+/+ cells only when the probe was transfected with SP. Based on these studies, it can be concluded that [18F]asODNs have the potential to image gene expression, however the focus may need to be directed to find an appropriate vector which can rapidly deliver [18F]-labelled asODNs to the target tissue in vivo.
Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/1267 |
Date | 11 1900 |
Creators | Koslowsky, Ingrid L |
Contributors | Mercer, John (Medicine), Mercer, John (Medicine), McQuarrie, Steve (Medicine), Suresh, Mavanur (Pharmacy & Pharmaceutical Sciences), Murray, David (Medicine), Lavasanifar, Afsaneh (Pharmacy & Pharmaceutical Sciences), Adam, Michael (Chemistry, University of British Columbia) |
Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
Language | English |
Detected Language | English |
Type | Thesis |
Format | 1738748 bytes, application/pdf |
Relation | Koslowsky I, Shahhosseini S, Wilson J, Mercer J. (2008) J Label Compd Radiopharm;51:352, Koslowsky I, Mercer J, Wuest F. (2010) Org Biomol Chem |
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