Browse

In vivo imaging of 64Cu-HSA-aptamer as a targeting agent for HER2 (Human Epidermal Growth Factor Receptor 2) expressing cancer cells
방사성구리-알부민-앱타머를 이용한 HER2 표적 암세포 생체영상

Cited 0 time in Web of Science Cited 0 time in Scopus
Authors
송명근
Advisor
강건욱
Major
의과대학 협동과정 종양생물학전공
Issue Date
2014-08
Publisher
서울대학교 대학원
Keywords
Human Epidermal Growth Factor Receptor 2 (HER2)AptamerHuman serum albumin (HSA)PET imagingCu-64Targeted therapy
Description
학위논문 (박사)-- 서울대학교 대학원 : 협동과정 종양생물학전공, 2014. 8. 강건욱.
Abstract
Objective: Human epidermal growth factor receptor-2(HER2) is one of the most important cancer biomarkers. It has been reported that HER2 overexpression in breast
cancer patients correlates with a poor prognosis and resistance to therapy. A humanized HER2 monoclonal antibody, trastuzumab (Herceptin), has been used for
HER2-targeted imaging and therapy. However, immunosintigraphy showed a relatively low target-to-background ratio, which induced low detectability. Therefore, there is a need for a new imaging probe to detect HER2 expression in vivo. To develop a new imaging probe using aptamer, single-stranded DNA or RNA oligonucleotides, 64Cu-labeled human serum albumin (HSA) with HER2-specific aptamer was designed and in vitro and in vivo properties of targeting HER2 expressing cancer cells were evaluated.
Methods: HER2-targeted aptamer was developed using systematic evolution of ligands by exponential enrichment technology (SELEX) and the aptamer was conjugated with human serum albumin (HSA) for prolonged blood circulation. FITC-labeled aptamer was used for confocal microscopy to monitor in vitro targeting. HSA was consecutively conjugated with SCN-Bn-NOTA and the bifunctional cross-linker sulfo-SMCC, which was then covalently conjugated with HER2-specific aptamer. The resulting NOTA-HSA-aptamer was further radiolabeled with 64CuCl2. Labeling efficiencies and purity of 64Cu-HSA-aptamer were determined by instant thin layer
chromatography (ITLC). For in vivo tumor imaging, 64Cu-HER2 aptamer and 64Cu-HSA-HER2 aptamer were intravenously injected to MDA-MB468 and KPL4 tumorbearing nude mice and their uptakes were imaged by PET from 0 to 46 h. The biodistribution of 64Cu-HER2 aptamer and 64Cu-HSA-HER2 aptamer was examined
in KPL4 and MDA-MB468 human breast tumor-bearing mice at 1 and 22 h postinjection. Autoradiography was performed to compare radiouptake between 64Cu-
HER2 aptamer and 64Cu-HSA-HER2 aptamer in tumor tissues.
Results: FITC-HER2 aptamer uptakes were visualized by confocal microscopy in HER2-positive cells (T47D and KPL4 cells), but not in HER2-negative cells (MCF7 and MDA-MB468 cells). Purity of both 68Ga-HER2 aptamer and 64Cu-HSA-HER2 aptamer was over 95% and in vitro uptake test showed their specific binding to HER2 in KPL4 cells (up to 3-fold increase). In KPL4 cells, the 50% inhibitory concentration (IC50) values for the HSA-aptamer, NOTA-aptamer and aptamer-biotin were estimated to be 1.44 ± 0.23, 12.97 ± 0.23 and 9.68 ± 0.26 nM, respectively. 68Ga-HER2 aptamer was quickly excreted in the urine or hepatic pathway within 46 h after tail vein injection. However, HSA conjugated HER2 aptamer was excreted slowly in the urine compared to HER2 aptamer. In tumor tissues, radio uptake of 64Cu-HSAHER2 aptamer was increased compared to HER2 aptamer. Serial PET imaging revealed that KPL4 tumor uptake of 64Cu-HSA-HER2 aptamer was 1.67 ± 0.15, 4.63 ± 2.66, 5.72 ± 3.22 and 6.63 ± 1.22 %ID/g at 5 min, 10, 22, and 46 h post-injection, respectively. In KPL4 tumor, 64Cu-HSA-HER2 aptamer biodistribution were also examined to be over 4.74 ± 1.60 and 8.98 ± 0.19 %ID/g at 1 and 22 h post-injection respectively, which was about 3.4-fold higher than that of 64Cu-HER2 aptamer. Moreover, the KPL4 tumor uptake of 64Cu-HSA-HER2 aptamer was about 4.2-fold higher than the MDA-MB468 tumor uptake. Autoradiograms revealed accumulation of HSA-HER2 aptamer in HER2-positive tumor tissues. The localizations of HSAHER2 aptamer-FITC in KPL4 tumor indeed corresponded to the distribution of the HER2-positive cells.
Conclusion: Tumor uptake of HSA-conjugated aptamer was increased compared to HER2 aptamer. 64Cu-HSA modified aptamer could be a promising probe for HER2-specific molecular imaging. The strategy using HSA as a carrier to prolong blood circulation of biomolecules such as aptamer could be applied in the design of many other radiolabeled probes and radionuclide therapy agents.
Language
English
URI
https://hdl.handle.net/10371/121767
Files in This Item:
Appears in Collections:
College of Medicine/School of Medicine (의과대학/대학원)Program in Cancer Biology (협동과정-종양생물학전공)Theses (Ph.D. / Sc.D._협동과정-종양생물학전공)
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse