Biomedical Imaging and Instrumentation
Sarah Lee (she/her/hers)
Summer Undergraduate Researcher
Nemours Children's Hospital
Fairfax, Virginia, United States
Wenqi Xu, PhD
Post Doctoral Fellow
NCH-RSCH-Research - DV, United States
Sigrid Langhans, PhD
Principal Research Scientist
NCH-RSCH-Research, United States
Heidi Kecskemethy, MSEd
Program Director
NDP-WIL-Radiology, United States
Thomas Shaffer, PhD
Director of Nemours Res. Lung
NCH-RSCH-Research - DV, United States
Lauren Averill, MD
Chair, Medical Imaging, DV
NDP-WIL-Radiology, United States
Xuyi Yue, PhD
Research Scientist/Lab Head, Department of Radiology
Nemours Children's Health, Delaware Valley, United States
Milroy Disease (MD) is an autosomal dominant disorder that causes defects in the lymphatic system, leading to lymphedema. MD is caused by mutations in vascular endothelial growth factor receptor-3 (VEGFR3, Figure 1) and mainly affects newborns and infants. Children with MD have an increased risk of developing lymphangiosarcoma, a rare cancer with a poor prognosis and a 5-year survival rate of less than 5%. The traditional imaging modalities of computed tomography (CT) and magnetic resonance imaging (MRI) are size-dependent anatomical imaging methods; they have limitations in probing functional changes of lymphatic disorders and progression. Positron emission tomography (PET) using radioactive imaging agents provides a noninvasive way to investigate the biochemical and metabolic processes in the body (Figure 1). PET imaging targeting VEGFR3 can early probe malignant changes of MD.
Unless otherwise noted, all chemicals and reagents used for organic synthesis were obtained from commercial sources. An active ester-based prosthetic group was synthesized by a condensation reaction. Thin-layer chromatography (TLC) analysis was used to monitor reaction progress. Silica gel chromatography was used to purify the synthesized compound (Figure 1). The prosthetic group was conjugated with a peptide with a known high binding affinity towards VEGFR3 under mild conditions (phosphate-buffered saline (PBS)/MeCN, pH 9.0). The resulting peptide was purified by high-performance liquid chromatography (HPLC). Compounds were characterized by high-resolution mass spectrometry (HRMS), proton nuclear magnetic resonance (1H NMR), carbon-13 NMR, fluorine-19 NMR, and analytical HPLC (Figures 2, 3). A stability test of the synthesized peptide was performed in water and PBS (pH 7.4) at 37 °C (Figure 5). Lipophilicity was determined by a shake flask method and HPLC determination using the partition coefficient (logD7.4) of the peptide between n-octanol and aqueous phase measurements (Figure 5).
The active ester F-Py-Ester was synthesized by condensation of carboxylic acid 1 with phenol 2 in 79% yield over 95% purity. F-Py-Ester was characterized by 1H NMR, 19F NMR, 13C NMR, HPLC, and HRMS (Figure 2). The VEGFR3-specific peptide AA187 was custom synthesized with high purity ( > 95%) and characterized by HPLC and mass spectrometry (Figure 3). The conjugation between the active ester F-Py-Ester and peptide AA187 proceeded smoothly and provided the resulting peptide with a 55% yield. The target peptide F-Py-AA187 was characterized by HPLC and HRMS with over 95% purity (Figure 4). Stability tests show the peptide was stable for up to 90 min in water and PBS. The peptide displays favorable water solubility determined by lipophilicity measurements (Figure 5). Based on the preliminary results, a new peptide targeting VEGFR-3 for lymphatic disorders was synthesized with high purity and stability. The next steps in this project are to determine the binding affinity of the new peptide and compare the results with the parent peptide using the VEGFR3 kinase assay kit and develop an efficient method for fluorine-18 incorporation. The imaging probe will provide a noninvasive and quantitative strategy to study MD that affects children.