저작자표시-비영리-변경금지 2.0 대한민국 이용자는 ... - S-Space

전체 글

(1)저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l. 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다.. 다음과 같은 조건을 따라야 합니다:. 저작자표시. 귀하는 원저작자를 표시하여야 합니다.. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다.. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.. l l. 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다.. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer. (2) 이학박사 학위논문. Development of a Multimodal Imaging Probe for Lymph Node Targeting Using Specific Amphiphile Encapsulation Technology on Iron Oxide Nanoparticles 산화철 나노입자에 특이적 엠피파일 인캡슐레이션 방법을 이용한 림프절 표적용 다중영상 프로브의 개발 2014 년 02 월. 서울대학교 대학원 협동과정 방사선응용생명과학전공 양 보 연. (3) A thesis of the Degree of Doctor of Philosophy. 산화철 나노입자에 특이적 엠피파일 인캡슐레이션 방법을 이용한 림프절 표적용 다중영상 프로브의 개발. Development of a Multimodal Imaging Probe for Lymph Node Targeting Using Specific Amphiphile Encapsulation Technology on Iron Oxide Nanoparticle February 2014. The Department of Interdisciplinary Program in Radiation Applied Life Science, Seoul National University College of Medicine. Bo Yeun Yang. (4) 산화철 나노입자에 특이적 엠피파일 인캡슐레이션 방법을 이용한 림프절 표적용 다중영상 프로브의 개발 지도교수 정 재 민. 이 논문을 이학 박사 학위논문으로 제출함 2014년 2월. 서울대학교 대학원 협동과정 방사성응용생명과학전공 양 보 연 양 보 연의 이학박사 학위논문을 인준함 2014년 2월 위 원 장 부위원장 위. 원. 위. 원. 위. 원. (5) Development of a Multimodal Imaging Probe for Lymph Node Targeting Using Specific Amphiphile Encasulation Technology on Iron Oxide Nanoparticle by Bo Yeun Yang A thesis submitted to the Department of Interdisciplinary Program in Radiation Applied Life Science in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Interdisciplinary Program in Radiation Applied Life Science at Seoul National University College of Medicine. February 2014 Approved by Thesis Committee: Professor. Chairman. Professor. Vice chairman. Professor Professor Professor. (6) ABSTRACT. Bo Yeun Yang Interdisciplinary Program in Radiation Applied Life Science The Graduate School Seoul National University College of Medicine. Introduction: Multimodal imaging probes could provide synergistic. effect through combining the benefits and supplementing the weaknesses from each imaging system. Nanoparticle is a good carrier and imaging probe itself and surface modification using huge surface area make it possible for multifunctional application. The aim of this study is to develop a multimodal iron oxide nanoparticle using an encapsulation method with specific amphiphiles (1). Methods: Specific amphiphiles were synthesized by conjugating. stearylamine with SCN-Bn-NOTA and SCN-Bn-Mannose. Mannosecontaining nanoparticle was designed for imaging lymph nodes (2). i. (7) NOTA is an excellent chelating agent for. 68. Ga labeling (3). Iron oxide. nanoparticles were encapsulated with prepared solution containing polysorbate 60 and functionalized amphiphiles by vigorous mixing and sonication. Reaction mixture was purified using size exclusion column and concentrated with centrifugal filter. Concentration of the final material was measured by colorimetric analysis at 457 nm. Size distribution was analysed using a DLS instrument. Stability was evaluated under various conditions (0.9, 1.8, 3.6% NaCl solution, rt, 1, 3, 24 h; human serum, 37°C, 2 h). Phantom study was performed for estimation of T2-weighted MR (3.0 T) signal of the final probes and was compared with that of commercialized iron oxide-based probes (Feridex® ). In vivo study was performed for 60 min dynamic imaging after s.c. injection of 68Ga labeled probe to the left footpad of a mouse (12.0 MBq/10 µL) and follow up with T2-weighted MRI imaging for 30 min. Microscopic histopathologic examination of popliteal lymph nodes after prussian blue staining was performed. Results: The size distribution of NOTA-IO-Man was 10.1±1.5 nm with. high encapsulation recovery ratio (92.0%). No significant aggregation ii. (8) or degradation was found under various harsh conditions. The radiochemical purity of 68Ga-NOTA-IO-Man was >99% and was stable in human serum for 120 min and no size transition (10.1±1.5 nm, P=.29) was observed. The calculated relaxivity coefficient (r2) of mM-1s-1 was 449.9 which was higher than Feridex® (180.38). The specificity of 68. Ga-NOTA-IO-Man was confirmed showing both PET/CT and MRI. signal at the same site. The tissue section of left popliteal lymph node resulted in a distinctive blue pigment demonstrating the existence of ferric iron. Conclusions: In this study, we developed lymph node targeting. PET/MRI dual-modality imaging probes successfully using facile encapsulation method. ------------------------------------Keywords:. PET/MRI,. Multimodal. Imaging,. Surface. Amphiphile, Encapsulation, Iron Oxide Nanoparticles Student number: 2008-2328. iii. Chemistry,. (9) CONTENTS Abstract ..........................................................................................................i Contents ........................................................................................................iv List of tables and figures .............................................................................. v List of abbreviations .................................................................................. vii Introduction ................................................................................................ 1 Material and Methods .................................................................................. 6 Results .......................................................................................................... 19 Discussion .................................................................................................... 45 References.................................................................................................... 48 Abstract in Korean ..................................................................................... 53. iv. (10) LIST OF TABLES AND FIGURES. Figure 1.. Synthesis scheme of functionalized amphiphiles. Figure 2.. Preparation of encapsulated IO NPs with polysorbate 60, NOTA-SA and Man-SA. Figure 3.. 1. Figure 4.. LC-MS data of functionalized amphiphiles. Figure 5.. ICP-MS results of encapsulated IO NPs with various. H NMR spectra of functionalized amphiphiles. polysorbate 60 solutions Figure 6.. Size distribution of NOTA-IO-Man using DLS and TEM. Figure 7.. Colorimetric methods of [Fe(SCN)2+]. Figure 8.. Radio TLC analysis of free 68Ga and 68Ga-NOTA-IO-Man. Figure 9.. Stability test of NOTA-IO-Man in high concentration NaCl solutions. Figure 10.. Stability test of 68Ga-NOTA-IO-Man in human serum. Figure 11.. In vitro T2-wighted MR images of the phantom. Figure 12.. In vivo small-animal PET/CT images. Figure 13.. In vivo MRI image v. (11) Figure 14.. Popliteal lymph node biopsy study. Table 1.. Hydrodynamic. size. of. concentration NaCl conditions. vi. NOTA-IO-Man. in. high. (12) LIST OF ABBREVIATIONS. IO, Iron Oxide NPs, Nanoparticles PEG, Polyethylene glycol NOTA, p-SCN-Bn-NOTA Man, Mannose SA, stearylamine NOTA-IO-Man, NOTA-Iron Oxide Nanoparticles-Mannose DLS, Dynamic Light Scattering TEM, Transmission Electron Microscopy ICP-MS, Inductively Coupled-Mass Spectrometer 68. Ga, Gallium-68. ITLC-SG, Instant Thin Layer Chromatography-Silica Gel PET/CT, Positron Emission Tomography/Computed Tomography MRI, Magnetic Resonance Imaging SLN, Sentinel Lymph Node. vii. (13) INTRODUCTION. Development of medical imaging systems accelerated the treatment timing by early diagnosis of diseases and contributed to the improvement of recovery rate. Various medical imaging systems, such as Positron Emission Tomography (PET), Computed Tomography (CT), Single Photon Emission Computed Tomography (SPECT), and Magnetic Resonance Imaging (MRI) have been successfully developed for clinical applications. However, using one modality for imaging a disease was not enough to provide all biological information for diagnosis. Hence, combination of two of more imaging modalities might be more appropriate to obtain images for diagnosis.. PET is a nuclear imaging modality, which uses positron emitters. PET can show functional differences in molecular levels using radiopharmaceuticals specific to target organs. It is also possible to detect the molecular events non-invasively with high sensitivity and specificity; however, it cannot provide exact anatomical information because of low spatial resolution.. PET/CT has replaced PET-only imaging systems. CT provides anatomical 1. (14) information, which results in improved diagnostic images. MRI is a powerful medical imaging technique in radiology, which provides excellent contrast bepolysorbate the soft tissues. Thus, it generally can give more detailed anatomical information than CT with higher resolution. Furthermore, it can reduce radiation exposure compared to CT because it uses radiofrequency with a large magnet instead of X-rays. PET/MRI system emerged about a decade ago for better diagnostic information, and then was improved with significant associated technological development (4). Recently, preclinical study reports using PET/MRI system are increasing (5-14). However, most of the PET/MRI studies used radiopharmaceuticals only for PET imaging not for MRI to date. So, it is necessary to develop multimodal imaging agents which provide both PET and MRI information simultaneously for PET/MRI dual modality imaging system. Medical applications of nanoparticles (NPs) have been actively studied due to numerous advantages. NPs are nanometer sized and spherical shaped objects that can be used for radioisotope delivery in living organism. In particular, iron oxide (IO) NPs has been applied for clinical trial as a MRI contrast agent because of magnetism and low toxicity. Compare to other NPs – for example, quantum dots (QDs) which are made of binary alloys such as cadmium 2. (15) selenide (CdSe) or cadmium sulfide (CdS) – IO NPs are safe because the components are biocompatible. This material is composed of iron and an oxygen core (mostly magnetite, Fe3O4, or maghemite, 𝛾-Fe2O3). IO NPs are relatively benign MRI contrast agents because small ferromagnetic or ferromagnetic NPs have superparamagentism. Superparamagentic iron oxide (SPIO) and USPIO (ultrasmall superparamagnetic iron oxide) is extensively studied IO NPs (15-17). Those are suspended colloids of IO NPs for T2 signals. Porous structured NPs are suitable for use as a carrier to circulate in the living body. Those are required to circulate in in vivo for adequate time to uptake in the targeted organ. In addition, they have large surface-to-volume ratio than single NPs, as they can be surface-modified with targeting molecules or imaging modalities in high capacity.. Unfortunately, NPs presents possible dangers in medical approaches. Uncontrolled agglomeration of powder NPs due to attractive Van Der Waals forces is a serious problem for controlling of NPs. In addition, NPs are very reactive and catalytic because of their high surface-to-volume ratio; they have high potential to aggregates in in vitro conditions. In addition, there is a need to protected degradation in physiological condition such as pH variation, body 3. (16) temperature, and enzymatic reaction. Thus, for biological applications, surface coating of NPs is necessary to give stability and solubility. Nevertheless, un-functionalized NPs could uptake in a passive way by enhance permeability and retention (EPR) effect (18, 19). Thus, to prevent non-specific uptake and for better diagnosis, imaging functionalization of NPs are required for active targeting.. Medical imaging of sentinel lymph node (SLN) has clinical significance. They become inflamed or enlarged in various conditions, which may range from trivial, such as a throat infection, to life threatening, such as cancer. These can also be diagnosed by biopsy whenever they are inflamed. Certain diseases affect lymph nodes with characteristic consistency and localization, such as melanoma and breast cancer (20-22). Mannose receptor is an immune receptor that participates in the endocytosis of glycoproteins by macrophages. In particular, mannose receptor on lymphatic endothelial cells contributes to the metastatic behavior of cancer cells (23). Therefore, imaging lymph nodes offers an effective way to early diagnosis of cancer.. Thus, the aim of the present study is to develop specified PET/MRI dual4. (17) imaging probe for targeting lymph nodes. A facile encapsulation method using specific amiphiphiles was used for surface modification of IO NPs.. 5. (18) MATERIALS AND METHODS. 1. General remarks Magnetic iron oxide nanoparticles (Fe3O4, [Iron(II, III) oxide]) (in non-polar solvent; chloroform, d=5 nm) were purchased from MKnano (MK Impex Corp., ON, Canada). All other reagents and solvents were purchased from Sigma-Aldrich (MO, USA).. 1. H-Nuclear Magnetic Resonance (NMR). spectroscopy was performed with a 600MHz, Avance-600 (Bruker Corporation, MA, USA). A Waters 3100 Liquid Chromatography-Mass Spectroscopy (LC-MS) was used. A FC203B fraction collector was used (Gilson, Inc., WI, USA). Hydrodynamic diameter and size distribution of nanoparticles was analyzed using Dynamic Light Scattering (DLS) system with Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK) and JEM-1400 transmission electron microscope (TEM) from JEOL (Tokyo, Japan). A Varian 820-MS was used for Inductively Coupled-Mass Spectrometer (ICP-MS) (Varian Inc., CA, USA). A Sinco S-3100 was used for UV/Vis spectrometer (SCINCO America, WI, USA). 68Ge-68Ga generator was purchased from ITG (itg GmbH, Munich, Germany). Instant TLC-silica 6. (19) gel (ITLC-SG) plates were purchased from Agilent Technologies Inc. (CA, USA). Radio-Thin Layer Chromatography (TLC) was counted using a Bio-Scan AR-2000 System imaging scanner (Bioscan, WI, USA). A Vista micro PET/CT scanner (eXplore Vista PET/CT, GE Healthcare, CT, USA) was used for Positron Emission Tomography (PET) imaging. A 3-T MR scanner (MAGNETOM Trio, Siemens, Munich, Germany) was used for Magnetic Resonance Imaging (MRI) system. An Olympus BX-51 (Olympus, PA, USA) and Leica DFC280 (Leica Microsystsems CMS GmbH, Wetzlar, Germany) were used for light microscope and digital imaging system. All animal experiments were performed in Seoul National University Hospital, Seoul, Korea, which is fully accredited by AAALAC International (2007, Association for Assessment and Accreditation of Laboratory Animal Care International).. 2. Encapsulation of IO NPs 2.1 Synthesis of specialized amphiphiles 2.1.1 NOTA-SA (NOTA-stearylamine) To a solution of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane1,4,7-triacetic acid (p-SCN-Bn-NOTA) (150 mg, 0.268 mmol, 1.0 eq) in 7. (20) 2 mL of CHCl3, added TEA (0.112 mL, 0.804 mmol, 3.0 eq) and stirred for 10 min at room temperature. Stearyl amine (145 mg, 0.536 mmol, 2.0 eq) was added to the reaction mixture and stirred for overnight at room temperature. The reaction mixture was concentrated using a rotary vacuum evaporator at 40°C. The resulting oil was purified using a silica gel (~20 g) column chromatography (90:10=MC:MeOH; Rf=0.1).. 2.1.2 Man-SA (Mannose-stearylamine) To a solution of α-D-Mannopyranosylphenyl isothiocyannate (100 mg, 0.319 mmol, 1.0 eq) in 2 mL of CHCl3, added TEA (0.133 mL, 0.957 mmol, 3.0 eq) and stirred until the mixture turns clear. Added stearyl amine (172 mg, 0.638 mmol, 2.0 eq) and stirred for overnight at room temperature. The reaction mixture was concentrated using a rotary vacuum evaporator at 40°C. The resulting organic layer was purified using a silica gel (~ 20 g) column chromatography (90:10=MC:MeOH; Rf=0.1).. 8. (21) Figure 1. Synthesis schemes of functionalized amphiphiles (A) NOTA-SA, (B) Man-SA.. 9. (22) 2.2 Encapsulation of IO NPs 2.2.1 Encapsulation of IO NPs with polysorbate 60 Prepared 1, 2, 3, … , 8, 9, 10% polysorbate 60 solutions (v/v) and sonicated for 30 min. To 4 mL glass vials added 10 μL of IO NPs (5 mg/mL in CHCl3) and evaporated CHCl3. Added polysorbate 60 solutions each and sonicated for 3 hrs. The reaction mixtures were centrifuged (15,000 rpm, 25°C, 5 min) to separate supernatants and pellets. Each supernatants and pellets were analyzed by ICP-MS to measure the concentration of ferric ion (Fe3+).. 2.2.2 Encapsulation of IO NPs with functionalized amphiphiles Prepared 8% polysorbate 60 solution (v/v) and sonicated for 30 min. To 4 mL glass vial added 2 mol% of NOTA-SA (0.94 mg, 1 mg/mL in CHCl3) and 5 mol% of Man-SA (1.95 mg, 1 mg/mL in 1:1=CHCl3:MeOH) and evaporated all of the organic solvents. Added 1 mL of 8% polysorbate 60 solution and sonicated for 30 min to make micelle based reaction mixture. Iron oxide (IO) in CHCl3 was also sonicated for 30 min. To micelle based reaction mixture, added 200 μL of IO NPs (5 mg/mL in CHCl3) was added during sonication, and sonicated for 30 min. The reaction mixture was heated at 80°C for 5 min and opened the crew cap during the sonication to 10. (23) evaporate CHCl3 and sonicated for 5 min. This was repeated for 3 times and until now all sonication was in water-bath sonicator. Remaining CHCl3 was eliminated using rotary vacuum evaporator until the reaction solution turns clear and sonicated using ultra sonicator for 3 hrs (Amplitute=70%, Cycle=1, 77.8W).. 2.3 Purification and Characterization 2.3.1 Size exclusion column purification Reaction mixture was purified using Sephacryl® S500-HR (14.5 x 150 mm, V0=2.37 mL) gel filtration with distilled water using fraction collector (0.5 mL x 80). Final compounds were concentrated using ultra-filtration (Amicon Ultra-0.5, 100 kDa, 5,000 g, 25°C, 5 min).. 2.3.2 Size analysis Encapsulated IO NPs were measured their hydrodynamic diameter and size distribution using dynamic light scattering (DLS) instrument. Final concentrated IO NPs solutions were diluted 100 times in distilled water and dispersed well using sonication for 1 min. The particle size and distribution were obtained in number-percent (%) value at 25°C at a scattering angle of 11. (24) 90°. Transmission electron microscopy (TEM) was used for shape examination and for size checking. Final product was dropped to grid and image was taken with an acceleration voltage of 80 keV.. 12. (25) Figure 2. Preparation of encapsulated IO NPs with polysorbate 60, NOTA-SA and Man-SA. 13. (26) 2.3.3 Determination of ferric ion (Fe3+) After encapsulation with 10 different polysorbate 60 solutions separated supernatants and pellets were diluted to 10 mL of distilled water. Ferric ion from each samples were analyzed using ICP-MS. Ferric ion from encapsulated iron oxide with functionalized amphiphiles, NOTA-IO-Man, was determined using ferric thiocyanate colorimetric method. For Beer’s law plot for [Fe(SCN)2+], 2x10-3 M of Fe(NO3)3 solution and 2x10-3 M KSCN solution was prepared for thiocyanatoiron(III) ion ([Fe(SCN)2+]. To 20 mL scintillation vials, labeled the 1 to 5. Pipetted 1, 2, 3, 4, 5 mL of 2x10-3 M of Fe(NO3)3 solution and 5, 4, 3, 2, 1 mL of 0.5 M HNO3 solution, respectively into vials 1 – 5. Pipetted 5 mL of 2x10-3 M KSCN solution to 5 vials each. Shook the vials and left at room temperature for 15 min. The absorbance of each solution was measured in the UV-Vis spectrophotometer using a 0.5 M HNO3 blank at a wavelength of 457 nm. Make a graph of absorbance vs. concentration of Fe3+ and performed linear regression. This equation was used for standard plot for [Fe(SCN)2+]. To determinate unknown concentration of Fe3+ from encapsulated IO NPs, 10 μL of NOTA-IO-Man was added to 490 μL of 0.1 M HNO3 solution and vortexed and left at room temperature for 30 min. Added 500 μL of 1 M 14. (27) KSCN solution and shook and left at room temperature for 30 min. Final. solution. was. measured. the. absorbance. in. the. UV-Vis. spectrophotometer using a 0.5 M HNO3 blank at a wavelength of 457 nm. The concentration of Fe3+ was calculated with measured absorbance value using standard plot.. 2.4 68Ga labeling To 100 μL of 1 M sodium acetate buffer (pH 5.6) added 50 μL of encapsulated iron oxide (0.200 mM) followed by 500 μL of 68GaCl3/0.05 M HCl. Shook and reacted at 50°C for 10 min. Radiochemical purity was measured using radioTLC system (0.1 M citric acid/ITLC-SG).. 2.5 Stability test Encapsulated iron oxide with functionalized amphiphiles was incubated in high concentration of sodium chloride (NaCl) solution for stability test. To 1 mL of 0.9, 1.8, 3.6% NaCl solution, 100 μL of encapsulated iron oxide was added during sonication. And kept samples at room temperature for 1, 3, 24 hrs. After selected time checked size distribution of iron oxide using DLS system. Another stability test was performed in human serum. After labeling 15. (28) Ga, 100 μL of labeled compound was added to 1 mL of human serum and. 68. incubated at 37°C in shaking incubator. Control sample was. 68. Ga labeled. compound in 1 mL distilled water in room temperature. After 1 hr, 500 μL incubated sample was loaded to Sephacryl® S-500 HR gel filtration and eluted with distilled water using fraction collector (0.5 mL x 80). Control group was also eluted using same gel filtration with sample method. Radioactivity of each fraction was measured using gamma counters. Also 68. Ga labeled compound in human serum incubation, radiochemical purity. was checked using radioTLC system at 0, 10, 30, 60, 120 min.. 2.6 Phantom study Encapsulated iron oxide and Feridex® were serially diluted from a concentration of 0.2 mM in an agarose phantom designed for T2 relaxivity measurements, which was done using a 3-T MR scanner. Fast spin echo MR images of the phantom were acquired using the following parameters: relaxation time=5000 ms, echo times=16, 32, 48, 64, 20, 40, 60, 80, 50, or 100 ms, flip angle=180, ETL=18 fields of view, FOV=77x110 mm2, matrix=256x117, slice thickness/gap = 1.4 mm/1.8 mm, and NEX=1.. 16. (29) 3. Animal study 3.1 In vivo study After labeling. 68. Ga, labeled compound was concentrated using ultra-. filtration (Amicon Ultra-0.5, 100 kDa, 5,000 g, 25°C, 5 min). Sprague Dawley® rat (M, 8 weeks, 303.16 g) was anesthetized with isoflurane/O2 (2:1) before PET/CT imaging. To right footpad of the rat 12.02 MBq/10 μL of. 68. Ga labeled encapsulated iron oxide was s.c. injected using Hamilton. syringe. PET/CT image was obtained for 60 min (list mode). After PET/CT imaging, 100 μL of anesthetized solution (30% Zoletil:Rompun=4:1 solution in normal saline) was i.m. injected to right thigh of the animal and MRI images were obtained using same scanner which was used in phantom study. T2-weighted MR image was obtained using following parameters: TR=51 ms, TE=20 ms and slice thickness of 0.6 mm.. 3.2 Ex vivo study The animal was sacrificed using CO2 inhalation after in vivo imaging. Both left and right popliteal lymph nodes were isolated, embedded in paraffin and dissected (4 μm). Sliced sections were deparaffinized and soaked with 17. (30) working solution (1:1=20% HCl:10% KSCN) for 90 min in black plastic case to keep out the light. The tissue was rinsed with distilled water for 10 min. For nuclear-fast red solution, 0.1 g of nuclear-fast red and 5 g of aluminum sulfate was dissolved in 100 mL of distilled water and slightly heated to dissolve completely and filtered with 0.2 μm syringe filtered. The tissue was counterstained with nuclear-fast red solution for 15 min and washed in tap water for 10 min. The specimen was dehydrated and mounted with coverslip and performed microscope analysis.. 18. (31) RESULTS. 1. Encapsulation of IO NPs 1.1 Synthesis of NOTA-SA and Man-SA Final product of NOTA-SA was white powder after drying using a rotary vacuum evaporator at 50°C. Yield=192.9 mg (58%). 1H NMR (600 MHz, CDCl3): δ 7.365 (10, 1H, ddd, J=8.230, J=5.367, J=0.000), 7.193 (11, 1H, ddd, J=8.230, J=5.374, J=0.000), 3.536 (23, 2H), 2.884 (26, 1H, ddd, J=7.838, J=7.210, J=1.610), 3.042 (28, 1H, ddd, J=10.843, J=7.760, J=5.310), 3.156 (28, 1H, ddd, J=10.843, J=4.670, J=3.390), 3.528 (29, 2H), 1.232 (30, 2H, quint, J=0.000), 2.856 (34, 1H, ddd, J=9.111, J=5.310, J=3.390), 3.029 (34, 1H, ddd, J=9.111, J=7.760, J=4.670), 1.227 (47, 2H, quint, J=7.000), 0.851 (50, 3H, t, J=7.000) (Figure 3, A). MS(ESI+) m/z calculated for C38H65N5O6S [M+H]+ 720.0176, found 721.3002 (Figure 4, A). Final compound of ManSA was obtained in white powder after drying using a rotary vacuum evaporator at 50°C. Yield=185.9 mg (75%). 1H NMR (600 MHz, CDCl3): δ 7.270 (7, 1H, ddd, J=8.704, J=4.627, J=0.000), 1.261 (14, 2H, tt, J=7.000, J=6.500), 1.238 (16, 2H, tt, J=6.866, J=6.500), 1.238 (18, 2H, tt, J=6.866, 19. (32) J=0.000), 3.587 (22, 1H, td, J=6.293, J=2.680) (Figure 3, B). MS(ESI+) m/z calculated for C31H54N2O6S [M+H]+ 582.3661, found 583.1211 (Figure 4, B).. 20. (33) Figure 3. 1H NMR spectra data of functionalized amphiphiles (A) NOTA-SA, (B) Man-SA.. 21. (34) Figure 4. LC-MS data of functionalized amphiphiles (A) NOTA-SA, (B) Man-SA.. 22. (35) 1.2 Encapsulation of IO NPs with Polysorbate60 IO NPs were dispersed in polysorbate 60 solution. Reaction mixture was brown transparent liquid. An absolute quantity of encapsulated IO NPs assumed as concentration of Fe (mg/L) in supernatants. As the polysorbate 60 percentages increase, the amount of encapsulated Fe (mg/L) increased (Figure 5, A). Recovery efficiency (%) ratio was calculated as following formula.. ⁄. Recovery efficiency (%) =. ⁄. x 100. From 1% to 7% polysorbate60 solutions pellet was formed after centrifugation, but did not formed from 8% to 10% polysorbate 60 solutions. And from 8% polysorbate 60 solutions, recovery ratio (%) was more than 90% (Figure 5, B). Thus, we selected 8% polysorbate 60 solution to encapsulated IO NPs to give water solubility.. 23. (36) Figure 5. ICP-MS results of encapsulated IO NPs with various polysorbate 60 solutions (A) Concentration of ferric ion (Fe3+) from supernatant of encapsulated IO NPs with various % of polysorbate 60. (B) Recovery efficiency (%).. 24. (37) 1.3 Encapsulation of IO NPs with Polysorbate60, NOTA-SA and Man-SA: NOTA-IO-Man Using encapsulation method, 5 nm IO NPs were surface modified with 8% polysorbate 60, NOTA-SA and Man-SA. The hydrodynamic diameter of NOTA-IO-Man was 10.12±1.46 nm in DLS data (Figure 6, A). The zetapotential was -2.87±4.47 mV and the polydispersity index was 0.611. TEM images showed NOTA-IO-Man was well dispersed in a narrow size distribution (Figure 6, B).. 25. (38) Figure 6. Size distribution of NOTA-IO-Man (A) Size distribution of NOTA-Man in number (%) was measured by DLS system. The hydrodynamic size represents 10.12±1.46 nm. (B) TEM image of NOTA-IO-Man. Scale bar indicates 10 nm. Data are expressed as means ± SDs of five independent experiments.. 26. (39) 1.4 Determination of ferric ion (Fe3+) Colorimetric methods of [Fe(SCN)2+] follows below equation.. Fe3+ (aq) + SCN (aq). [Fe(SCN)2+] (aq). IO NPs were all decomposed to Fe3+ ion in HNO3 solution and formed ferric thiocyanate ([Fe(SCN)2+]) chelator after adding KSCN solution. Final solutions were red color and showed peak valley at 457 nm in UV-Vis spectrometer analysis (Figure 7, A). Five different concentration of [Fe(SCN)2+] solutions were used to make a Beer’s law plot, which showed high correlation bepolysorbate absorbance and concentration (r2=0.9993) (Figure 7, B). This standard equation was used to measure the concentration of Fe3+ from unknown sample after encapsulation of IO NPs.. 27. (40) Figure 7. Colorimetric method of [Fe(SCN)2+] (A) UV-Vis spectrum of. [Fe(SCN)2+] in 5 different concentration at. 457 nm, (B) Standard equation of [Fe(SCN)2+].. 28. (41) 1.5 68Ga labeling of NOTA-IO-Man NOTA-IO-Man was labeled with 68Ga in high radiochemical yield (>95%). Using radio TLC system 68Ga labeled NOTA-IO-Man could certainly sorted with free 68Ga. With same radio TLC condition, 0.1 M citric acid/ITLC-SG, free 68Ga showed peak in Rf =1.0 (Figure 8, A) and 68Ga-NOTA-IO-Man was in Rf =0.1 (Figure 8, B).. 29. (42) Figure 8. Radio TLC analysis of free 68Ga and 68Ga-NOTA-IO-Man (A) Free. 68. Ga in Rf=1.0, (B). 68. Ga-NOTA-IO-Man showed 100%. radiochemical purity in Rf =0.1. Both were in same 0.1 M citric acid/ITLCLC conditions.. 30. (43) 2. In vitro study of NOTA-IO-Man 2.1 Stability test of NOTA-IO-Man in high concentration of NaCl solution NOTA-IO-Man was tested their stability in high concentrated NaCl solutions. NOTA-IO-Man was incubated in 0% (control), 0.9%, 1.8%, 3.6% NaCl solution at rt. At 0, 1, 3, 24 hrs later, checked the hydrodynamic size distribution of incubated NOTA-IO-Man using DLS system (Figure 9). Measured values at each time point was performed two-way ANOVA with the control (0% NaCl, 0 hr) values. From 0.9% to 3.6% NaCl solution until 24 hrs, NOTA-IO-Man was stable which showed no aggregation or decomposition in size distribution. (P>.05) (Table 1).. 31. (44) Figure 9. Stability test of NOTA-IO-Man in high concentration NaCl solutions The hydrodynamic size of NOTA-IO-Man was analyzed using DLS system. Data are expressed as means ± SDs of four independent experiments.. 32. (45) Table 1. Hydrodynamic size of NOTA-IO-Man in high concentration of NaCl solutions Time (h) 0 1 3 24. NaCl(%) 1.8. 0.9. 3.6. 9.71. ±. 1.33. 10.24. ±. 0.27. 9.46. ±. 0.71. 9.63. ±. 0.37. 10.12. ±. 0.91. 10.53. ±. 0.51. 10.20. ±. 0.36. 10.44. ±. 0.79. 10.69. ±. 0.64. 10.38. ±. 0.63. 10.27. ±. 0.64. 10.65. ±. 0.27. Values for size (d, nm) represent mean±S.D (n=4) * P>.05 (two-way ANOVA). The hydrodynamic size of NOTA-IO-Man after incubation in 0.9, 1.8, 3.6% NaCl solutions. Number (%) average particle size was determined by DLS system. Data are expressed as means ± SDs of five independent experiments.. 33. (46) 2.2 Stability test of 68Ga-NOTA-IO-Man in human serum NOTA-IO-Man was tested in human serum for stability test. Both control and human serum incubated 68Ga-NOTA-IO-Man was checked the hydrodynamic size using DLS system (Figure 10, A). Student’s t-test showed that there was no significant difference between two groups (P>.29). And 68Ga-NOTA-IOMan did not aggregated in human serum, which showed same elution profile in same size exclusion column (Figure 10, B). Until 2 hrs,. 68. Ga-NOTA-IO-. Man was stable that showed high radiochemical purities (%) in radio TLC system (Figure 10, C).. 34. (47) 35. (48) Figure 10. Stability test of 68Ga-NOTA-IO-Man in human serum (A) Hydrodynamic size distribution analysis of control and human serum incubated. 68. Ga-NOTA-IO-Man using DLS system (*P=.25), Data are. expressed as means ± SDs of four independent experiments, (B) Elution profile of control and 68Ga-NOTA-IO-Man using same size exclusion column, (C) Radiochemical purity (%) of. 68. Ga-NOTA-IO-Man in human serum. analysed using radio TLC system.. 36. (49) 2.3 T2-weighted MR images of the phantom Phantom study was performed for estimation of T2-weighted MR (3.0 T) signal of the NOTA-IO-Man and was compared with that of commercialized IO NPs basted probes (Feridex® ). NOTA-IO-Man showed more negative signal compared to Feridex® in same concentration (Figure 11, A). The calculated relaxivity coefficient (r2) of mM-1s-1 was 449.9 which was higher than Feridex® (180.38) (Figure 11, B).. 37. (50) Figure 11. In vitro T2-weighted MR images of phantom (A) T2-weighted MR images of phantom, (B) Calculated relaxivity coefficient value of NOTA-IO-Man vs. Feridex® .. 38. (51) 3. In vivo study of 68Ga-NOTA-IO-Man 3.1 PET/CT study of 68Ga-NOTA-IO-Man In vivo study was performed with 68Ga-NOTA-IO-Man with PET/CT imaging system. SLN image was obtained in high signal. Popliteal lymph node was showed most highly signal (Figure 12) and inguinal, mesenteric lymph node was shown successively. This represent that 68Ga-NOTA-IO-Man was uptake specifically to mannose receptor. And the imaging probes did not aggregated in in vivo system that drains to next lymph node successfully.. 39. (52) Figure 12. In vivo small-animal PET/CT images The PET/CT study was performed for 60 min dynamic imaging after s.c injection of 12.02 MBq. 68. Ga labeled probe to the left footpad of a mouse.. White arrow indicates injection site and red arrow indicates popliteal lymph node area.. 40. (53) 3.2 MRI study of 68Ga-NOTA-IO-Man After PET/CT imaging MRI study was performed with same animal. Resulted MRI image showed significant uptake of. 68. Ga-NOTA-IO-Man in. same popliteal lymph node site (Figure 13). Compare to the control MRI image that was performed before injection of the imaging probe, shows negative image in popliteal lymph node that represent the uptake of IO NPs in targeted organ.. 41. (54) Figure 13. In vivo MRI image T2-weighted MRI imaging was follow up for 30 min after PET/CT imaging. Red arrow indicates lymph node area. The negative MR signal demonstrates the existence of ferric ion.. 42. (55) 4. Ex vivo study of 68Ga-NOTA-IO-Man Microscopic histopathology examination of popliteal lymph nodes after Prussian blue staining was performed. The tissue section of left popliteal lymph node resulted in a distinctive blue pigment demonstrating the existence of ferric iron (Fe3+) (Figure 14, A), but not in right popliteal lymph node (Figure 14, B).. 43. (56) Figure 14. Popliteal lymph node biopsy study Microscopic histopathologic examination of popliteal lymph nodes after Prussian blue staining was performed. The blue pigment demonstrates the existence of ferric ion. (A) Left, (b) Right popliteal lymph node.. 44. (57) DISCUSSION. Surface modification is the most important aspect of NPs for imaging applications. In order to conjugate several materials to the surface of single NPs to allow multispecificity, modification requires multistep in conventional methods.. Usually,. the. reactions. are. performed. in. liquid. state.. Thus, concentration of NPs using centrifugation is always required at the end of a reaction at each step to decrease the volume for the next reaction. The amount. of. NPs. will. aggregate. through. repeated. centrifugation.. Consequentially, the final yield is very low.. Aim of this study was to encapsulate IO NPs using specially designed amphiphiles. Polysorbate 60 was used as a surfactant. It is a nonionic detergent that is a mixture of polyoxyethylene ethers of mixed partial stearic (47.0–55.0%) and palmitic acid (35.0–50.0%) ester of sorbitol anhydrides. The U.S Food and Drug Administration (FDA) approved polysorbate 60 as food additive emulsifier at the 17th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1973, with an ADI of 0–25 mg/kg body weight/day. As I mentioned before, polysorbate 60 contains 45. (58) 20 polyethylene glycol (PEG) in its structure (Figure 2). PEG is preferred material to encapsulate NPs for solubility and stability (24-27). This is because it is water-soluble and easily coupled to hydrophobic molecules to produce non-ionic surfactants. Although advanced research was already reported using polysorbate 60 to encapsulate quantum dot NPs (1), encapsulation condition using polysorbate 60 has changed in order to apply for IO NPs. IO NPs were encapsulated with 8% polysorbate 60 solution with high recovery efficiency (%).. To give multimodality, NOTA-SA was used for labeling with 68Ga. And for multispecificity, Man-SA was used for SLN targeting. Those functionalized amphiphiles. were. mixed. with. 8%. polysorbate. 60. solutions. and. simultaneously encapsulated the IO NPs. Reaction mixture was purified twice using Sephacry® S-500 HR packed size exclusion column. Micelles composed of polysorbate 60, NOTA-SA, and Man-SA, which were not used to encapsulate the IO NPs, were eliminated completely.. In order to use IO NPs as imaging probes, it is necessary to measure the concentration before in vivo study. In the present study, colorimetric methods 46. (59) of [Fe(SCN)2+] were performed. The [Fe(SCN)2+] complex solutions are red, with significant absorbance at 457 nm. This method is convenient to measure the concentration of IO NPs on site. Radiochemical yield (%) was high after 68. Ga labeling. No significant aggregation or degradation was found under. various harsh conditions. The radiochemical purity of. 68. Ga-NOTA-IO-Man. was >99% and was stable in human serum for 120 min; in addition, no size transition was observed.. The calculated relaxivity coefficient (r2) of mM-1s-1 was 449.9, which was higher than Feridex® (180.38). The specificity of. 68. Ga-NOTA-IO-Man was. confirmed showing both PET/CT and MRI signals at the same site. The tissue section of the left popliteal lymph node resulted in a distinctive blue pigment, demonstrating the existence of ferric iron.. In conclusion, I modified surface of IO NPs with special lamphiphiles. IO NPs provided specificity and multimodality using this one-pot encapsulation method. This method can be applied for preparation of other hydrophobic NPs effectively.. 47. (60) REFERENCES. 1.. Lee YK, Jeong JM, Hoigebazar L, Yang BY, Lee YS, Lee BC, et al.. Nanoparticles modified by encapsulation of ligands with a long alkyl chain to affect multispecific and multimodal imaging. J Nucl Med. 2012 Sep;53(9):1462-70.. 2.. Choi JY, Jeong JM, Yoo BC, Kim K, Kim Y, Yang BY, et al. Development. of 68Ga-labeled mannosylated human serum albumin (MSA) as a lymph node imaging agent for positron emission tomography. Nucl Med Biol. 2011;38(3):371-9.. 3.. Jeong JM, Hong MK, Chang YS, Lee YS, Kim YJ, Cheon GJ, et al.. Preparation of a promising angiogenesis PET imaging agent: Ga-68-labeled c(RGDyK)-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic. acid. and. feasibility studies in mice. J Nucl Med. 2008 May;49(5):830-6.. 4.. Judenhofer MS, Cherry SR. Applications for preclinical PET/MRI. Semin. Nucl Med. 2013 Jan;43(1):19-29.. 5.. Platzek I, Beuthien-Baumann B, Schneider M, Gudziol V, Langner J,. Schramm G, et al. PET/MRI in head and neck cancer: initial experience. Eur J Nucl Med Mol Imaging. 2013 Jan;40(1):6-11.. 6.. Moodley KK, Minati L, Barnes A, Dickson JC, Ell PJ, Chan D.. Simultaneous PET/MRI in frontotemporal dementia. Eur J Nucl Med Mol Imaging. 2013 Feb;40(3):468-9. 48. (61) 7.. Kjaer A, Loft A, Law I, Berthelsen AK, Borgwardt L, Lofgren J, et al.. PET/MRI in cancer patients: first experiences and vision from Copenhagen. MAGMA. 2013 Feb;26(1):37-47.. 8.. Keller SH, Holm S, Hansen AE, Sattler B, Andersen F, Klausen TL, et al.. Image artifacts from MR-based attenuation correction in clinical, whole-body PET/MRI. MAGMA. 2013 Feb;26(1):173-81.. 9.. Heusch P, Buchbender C, Beiderwellen K, Nensa F, Hartung-Knemeyer V,. Lauenstein TC, et al. Standardized uptake values for [(18)F] FDG in normal organ tissues: Comparison of whole-body PET/CT and PET/MRI. Eur J Radiol. 2013 Feb 7.. 10.. Garibotto V, Heinzer S, Vulliemoz S, Guignard R, Wissmeyer M, Seeck M,. et al. Clinical applications of hybrid PET/MRI in neuroimaging. Clin Nucl Med. 2013 Jan;38(1):e13-8.. 11.. Cho ZH, Son YD, Choi EJ, Kim HK, Kim JH, Lee SY, et al. In-vivo human. brain molecular imaging with a brain-dedicated PET/MRI system. MAGMA. 2013 Feb;26(1):71-9.. 12.. Arce-Calisaya P, Souvatzoglou M, Eiber M, Beer A, Scheidhauer K,. Geinitz H, et al. Sensitivity of PET/MRI to detect recurrence of prostate cancer. Eur J Nucl Med Mol Imaging. 2013 Feb 22.. 49. (62) 13.. Yoon HS, Ko GB, Kwon SI, Lee CM, Ito M, Song IC, et al. Initial results of. simultaneous PET/MRI experiments with an MRI-compatible silicon photomultiplier PET scanner. J Nucl Med. 2012;53(4):608-14.. 14.. Yamamoto S, Watabe T, Watabe H, Aoki M, Sugiyama E, Imaizumi M, et. al. Simultaneous imaging using Si-PM-based PET and MRI for development of an integrated PET/MRI system. Physics in Medicine and Biology. 2012;57(2):N1.. 15.. Huang. C,. Neoh. KG,. Kang E-T,. Shuter. B.. Surface. modified. superparamagnetic iron oxide nanoparticles (SPIONs) for high efficiency folatereceptor. targeting. with. low. uptake. by. macrophages.. J. Mater. Chem.. 2011;21(40):16094-102.. 16.. Mahmoudi M, Sant S, Wang B, Laurent S, Sen T. Superparamagnetic iron. oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Adv Drug Deliv Rev. 2011 Jan-Feb;63(1-2):24-46.. 17.. Mahajan S, Koul V, Choudhary V, Shishodia G, Bharti AC. Preparation and. in vitro evaluation of folate-receptor-targeted SPION-polymer micelle hybrids for MRI contrast enhancement in cancer imaging. Nanotechnology. 2013 Jan 11;24(1):015603.. 18.. Xiao K, Luo JT, Li YP, Xiao WW, Lee JS, Gonik AM, et al. The Passive. Targeting of Polymeric Micelles in Various Types and Sizes of Tumor Models. Nanosci Nanotech Let. 2010 Jun;2(2):79-85.. 50. (63) 19.. Prabhakar U, Maeda H, Jain RK, Sevick-Muraca EM, Zamboni W,. Farokhzad OC, et al. Challenges and Key Considerations of the Enhanced Permeability and Retention Effect for Nanomedicine Drug Delivery in Oncology. Cancer Research. 2013 Apr 15;73(8):2412-7.. 20.. Bluemel C, Schnelzer A, Okur A, Ehlerding A, Paepke S, Scheidhauer K, et. al. Freehand SPECT for image-guided sentinel lymph node biopsy in breast cancer. Eur J Nucl Med Mol I. 2013 Oct;40(11):1656-61.. 21.. Abe H, Schacht D, Kulkarni K, Shimauchi A, Yamaguchi K, Sennett CA, et. al. Accuracy of Axillary Lymph Node Staging in Breast Cancer Patients: An Observer-Performance Study Comparison of MRI and Ultrasound. Acad Radiol. 2013 Nov;20(11):1399-404.. 22.. Radziszewski J, Kowalewska M, Jedrzejczak T, Kozlowicz-Gudzinska I,. Nasierowska-Guttmejer A, Bidzinski M, et al. The accuracy of the sentinel lymph node concept in early stage squamous cell vulvar carcinoma. Gynecol Oncol. 2010 Mar;116(3):473-7.. 23.. Marttila-Ichihara F, Turja R, Miiluniemi M, Karikoski M, Maksimow M,. Niemela J, et al. Macrophage mannose receptor on lymphatics controls cell trafficking. Blood. 2008 Jul 1;112(1):64-72.. 24.. Chen YJ, Tao J, Xiong F, Zhu JB, Gu N, Geng KK. Characterization and in. vitro cellular uptake of PEG coated iron oxide nanoparticles as MRI contrast agent. Pharmazie. 2010 Jul;65(7):481-6. 51. (64) 25.. Cole AJ, David AE, Wang J, Galban CJ, Hill HL, Yang VC. Polyethylene. glycol modified, cross-linked starch-coated iron oxide nanoparticles for enhanced magnetic tumor targeting. Biomaterials. 2011 Mar;32(8):2183-93.. 26.. Knop K, Hoogenboom R, Fischer D, Schubert US. Poly(ethylene glycol) in. Drug Delivery: Pros and Cons as Well as Potential Alternatives. Angew Chem Int Ed Engl. 2010;49(36):6288-308.. 27.. Larsen EK, Nielsen T, Wittenborn T, Birkedal H, Vorup-Jensen T,. Jakobsen MH, et al. Size-Dependent Accumulation of PEGylated Silane-Coated Magnetic Iron Oxide Nanoparticles in Murine Tumors. Acs Nano. 2009 Jul 2.. 52. (65) 국문 초록. 서론: 다중영상용 프로브는 각 영상 시스템의 장점을 융합하고 단점 을 보완함으로써 상승효과를 기대할 수 있다. 나노입자는 그 자체로 서 매우 좋은 조영제이자 운반체이며, 표면적이 넓어서 다양한 표면 처리가 가능하여 다중영상 프로브로 사용하기 좋다. 이 연구에서는 특수한 엠피파일로 인캡슐레이션 하는 방법을 사용하여 다중영상용 산화철 나노입자를 개발하였다.. 방법: 특이적 엠피파일들은 스테아릴아민을 SCN-Bn-NOTA 및 SCN-Bn-만노즈와 결합시켜 합성하였다. 만노즈는 림프절을 영상 화 하기 위한 표적용 물질로 사용되었다. NOTA 는 갈륨-68 을 표 지 하기 위한 킬레이트제로 사용되었다. 산화철 나노입자는 폴리소 르베이트 60 과 특이적 엠피파일들이 함께 섞인 용매와 초음파 분 산기를 이용하여 인캡슐레이션 하였다. 반응 용매는 분자체 크로마. 53. (66) 토그래피를 통하여 분리하고 원심 필터를 이용하여 농축시켰다. 최 종 농도는 457 nm 에서 UV/Vis 분광광도계를 이용한 비색분석을 통하여 구하였다. 인캡슐레이션 된 나노입자의 크기 분포는 DLS 기 기를 이용하여 분석하였다. 안정성 검사는 다양한 조건에서 시행되 었다 (0.9, 1.8, 3.6% NaCl 용액, rt, 1, 3, 24 시간; 사람 혈청, 37℃, 2 시간). 최종 물질의 자성 정도를 확인하기 위해서는 팬텀을 만들어 T2 강조신호를 확인하였고 (3.0 T) 이 수치는 상용화 된 산화철 나노입자 조영제인 페리덱스와 비교하였다. 생체 내 실험을 위해서 갈륨-68 이 표지 된 물질은 랫드의 왼쪽 발바닥에 피하 주 사하여 (12.0 MBq/10 μL) 60 분간 dynamic PET 영상을 촬영하 였고 그 직후에 MRI 를 이용하여 T2 강조영상을 30 분간 얻었다. 영상을 찍은 후 실험동물은 희생하고 양쪽 림프절을 각각 적출하여 산화철 나노입자의 존재여부를 확인하기 위한 프러시안 블루 염색 을 통한 병리조직학적 검사를 시행하였다.. 결과: 최종 물질인 NOTA-IO-Man 의 크기는 10.1±1.5 nm 였으 며 이는 높은 인캡슐레이션 수득률로 얻어졌다 (92%). 다양한 가 54. (67) 혹 조건 하에서 실시한 안정성 시험 결과 최종 물질의 특이적인 뭉 침이나 분해현상은 관찰되지 않았다.. 68. Ga-NOTA-IO-Man 의 방. 사화학적 순도는 99% 이상이었으며 이 또한 사람 혈청 내에서 120 분간 크기 변화 없이 안정하였다 (10.1±1.5 nm, P=.29). 측 정된 mM-1s-1 에 대한 이완성 계수(r2) 값은 449.9 로 페리덱스 (180.38)보다 약 2 배 이상 높은 수치였다. PET/CT 와 MRI 영상 실험에서는. 68. Ga-NOTA-IO-Man 가 같은 림프절 자리에 선택적. 으로 섭취된 것을 확인할 수 있었다. 적출된 왼쪽 오금 부위의 림프 절은 파란색으로 확연히 조직이 염색되는 것으로 보아 철 이온의 존재를 확인할 수 있었다. 결론: 이 연구에서는 특수 엠피파일로 인캡슐레이션하는 방법으로 림프절을 영상화 할 수 있는 PET/MRI 이중영상용 프로브를 개 발하는데 성공하였다. ------------------------------------주요어 : PET/MRI, 다중영상, 표면화학, 엔피파일, 인캡슐레이션, 산화철 나노입자 학 번 : 2008-23288 55. (68)