Development of Pinhole X-ray Fluorescence Imaging System to Measure in vivo Biodistribution of Gold Nanoparticles

학위논문(박사)--서울대학교 대학원 :융합과학기술대학원 융합과학부,2019. 8. 예성준.목적: 본 연구의 목표는 금나노입자의 체내 농도 분포 측정을 위한 핀홀 엑스선 형광 영상시스템을 개발하고, 시간에 따른 쥐의 체내 금나노입자 분포 영상을 획득하여 개발 영상시스템이 전임상시험에 활용 가능함을 실험적으로 증명하는 것이다. 2차원 cadmium zinc telluride (CZT) 감마 카메라를 사용하여 K-shell 엑스선 형광 신호를 측정함으로써, 영상 획득 시간과 피폭 방사선량을 줄일 수 있다. 또한, 본 연구는 샘플의 복잡한 전처리 과정 없이 금나노입자의 체외 농도를 측정할 수 있는 silicon drift detector (SDD)를 사용한 L-shell 엑스선 형광 측정 시스템을 개발하고자 한다. 방법: 금나노입자의 농도와 K-shell 엑스선 형광 신호 사이의 교정 곡선을 획득하기 위해 0.0 wt%, 0.125 wt%, 0.25 wt%, 0.5 wt%, 1.0 wt%, 2.0 wt%의 금나노입자 샘플을 반지름 2.5 cm인 아크릴 팬톰에 삽입하여 140 kVp 엑스선을 1분씩 조사하였다. K-shell 엑스선 형광 신호는 금나노입자가 삽입되어 있는 아크릴 팬톰으로부터 측정한 엑스선 스펙트럼에서 금나노입자가 삽입되어 있지 않은 아크릴 팬톰으로부터 측정한 엑스선 스펙트럼의 차이를 통해 추출하였다. 금나노입자 주입 후 측정 데이터만으로 금나노입자의 엑스선 형광 영상을 획득하기 위해 인공지능 convolutional neural network (CNN) 모델을 개발하고 적용하였다. 실험용 쥐로부터 추출한 장기의 금나노입자 농도 측정을 위해 L-shell 엑스선 형광 시스템측정을 개발하였으며, 이 시스템은 SDD 측정기와 40 kVp의 선원을 이용하여 2.34 μg – 300 μg (금나노입자)/30 mg (물) (0.0078 wt%-1.0 wt%)의 금나노입자와 L-shell 엑스선 형광 신호 사이의 교정 곡선을 얻어 장기 내 축적된 금나노입자의 질량을 측정하였다. 핀홀 엑스선 형광 영상시스템을 이용하여 실험용 쥐에 금나노입자를 주입 후 시간에 따른 신장 내 금나노입자 농도 영상을 획득하였다. 안락사 후 적출한 양쪽 신장, 간, 비장, 혈액의 금나노입자 농도를 L-shell 엑스선 형광 체외 측정 시스템과 ICP-AES를 사용하여 측정하였고 영상시스템을 통해 획득한 농도와 비교·검증하였다. 영상 획득 시 실험용 쥐에 조사되는 방사선량은 TLD를 실험용 쥐의 피부에 붙여 측정하였다. 결과: 엑스선 형광 영상 분석을 통해 측정한 실험용 쥐의 오른쪽 신장 내 금나노입자의 농도는 주입 직후 1.58±0.15 wt%였으며, 60분 후 그 농도는 0.77±0.29 wt%로 감소하였다. 개발한 인공지능 CNN 모델을 적용해 금나노입자 주입 전 영상의 획득 없이 금나노입자의 엑스선 형광 영상을 생성할 수 있었다. 적출한 장기에서 측정된 금나노입자의 신장 내 농도는 L-shell 엑스선 형광 측정법으로 0.96±0.22 wt%, ICP-AES로는 1.00±0.50 wt% 였다. 영상 획득 시 실험용 쥐의 피부에 전달된 방사선량은 금나노입자 주입 전과 후 영상을 모두 획득 시(총 2분) 107±4 mGy, CNN 모델 적용 시(1분) 53±2 mGy로 측정되었다. 결론: 2차원 CZT 감마 카메라와 핀홀 콜리메이터를 사용한 엑스선 형광 영상시스템은 영상 획득 시간과 피폭 방사선량을 크게 감소시켰으며, 살아있는 쥐의 시간에 따른 체내 금나노입자 분포 변화를 영상화 할 수 있음을 증명하였다. 또한 L-shell 엑스선 형광 측정 시스템은 복잡한 전처리 과정 없이 체외 금나노입자의 농도를 정확하게 측정할 수 있었다. 본 개발 시스템을 금속나노입자의 체내 분포 연구를 위한 전임상시험용 분자영상장비로서 활용할 수 있을 것으로 기대한다.Purpose: This work aims to show the experimental feasibility for a dynamic in vivo X-ray fluorescence (XRF) imaging of gold in living mice exposed to gold nanoparticles (GNPs) using polychromatic X-rays. By collecting K-shell XRF photons using a 2D cadmium zinc telluride (CZT) gamma camera, the imaging system was expected to have a short image acquisition time and deliver a low radiation dose. This study also investigated the feasibility of using an L-shell XRF detection system with a single-pixel silicon drift detector (SDD) to measure ex vivo GNP concentrations from biological samples. Methods: Six GNP columns of 0 % by weight (wt%), 0.125 wt%, 0.25 wt%, 0.5 wt%, 1.0 wt% and 2.0 wt% inserted in a 2.5 cm diameter polymethyl methacrylate (PMMA) phantom were used for acquiring a linear regression curve between the concentrations of GNPs and the K-shell XRF photons emitted from GNPs. A fan-beam of 140 kVp X-rays irradiated the phantom for 1 min in each GNP sample. The photon spectra were measured by the CZT gamma camera. The K-shell XRF counts were derived by subtracting the photon counts of the 0 wt% PMMA phantom (i.e., pre-scanning) from the photon counts of the GNP-loaded phantom (i.e., post-scanning). Furthermore, a 2D convolutional neural network (CNN) was applied to generate the K-shell XRF counts from the post-scanned data without the pre-scanning. For a more sensitive detection of the ex vivo concentrations of GNPs in the biological samples, the L-shell XRF detection system using the single-pixel SDD was developed. Six GNP samples of 2.34 μg–300 μg Au/30 mg water (i.e., 0.0078 wt%–1.0 wt% GNPs) were used for acquiring a calibration curve to correlate the GNP mass to the L-shell XRF counts. The kidney slices of three Balb/C mice were scanned at various periods after the injection of GNPs in order to acquire the quantitative information of GNPs. The concentrations of GNPs measured by the CZT gamma camera and the SDD were cross-compared and then validated by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The radiation dose was assessed by the measurement of TLDs attached to the skin of the mice. Results: The K-shell XRF images showed that the concentration of GNPs in the right kidneys from the mice was 1.58±0.15 wt% at T = 0 min after the injection. At T = 60 min after the injection, the concentration of GNPs in the right kidneys was reduced to 0.77±0.29 wt%. The K-shell XRF images generated by the 2D CNN were similar to those derived by the direct subtraction method. The measured ex vivo concentration of GNPs was 0.96±0.22 wt% by the L-shell XRF detection system while it was 1.00±0.50 wt% by ICP-AES. The radiation dose delivered to the skin of the mice was 107±4 mGy for acquiring one slice image by using the direct subtraction method while it was 53±2 mGy by using the 2D CNN. Conclusions: A pinhole K-shell XRF imaging system with a 2D CZT gamma camera showed a dramatically reduced scan time and delivered a low radiation dose. Hence, a dynamic in vivo XRF imaging of gold in living mice exposed to GNPs was technically feasible in a benchtop configuration. In addition, an L-shell XRF detection system can be used to measure ex vivo concentrations of GNPs in biological samples. This imaging system could provide a potential in vivo molecular imaging for metal nanoparticles to emerge as a radiosensitizer and a drug-delivery agent in preclinical studies.CHAPTER I. INTRODUCTION 1 I.1 Applications of Metal Nanoparticles in Medicine 1 I.2 Molecular Imaging of Metal Nanoparticles 3 I.3 X-ray Fluorescence Imaging 5 I.3.1 Principle of X-ray Fluorescence Imaging 5 I.3.2 History of X-ray Fluorescence Imaging 8 I.3.3 Specific Aims 12 CHAPTER II. MATERIAL AND METHODS 15 II.1 Monte Carlo Model 15 II.1.1 Geometry of Monte Carlo Simulations 15 II.1.2 Image Processing 21 II.1.3 Radiation Dose 27 II.2 Development of Pinhole K-shell XRF Imaging System 28 II.2.1 System Configuration and Operation Scheme 28 II.2.2 Pinhole K-shell XRF Imaging System 31 II.2.2.1 Experimental Setup 31 II.2.2.2 Measurement of K-shell XRF Signal 36 II.2.2.3 Signal Processing: Correction Factors 39 II.2.2.4 Application of Convolutional Neural Network 42 II.2.3 K-shell XRF Detection System 45 II.2.3.1 Experimental Setup 45 II.2.3.2 Signal Processing 47 II.2.4 L-shell XRF Detection System 49 II.2.4.1 Experimental Setup 49 II.2.4.2 Signal Processing 51 II.3 In vivo Study in Mice 53 II.3.1 Experimental Setup 53 II.3.2 Dose Measurement 56 CHAPTER III. RESULTS 57 III.1 Monte Carlo Model 57 III.1.1 Geometric Efficiency, System and Energy Resolution 57 III.1.2 K-shell XRF Image by Monte Carlo Simulations 59 III.1.3 Radiation Dose 69 III.2. Development of Pinhole XRF Imaging System 70 III.2.1 Pinhole K-shell XRF Imaging System 70 III.2.1.1 Energy Calibration and Measurement of Field Size 70 III.2.1.2 Raw K-shell XRF Signal 73 III.2.1.3 Correction Factors 78 III.2.1.4 K-shell XRF Image 81 III.2.2 K-shell XRF Detection System 85 III.2.3 L-shell XRF Detection System 89 III.3 In vivo Study in Mice 92 III.3.1 In vivo K-shell XRF Image 92 III.3.2 Quantification of GNPs in Living Mice 96 III.3.3 Dose Measurement 101 CHAPTER IV. DISCUSSION 102 IV.1 Monte Carlo Model 102 IV.2 Development of Pinhole K-shell XRF Imaging System 104 IV.2.1 Quantification of GNPs 105 IV.2.2 Comparison between MC and Experimental Results 107 IV.2.3 Limitations 108 IV.2.3.1 Concentration 108 IV.2.3.2 System Resolution 110 IV.2.3.3 Radiation Dose 111 IV.2.4 Application of CNN 112 IV.2.5 Future Work 114 CHAPTER V. CONCLUSIONS 115 REFERENCES 116 ABSTRACT (in Korean) 123Docto

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