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

Accomplishments and challenges in stem cell imaging in vivo.

Stem cell therapies have demonstrated promising preclinical results, but very few applications have reached the clinic owing to safety and efficacy concerns. Translation would benefit greatly if stem cell survival, distribution and function could be assessed in vivo post-transplantation, particularly in patients. Advances in molecular imaging have led to extraordinary progress, with several strategies being deployed to understand the fate of stem cells in vivo using magnetic resonance, scintigraphy, PET, ultrasound and optical imaging. Here, we review the recent advances, challenges and future perspectives and opportunities in stem cell tracking and functional assessment, as well as the advantages and challenges of each imaging approach

Liposomal nanotheranostics for multimode targeted in vivo bioimaging and near‐infrared light mediated cancer therapy

Developing a nanotheranostic agent with better image resolution and high accumulation into solid tumor microenvironment is a challenging task. Herein, we established a light mediated phototriggered strategy for enhanced tumor accumulation of nanohybrids. A multifunctional liposome based nanotheranostics loaded with gold nanoparticles (AuNPs) and emissive graphene quantum dots (GQDs) were engineered named as NFGL. Further, doxorubicin hydrochloride was encapsulated in NFGL to exhibit phototriggered chemotherapy and functionalized with folic acid targeting ligands. Encapsulated agents showed imaging bimodality for in vivo tumor diagnosis due to their high contrast and emissive nature. Targeted NFGL nanohybrids demonstrated near infrared light (NIR, 750 nm) mediated tumor reduction because of generated heat and Reactive Oxygen Species (ROS). Moreover, NFGL nanohybrids exhibited remarkable ROS scavenging ability as compared to GQDs loaded liposomes validated by antitumor study. Hence, this approach and engineered system could open new direction for targeted imaging and cancer therapy.publishersversionpublishe

Recent advances in cancer photo-theranostics: the synergistic combination of transition metal complexes and gold nanostructures

AbstractIn this mini review, we highlight advances in the last five years in light-activated cancer theranostics by using hybrid systems consisting of transition metal complexes (TMCs) and plasmonic gold nanostructures (AuNPs). TMCs are molecules with attractive properties and high potential in biomedical application. Due to their antiproliferative abilities, platinum-based compounds are currently first-choice drugs for the treatment of several solid tumors. Moreover, ruthenium, iridium and platinum complexes are well-known for their ability to photogenerate singlet oxygen, a highly cytotoxic reactive species with a key role in photodynamic therapy. Their potential is further extended by the unique photophysical properties, which make TMCs particularly suitable for bioimaging. Recently, gold nanoparticles (AuNPs) have been widely investigated as one of the leading nanomaterials in cancer theranostics. AuNPs—being an inert and highly biocompatible material—represent excellent drug delivery systems, overcoming most of the side effects associated with the systemic administration of anticancer drugs. Furthermore, due to the thermoplasmonic properties, AuNPs proved to be efficient nano-sources of heat for photothermal therapy application. Therefore, the hybrid combination TMC/AuNPs could represent a synergistic merger of multiple functionalities for combinatorial cancer therapy strategies. Herein, we report the most recent examples of TMC/AuNPs systems in in-vitro in-vivo cancer tharanostics application whose effects are triggered by light-exposure in the Vis–NIR region, leading to a spatial and temporal control of the TMC/AuNPs activation for light-mediated precision therapeutics

Standardization of research methods employed in assessing the interaction metallic-based nanoparticles and the blood-brain barrier: present and future perspectives

peer-reviewedThe full text of this article will not be available until the embargo expires on the 18/01/2020.Treating diseases of the central nervous system (CNS) is complicated by the presence of the blood-brain barrier (BBB), a semipermeable boundary layer protecting the CNS from toxins and homeostatic disruptions. However, this layer also excludes almost 100% of therapeutics, impeding the treatment of CNS diseases. The advent of nanoparticles, in particular metallic-based nanoparticles, presents the potential to overcome this barrier and transport drugs into the CNS. Recent interest in metallic-based nanoparticles has generated an immense array of information pertaining to nanoparticles of different materials, sizes, morphologies, and surface properties. Nanoparticles with different physico-chemical properties lead to distinct nanoparticle-host interactions; yet, comprehensive characterization is often not completed. Similarly, in vivo testing has involved a mixed evaluation of parameters, including: BBB permeability, integrity, biodistribution, and toxicity. The methods applied to assess these parameters are inconsistent; this complicates the comparison of different nanoparticle-host system responses. A systematic review was conducted to investigate the methods by which metallic-based nanoparticles are characterized and assessed in vivo. The introduction of a standardized approach to nanoparticle characterization and in vivo testing is crucial if research is to transition to a clinical setting. The approach suggested, herein, is based on equipment and techniques that are accessible and informative to facilitate the routine incorporation of this standardized, informative approach into different research settings. Thorough characterization could lead to improved interpretation of in vivo responses, which could clarify nanoparticle properties that result in favorable in vivo outcomes whilst exposing nanoparticle-specific weaknesses. Only then will researchers successfully identify nanoparticles capable of delivering life-saving therapeutics across the blood-brain barrier

Nanoparticle toxicity in Drosophila melanogaster: a case study with nickel, nickel oxide, and iron-nickel alloy nanoparticles

With an increase use of nanomaterials, growing concerns have risen about their potential exposure to environment and the risk of side effects on human health. My research investigates the ecological and mechanistic insights of in vivo nanoparticle toxicology via oral exposure, specifically metallic nickel, nickel-iron alloy, and nickel oxide particles, using the Drosophila melanogaster model system. In order to understand the physical and chemical behavior of the nickel-based nanoparticles that were used in this study, I characterized the particle size, morphology, aqueous aggregation state, and hydrodynamic zeta potential of these nanomaterials. I found that these nanomaterials displayed a distinct set of physicochemical properties and that these properties appear to have a significant role in the toxicological effects that I observed. Metallic nickel nanoparticles were toxic to the larvae D. melanogaster, having a dose-dependent mortality, a development delay in pupariation, inhibition of tissue growth, reduction of wing size, as well as the induction of the stress protein Hsp70 and ROS production. I also found unique fluorescent mineral depositions form in the Malpighian tubules after oral exposure to nickel nanoparticles. Energy-dispersive X-ray spectroscopy reveals that the chemical composition of these mineral crystals was calcium carbonate. The experimental evidence of the toxic effects of nickel nanoparticle effect via the oral route provides valuable information of risk and biohazard to the community

NANOCRYSTALS OF CHEMOTHERAPEUTIC AGENTS FOR CANCER THERANOSTICS: DEVELOPMENT AND IN VITRO AND IN VIVO EVALUATION

The majority of pharmacologically active chemotherapeutics are poorly water soluble. Solubilization enhancement by the utilization of organic solvents often leads to adverse side effects. Nanoparticle-based cancer therapy, which is passively targeted to the tumor tissue via the enhanced permeation and retention effect, has been vastly developed in recent years. Nanocrystals, which exist as crystalline and carry nearly 100% drug loading, has been explored for delivering antineoplastic agents. Additionally, the hybrid nanocrystal concept offers a novel and simple way to integrate imaging agents into the drug crystals, enabling the achievement of theranostics. The overall objective of this dissertation is to formulate both pure and hybrid nanocrystals, evaluate their performance in vitro and in vivo, and investigate the extent of tissue distribution and tumor accumulation in a murine model. Pure and hybrid nanocrystals of several model drugs, including paclitaxel (PTX), camptothecin, and ZSTK474, were precipitated by the antisolvent method in the absence of stabilizer, and their size was further minimized by homogenization. The nanocrystals of PTX, which is the focus of the study, had particle size of approximately 200 nm and close-to-neutral surface charge. Depending on the cell type, PTX nanocrystals exerted different level of cytotoxicity. In human colon and breast cancer xenograft models, nanocrystals yielded similar efficacy as the conventional formulation, Taxol, at a dose of 20 mg/kg, yet induced a reduced toxicity. Biodistribution study revealed that 3H-PTX nanocrystals were sequestered rapidly by the macrophages upon intravenous injection. Yet, apparent toxicity was not observed even after four weekly injections. The sequestered nanocrystals were postulated to be released slowly into the blood circulation and reached the tumor. Tritium-labeled-taxol, in contrast, was distributed extensively to all the major organs, inducing systemic toxicity as observed in significant body weight loss. The biodistribution results obtained from radioactive analysis and whole-body optical imaging was compared. To some degree, the correlation was present, but divergence in the quantitative result, due to nanocrystal integrity and limitations associated with the optical modality, existed. Despite their promising properties, nanocrystal suspensions must be securely stabilized by stealth polymers in order to minimize opsonization, extend blood-circulation time, and efficiently target the tumor

Multiphoton imaging of melanoma 3D models with plasmonic nanocapsules

We report the synthesis of plasmonic nanocapsules and the cellular responses they induce in 3D melanoma models for their perspective use as a photothermal therapeutic agent. The wall of the nanocapsules is composed of polyelectrolytes. The inner part is functionalized with discrete gold nanoislands. The cavity of the nanocapsules contains a fluorescent payload to show their ability for loading a cargo. The nanocapsules exhibit simultaneous two-photon luminescent, fluorescent properties and X-ray contrasting ability. The average fluorescence lifetime (τ) of the nanocapsules measured with FLIM (0.3 ns) is maintained regardless of the intracellular environment, thus proving their abilities for bioimaging of models such as 3D spheroids with a complex architecture. Their multimodal imaging properties are exploited for the first time to study tumorspheres cellular responses exposed to the nanocapsules. Specifically, we studied cellular uptake, toxicity, intracellular fate, generation of reactive oxygen species, and effect on the levels of hypoxia by using multi-photon and confocal laser scanning microscopy. Because of the high X-ray attenuation and atomic number of the gold nanostructure, we imaged the nanocapsule-cell interactions without processing the sample. We confirmed maintenance of the nanocapsules’ geometry in the intracellular milieu with no impairment of the cellular ultrastructure. Furthermore, we observed the lack of cellular toxicity and no alteration in oxygen or reactive oxygen species levels. These results in 3D melanoma models contribute to the development of these nanocapsules for their exploitation in future applications as agents for imaging-guided photothermal therapy. Statement of Significance: The novelty of the work is that our plasmonic nanocapsules are multimodal. They are responsive to X-ray and to multiphoton and single-photon excitation. This allowed us to study their interaction with 2D and 3D cellular structures and specifically to obtain information on tumor cell parameters such as hypoxia, reactive oxygen species, and toxicity. These nanocapsules will be further validated as imaging-guided photothermal probe

Dual-Targeted Multifunctional Nanoparticles for Magnetic Resonance Imaging Guided Cancer Diagnosis and Therapy

Hybrid nanostructures with combined functionalities can be rationally designed to achieve synergistic effects for efficient cancer treatment. Herein, a multifunctional nanoplatform is constructed, containing an inner core of an anticancer drug MTX surrounding by a nanometer-thin layer of gold as the shell with Fe₃O₄ magnetic nanoparticles (NPs) evenly distributed in the gold layer, and the outermost hybrid LA-PEG-MTX molecules as surface coating agent (denoted as MFG-LPM NPs). This nanocomposite possesses very high drug loading capacity as the entire core is MTX and integrates magnetic- and active- targeting drug delivery, light-controlled drug release, magnetic resonance imaging (MRI), as well as photothermal and chemotherapy. With a strong near-infrared (NIR) absorbance at 808 nm, the nanocomposite enables temperature elevation and light-triggered MTX release. In vitro cytotoxicity studies indicate that the strategy of combining therapy leads to a synergistic effect with high cancer cell killing efficacy. In consistency with this, due to the high accumulation of MFG-LPM NPs at tumor site and their combinatorial chemo-photothermal effects, 100% in vivo tumor elimination can be achieved. Additionally, in vivo MRI of tumor-bearing mice demonstrates an impressive performance of MFG-LPM NPs as a <i>T</i>₂ contrast agent. Therefore, such multifunctional nanocomposite has the potential to serve as an excellent theranostic agent that collectively integrates multiple functions for efficient MRI guided cancer diagnosis and treatment

Current concepts in nanostructured contrast media development for In vivo photoacoustic imaging

Photoacoustic (PA) imaging is indeed one of the most promising bioimaging techniques for theranostics applications in humans, allowing for the visualization of blood vessels and melanomas with high spatial resolution. However, in order to overcome the endogenous contrast arising from interfering endogenous species such as haemoglobin and melanin, specific contrast agents need to be developed, allowing PAI to successfully identify targeted contrast in the range of wavelengths in which interference from the biomatrix is minimized. This has been first performed by small molecule dyes, which, however, suffer from some important limitations such as low hydrophilicity and short circulation times. For this reason, scientific research has recently directed its efforts towards the development of nanostructured contrast agents capable of providing efficient PA contrast at low concentrations with low toxicity and high biocompatibility. The principal nanostructures are based on (1) metal and semiconducting nanoparticles, amongst which variously shaped nano-gold plays the main role, (2) carbon nanomaterials, such as carbon nanotubes and graphene, and (3) conjugated polymer nanoparticles. In this review, the principal characteristics of this class of materials are reported and greater focus is directed towards in vivo studies. A detailed analysis is performed on various physical-chemical parameters that define the PA response of reported contrast agents, like absorption coefficients and photoacoustic efficiencies. By comparing the experimental data, this review provides a comprehensive tool for the evaluation of new nanostructured contrast agents for PA imaging