Gold nanoparticle contrast agents in advanced X-ray imaging technologies

Abstract

Recently, there has been significant progress in the field of soft- and hard-X-ray imaging for a wide range of applications, both technically and scientifically, via developments in sources, optics and imaging methodologies. While one community is pursuing extensive applications of available X-ray tools, others are investigating improvements in techniques, including new optics, higher spatial resolutions and brighter compact sources. For increased image quality and more exquisite investigation on characteristic biological phenomena, contrast agents have been employed extensively in imaging technologies. Heavy metal nanoparticles are excellent absorbers of X-rays and can offer excellent improvements in medical diagnosis and X-ray imaging. In this context, the role of gold (Au) is important for advanced X-ray imaging applications. Au has a long-history in a wide range of medical applications and exhibits characteristic interactions with X-rays. Therefore, Au can offer a particular advantage as a tracer and a contrast enhancer in X-ray imaging technologies by sensing the variation in X-ray attenuation in a given sample volume. This review summarizes basic understanding on X-ray imaging from device set-up to technologies. Then this review covers recent studies in the development of X-ray imaging techniques utilizing gold nanoparticles (AuNPs) and their relevant applications, including two- and three-dimensional biological imaging, dynamical processes in a living system, single cell-based imaging and quantitative analysis of circulatory systems and so on. In addition to conventional medical applications, various novel research areas have been developed and are expected to be further developed through AuNP-based X-ray imaging technologies.

Figure 1

(A) Cryo X-ray microscopy of 3T3 cells [31]. (B) Direct comparison between the phase-contrast radiological image based on coherence of an optic fiber (on the left) and the corresponding absorption-contrast image (right) [34]. Adapted by permission from John Wiley and Sons and IOP Publishing.

Figure 2

(A) A dynamic flat-panel volumetric preclinical micro-CT scanner (eXplore Ultra, GE Healthcare, London, ON, Canada) images of rat lungs. Top left frame shows a minimum intensity projection of an oxygen-filled rat lung. Subsequent frames show the evolution of Xe with baseline image subtracted at 1s intervals. The field-of-view for each frame is 36.5 mm × 36.5 mm [36] (B) (a) Typical X-ray images of an (I) empty tube (II) blood flow, and (III) blood flow seeded with Iopamidol-incorporated microparticle inside the tube. (b) Comparison of absolute X-ray absorption of (I) stationary pure blood and (II) blood mixed with pure iopamidol. Images are obtained by 3^rd generation synchrotron X-ray source with unmonochromated beamline (bending magnet; energy range of 4–15 keV) [40]. Adapted by permission from the American Physiological Society and Elsevier.

Figure 3

Energy-dependent X-ray absorption coefficients of iodine (I) and gold (Au) [51]. Adapted by permission from National Institute of Standards and Technology (NIST).

Figure 4

(A) Various types of plasmon-resonant nanoparticles: 16 nm nanospheres (a) Au nanorods (b) Au bipyramids (c) Au nanorods surrounded by silver nanoshells (d) nanorice (Au-coated Fe₂O₃ nanorods) (e) SiO₂/Au nanoshells (f) (the inset shows a hollow nanoshell); nanobowls with bottom cores (g) spiky SiO₂/Au nanoshells (h) (the inset shows a Au nanostar); Au tetrahedra, octahedra, and cubooctahedra (i) Au nanocubes (j) Ag nanocubes and Au-Ag nanocages (obtained from those in the insets) (k) as well as Au nanocrescents (l) [9]. (B) Properties and possible biomedical applications of plasmonic nanoparticles. (C) Confocal image of HeLa cells in the presence of AuNPs [77]. (a) Blue indicates the nuclei stained with Hoechst 33258. Red indicates the actin cytoskeleton labeled with Alexa Fluor 488 phalloidin. Green indicates unlabeled AuNPs. The image is taken by two-photon microscopy. Dark-field microscopy of cancerous (b) and healthy (c) cells using AuNPs conjugated with antibodies for epidermal growth factor [78]. Adapted from the data of the cited papers by permission from The Royal Society of Chemistry, and The American Chemical Society.

Figure 5

(a) General anatomical diagram of a mosquito. The dorsal diverticulum [DD], foregut [FG], hindgut [HG], midgut [MD], malpighian tubules [MT], esophagus [ES], proboscis [Pr], salivary glands [SG], and ventral diverticulum [VD] are indicated. (b) Synchrotron X-ray images of a female mosquito that has taken up the AuNP-incorporated chitosan microparticles. A white beam X-ray source (8–30 keV) is utilized to maximize the absorption capability of the designed X-ray contrast flow tracer. The images are captured at 5 h after the consumption of the microparticles. Scale bar 1000 μm in the side view of the mosquito. The scale bar is 200 μm for three magnified images (i, ii, and iii). The three white boxes indicate the thorax (i), upper abdomen (ii), and hypogastrium (iii) of a mosquito. (i) Side, (ii and iii) front views of the mosquito. Paths 1 and 2 in (i) indicate the two digestive routes specialized for the different foods consumption of a mosquito. The locations of the particles are highlighted by circles [81]. Adapted by permission from Elsevier.

Figure 6

(A) AuNP incorporation into chitosan microparticles. (a) The Au ions are reduced in the empty microparticles (Method I). (b) Surface-modified AuNPs diffuse into the empty microparticles (Method II). (c) Surface-modified AuNPs used in Method II [83]. (B) (a) X-ray images of the dried microparticles fabricated by Method I (Figure 5B) with 6.35 × 10⁻² mmol Au ions. The size of the microparticles increases according to the molecular weight of chitosan: #1 (20,000 Da), #2 (30,000 Da) and #3 (85,000 Da). (b) X-ray images of the microparticles containing reduced AuNPs. AuNPs are reduced in the chitosan microparticles (20,000 Da) at 6.35 × 10⁻³ [I], 3.15 × 10⁻² [II], and 6.35 × 10⁻² [III] mmol Au [60]. (C) A typical raw (a), averaged (b), and preprocessed (c) X-ray image of the blood flow in a rat cranial vena cava. (d) The velocity vectors obtained from the rat vein. The dotted line represents the velocity profile of the blood flows as suggested by Casson model. The solid line denotes the parabolic curve fitting on the experimental data. R_ave is the vessel radius averaged from X-ray images [6]. Adapted by permission from Springer.

Figure 7

Pharmacokinetics of AuNPs (a–d) and iodine contrast agent (e–h) in mice. (a,e) Before injection. (b,f) 2 min after injection; (c,g) 10 min after injection; (d,h) 60 min after injection. The AuNPs show low liver and spleen uptake as well as clearance via kidneys and bladder (b–d). At 60 min (d), the contrast in the Au-injected mouse is similar to the uninjected mouse (a), indicating efficient clearance. A Lorad Medical Systems mammography unit (Hologic, Inc., Danbury, CT, USA; model XDA101827) is used with 8 mAs exposures (0.4 s at 22 kVp). Kodak Min-R2000 mammography film, 18 cm × 24 cm (Eastman Kodak, Rochester, NY, USA) is used [50]. Adapted by permission from The British Institute of Radiology.

Figure 8

(A) Six types of AuNPs of 20 nm diameter are designed for incorporation into RBCs. The AuNPs are reduced by sodium citrate tribasic dihydrate (AuNP 1). For surface-modification, each AuNP are covered with thioglycolic acid (SH-CH₂COOH) (AuNP 2), 4-mercaptobenzoic acid (SH-Ph-COOH) (AuNP 3), 6-thioguanine (SH-C₅H₄N₅) (AuNP 4), 2-mercaptoethanol (SH-CH₂CH₂OH) (AuNP 5), and 1-propanthiol (SH-CH₂CH₂CH₃) (AuNP 6) [83]. (B) (left) X-ray images of selected RBC 0 (without AuNP incorporation), RBC 4 (AuNP 4-incorporated RBC), RBC 5 (AuNP 5-incorporated RBC), and RBC 6 (AuNP 6-incorporated RBC) are detected by the enhanced phase contrast together with enhanced absorption contrast. The upper images are flat-field-correction images expressed by the absolute X-ray absorption (I). The lower images show the X-ray absorption intensities subtracted by the corresponding background (I–I₀). The scale bars indicate the degree of absorption. The size of the images is 500 mm × 500 mm. A short video of X-ray images capturing the time-dependent flow motion of RBC 6 is available in Ref. [63]. Adapted by permission from Elsevier.

Figure 9

(A) X-ray micrographs of the microvasculature of normal tissue and tumors at different time period after the tumor inoculation with AuNPs and heparin injection. Micro-radiology is implemented with unmonochromated (white) synchrotron X-rays.(a) In vivo image of the lateral thigh, 7 days after inoculation. (b and c) Magnified images of the left square in (a), near the tumor area, and the right square, which correspond to normal tissue area (medial thigh). The arrowheads in (a) and (b) mark vessels showing AuNP agglomeration. The yellow arrows mark vessels of < 6 μm diameter. (d) In vivo image of the normal lateral thigh. (e, f) Magnified images of the left and right squares in (d). The inset in the lower left corner of (e) is also magnified image. The yellow arrow marks a < 6 μm diameter vessel. (g) In vivo image of the lateral thigh, 16 days after inoculation. (h) Magnified image of the square in (g). (i) Image of a 1 mm thick tissue removed from the thigh shown in (g). (j) Magnified image of the rectangle in (i). The yellow arrowheads mark abnormal vessels. The white arrowheads mark a vessel with ~2 μm diameter. The yellow arrows show areas with diffusion of AuNPs. Scale bar for (a), (d) and (g): 2 mm. Scale bar for (b), (c), (e), (f), and (i): 500 μm. Scale bar for inset of (e) and (h): 50 μm. Scale bar for (j): 10 μm. [58]. (a) Mouse model with two implanted tumors: Her2+ in one thigh and Her2– in the contralateral thigh. (b, c) The same mouse with a Her2– tumor (b) and Her2+ tumor (c) 20 h after intravenous injection of 15 nm AuNP–Herceptin (0.45 g Au kg⁻¹ body weight). Tumor volumes (by caliper measurements) are nearly identical, but a distinction can be seen owing to the targeted Au localization. Bar = 55 mm [66]. Adapted by permission from BioMed Central and The British Institute of Radiology.

Figure 10

Application of AuNPs to the visualization of fluid dynamics in a living organism. (a) Time-dependent flow motions of the concentration-controlled hydrophilic AuNP aqueous solution captured by synchrotron X-ray imaging. (b) Four consecutive images showing the uptake and transport of hydrophilic AuNPs inside the xylem vessels of a rice leaf sheath visualized by synchrotron X-ray imaging. The arrow indicates the meniscus between water and air. (1) and (2) are initial positions of hydrophilic AuNP clusters in the sap. At t₀ = 0 s and t₀ = 0.3 s, only cluster (2) moves upward. At t₀ = 1.3 s and t₀ = 1.7 s, cluster (1) moves up, cluster (2) is simultaneously separated into two distinct clusters (2−1) and (2−2) with different velocity. The images are captured consecutively by the synchrotron X-ray source using a bending magnet. X-rays with a peak energy of 20.3 keV (8–30 keV range) are applied as a function of time without monochromator to obtain high energy [80]. Adapted by permission from The American Chemical Society.