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Imaging nanoprobes are a group of nano-sized contrast agents devised for providing improved contrast and spatial resolution for bioimaging. Among various imaging nanoprobes, optical nanoprobes capable of monitoring biological events or progresses in the cellular and molecular levels have been developed for early detection, accurate diagnosis, and personalized image-guided treatment of diseases. The optical activities of nanoprobes can be tuned on demand for specific applications by engineering their size, surface nature, morphology, and composition. In addition, by virtue of the nanostructure, nanoprobes have displayed favorable pharmacokinetic features and target specificity reflecting clinical demands. In this review, we focus on typical approaches and recent trends in development of nanoprobe-mediated optical imaging and their potential as a clinical diagnostic modality.
© 2016 Optical Society of America
1. Introduction
Bioimaging is one of the most reliable tools for diagnosis of numerous diseases. With tremendous advances in imaging technology in the past few decades, various imaging modalities have been successfully applied to clinical diagnosis leading to dramatic improvement in human healthcare. However, the imaging modalities including X-ray imaging, computer-assisted tomography (CT), ultrasound (US) imaging, and magnetic resonance (MR) imaging have been mainly employed to obtain structural and anatomical information at the tissue or organ level [1]. For early detection, accurate diagnosis, and personalized image-guided treatment of diseases, there is a clear need to develop imaging technologies that can provide information at the cellular and molecular levels. Optical imaging with suitable contrast agents can offer the information of specific biological events or progresses for in vitro and in vivo applications.
Among various contrast agents for optical imaging, nano-sized agents (nanoprobes) with specific optical properties such as fluorescence (FL), chemiluminescence (CL), and photoacoustic (PA) effect have received significant attention due to their inherent nanostructure-motivated features for clinical applications. The optical properties of nanoprobes are typically modulated by engineering material parameters such as size, surface nature, morphology, etc. Nanoprobes with compact size can be effectively utilized in monitoring biological events occurring both on the surface and inside of cells. Further, nanoprobes can overcome many biological barriers such as clearance by reticuloendothelial system (RES) with modification of size or surface properties [2], allowing target-selective imaging for in vivo applications. Moreover, the recently devised multifunctional nanoprobes can offer additional advantageous features for the clinically specific applications [3,4], and the nanoprobe-mediated optical imaging has emerged as a promising diagnostic tool for clinical uses. In this review, we focus on recent advances and potential of nanoprobes as a contrast agent of optical imaging by exploring basic principle, research strategies, cases, and state-of-the-art clinical applications.
2. Nanomaterials for various optical imaging modalities
2.1. Fluorescence imaging
Fluorescence (FL) is, in general, referred to as an optical phenomenon of photon-to-photon energy conversion; that is, emission of light from any matter after photoexcitation. In a strict view, photoluminescence is a precise terminology that describes such a photophysical process. In this review, however, the term FL will be used as photoluminescence for the sake of simplicity. FL is an intrinsic property of materials whose electronic structure specifies photon energies of both excitation input and emission output. When FL is brought as contrast signal, this material-specific attribute of probes can provide quite sensitive and selective images with high resolution up to the nanometer scale [5]. Based on such beneficial attractions, a number of researches and developments have been conducted in FL imaging for visualizing biological matters and events. Main concerns (or requisites) for FL imaging probes are to achieve high image contrast, high photo/chemical stability of the signal, and low toxicity. FL nanomaterials as an imaging agent can be featured by their versatility in giving beneficial variations to the physicochemical, optical or pharmaceutical characteristics in relatively facile manners, being discriminated from small-molecule FL probes. Regardless of nanoprobe types, there have been some typical routes in dealing with nanomaterials for biological applications: for example, surface coating/modification, encapsulation and bioconjugation techniques for improving dispersion, enhancing biocompatibility, or giving target specificity (e.g. cell-penetrating peptide) if needed. This section will not cover details on those parts. Focusing on the FL property of fluorophores and imaging modes, upcoming contents offer an overview, with several issues and examples, on types of FL nanoprobes that have been widely applied for bioimaging.
2.1.1. Stokes fluorescence
The majority of researches in FL imaging is based on Stokes FL in which emitted light has a lower energy (i.e. spectral band maxima) than the absorbed light. This energy gap, called Stokes shift, stems from energy loss through vibrational relaxations in the electronic ground and excited states. Imaging with Stokes FL is, in principle, to capitalize on the absorption and emission wavelengths of a given material for excitation and detection ranges, respectively. Stokes FL nanoprobes widely used in bioimaging can be classified into two types in a standpoint of the fluorophore: (i) nanostructures wherein multiple molecular fluorophores are integrated, and (ii) nanomaterials themselves as an intrinsic fluorophore. The former includes silica-based nanoparticles (NPs), polymeric NPs, polymeric micelles, liposomes, etc., providing nanoscale matrices where FL molecules are incorporated physically or chemically. This approach retains the merits of high brightness per single probe versus a molecular fluorophore while some of the structural designs are capable of protecting fluorophores from biological environment. With well-established techniques on fabrication and surface engineering of those platforms as well as bioconjugation library, numerous probes have been demonstrated for efficacious imaging of cellular structures, lymph node or tumor regions in vivo, etc. In the dyed nanomaterials, there have been some issues for the choice of dopant fluorophores. In terms of their spectral regions, biological conditions that can perturb probes’ real signal should be considered. Biological constituents such as tissues and blood absorb and/or scatter light exerted on the specimen as well as that generated from fluorophores. In addition, there exist fluorescent biomolecules (autofluorescence). Taken together, it is known that the ideal wavelength for FL imaging for attenuating such signal distortion is 650-950 nm and 1000-1350 nm, called near-infrared (NIR) windows [6,7]. Another strategy to overcome biological interference is to utilize aggregation-enhanced fluorescence (or solid-state fluorescence, SSF) for maximizing brightness of a single probe by overcoming concentration quenching typical in organic dyes [8]. Singh et al. demonstrated that NIR-emitting micellar nanoprobes in which SSF molecules (designed by bandgap tuning of dipolar arylvinyl derivatives through adjusting the intramolecular charge transfer) are highly concentrated are capable of imaging lymph nodes and tumor with a high signal-to-noise ratio in vivo [9] (Fig. 1).
Fig. 1 a) Schematic diagram of SSF-based NIR-emitting micellar nanoprobe and HOMO/LUMO electron distributions of the loaded dye. b) FL spectra of the dye in THF solution (dotted) and in self-aggregated NP dispersion (10% v/v THF in DW, solid) with corresponding NIR fluorescence imaging. c) FL images of a SCC7 tumor-bearing mouse after intravenous administration of the probe (left, red arrow indicates tumor region) and pseudo-color image at 12 h post-injection with FL spectra for tissue and tumor (upper right corner) and corresponding fluorescence intensity (lower right corner). (reprinted with permission from [9], ©2013 American Chemical Society)
The other types of FL nanoprobes are so-called “dots” having intrinsic FL properties: semiconducting polymer dots (P-dots) and quantum dots (Q-dots) as representatives. Though nonfluorescent materials like surfactants and functional moieties exist on their surface, the major fluorescing component is the semiconducting nanoscale core. Dots can be understood as continuous matrices of fluorophores, thus, they have fundamentally large molar extinction coefficients when compared to molecular probes and even to dyed nanoprobes. It means that the use of dots is also in the strategy of enhancing single probe brightness. Furthermore, they are generally known to exhibit good photostability, compared to molecular fluorophores. The strategy of color tuning toward NIR for optimizing image contrast is still adopted in this material category [10,11]. P-dots are referred to as sub-10 nm NPs of semiconducting π-conjugated polymers, such as derivatives of poly(phenylene vinylene), polyfluorene, and poly(phenylene ethynylene) [12,13]. In addition to controllability on their size and FL properties (emission from visible to NIR ranges with reasonable quantum yield), P-dots have low toxicity in comparison with Q-dots that will be discussed subsequently. Because of these merits, P-dots are considered as an attractive nanoprobe candidate for FL bioimaging. Q-dots are inorganic semiconducting nanocrystals, characterized by wide absorption spectra and size-dependent shifts in absorption/emission spectra due to the quantum confinement effect. Typical composition of Q-dots is cadmium chalcogenide (i.e. CdS, CdSe, CdTe) cores with higher bandgap shell, exhibiting high quantum yield and narrow emission color purity without photobleaching. In spite of their unique optical properties, potential toxicity from cadmium ions has been a critical issue that limits clinical translation. Ye et al. showed that intravenously injected CdSe/CdS/ZnS Q-dots (encapsulated by phospholipid) in primates do not show any toxic effect for several months [14]. Though toxicity of Q-Dots is, in part, a matter of passivation and dose, the presence of cadmium itself can potentially have adverse clinical influence for patients. Cd-free compositions such as InP/ZnS, CuInS2/ZnS, or CuInSeS/ZnS, etc [15–17], have also been utilized for bioimaging researches as alternatives.
Besides the strategies of maximizing quantum yield and brightness of nanoprobes as well as using the NIR window, an approach based on time-gated FL imaging technique was proposed to enhance contrast signals. This technique introduces a temporal delay between pulse excitation and detection time points. Gu et al. fabricated NIR luminescent porous silicon NPs whose emission lifetime is 5-13 μs, far longer than those of conventional molecular fluorescence and tissue autofluorescence (< 10 ns). It was found that the in vivo signal-to-background ratio increased more than 20-fold with time gating of 18 ns in comparison with normal fluorescence imaging [18].
FL imaging can be applied for sensing of chemical analytes, especially indicators (or biomarkers) of certain cellular processes or diseases. In such a dynamic imaging mode, FL signals are altered or turned on in response to the corresponding analytes. Shuhendler et al. demonstrated a nanoprobe that is capable of sensing reactive nitrogen species (RNS) involved in hepatotoxicity in real time by utilizing fluorescence resonance energy transfer (FRET) as a tool to induce variation in emission spectra of the probe [19]. Briefly, this nanoprobe is composed of a P-dot core matrix (FRET donor) and a RNS-degradable dye (FRET acceptor) that loses fluorescence when reacted with RNS to result in increment of P-dot emission (Fig. 2). For a fluorogenic probe, Heo et al. designed a micellar nanoprobe in which H2O2-responsive fluorogenic molecules and catalysts for the reaction are integrated together. It was shown that this reactor-like probe is capable of visualizing H2O2 involved in cellular activities (ligand-receptor binding, proliferation, hypoxia, autophagy, and phargocytosis) with high spatiotemporal resolution [20].
Fig. 2 a) Molecular structures of constituents of a RNS-responsive FRET-based nanoprobe (the first, second and third quadrants) and its schematic diagram for RNS sensing mechanism (the fourth quadrant). b) Ratiometric change in FL spectrum of the same probe with respect to ONOO- concentration. c) FL images of hepatotoxicity-induced mice (by intraperitoneal administration of anti-pyretic acetaminophen, APAP) followed by intravenous administration of the same probe. (Reprinted by permission from Macmillan Publishers Ltd, Nature Biotechnology [19], ©2014)
2.1.2. Anti-Stokes (upconversion) fluorescence
Anti-Stokes FL generally corresponds to photonic phenomena where emission light has higher energy than the adsorbed light. Most of the anti-Stokes phenomena utilized in bioimaging researches belong to nonlinear optics such as multiphoton absorption [21] and second harmonic generation [22,23]. Such nonlinear optical processes, in terms of quantum yield, are less efficient than usual linear Stokes-shifted FL because sequential or simultaneous association of photons is required within given lifetime of intermediate or virtual excited states. Considering biological conditions, however, anti-Stokes excitation energy that falls in the range of NIR window, offers benefits of imaging with deep tissue penetration and no autofluorescence. Among suitable material classes for that purpose, especially for sequential multiphoton absorption, are lanthanide-doped upconversion nanoparticles (UCNPs) with ladder-like electronic structures of lanthanide ions that assist stepwise light absorption (generally ~980 nm for UCNP alone). Numerous compositions have been reported with core or core/shell combinations of host lattice (NaYbF4, NaYF4, Y2O3, etc.) and lanthanide dopant (Yb3+, Er3+, Tm3+), by which their optical properties are determined. The state of the art in developing UCNPs for bioimaging has been thoroughly reviewed by Chen et al., covering wide realms of designs, syntheses and control over optical properties, as well as strategies for bioimaging applications [21]. In the view of the NIR window, NIR-to-NIR upconversion is, indeed, one of the efficacious photonic attributes [24–26]. For example, α-NaYbF4:Tm3+/CaF2 core/shell UCNPs that exhibit NIR-to-NIR upconversion properties were successfully synthesized with relatively high quantum yield. The signal-to-background ratio reached up to 310 when those nanoprobes were intravenously administrated to a small animal model with deep penetration through 3.2 cm-thick animal tissue [26]. (Fig. 3)
Fig. 3 a) Optical spectra and b) TEM images of α-(NaYbF4:0.5% Tm3+)/CaF2 core/shell UCNPs. c) Merged upconversion FL (980-nm laser excitation) and bright-field image of a cuvette filled with the same UCNPs under pork tissue (left) and bright-field image of the pork tissue in a side view (right). d) Upconversion FL (980-nm laser excitation, left) and merged (right) images of a BALB/c mouse injected intravenously with the same UCNPs coated by hyaluronic acid. (Reprinted from permission from [26], ©2010 American Chemical Society)
Another approach for anti-Stokes FL imaging is triplet-triplet annihilation (TTA)-based light upconversion. This system consists of two types of molecules: (i) sensitizer, responsible for low-energy excitation and sequential intersystem crossing that generates the triplet excited-state species, and (ii) annihilator (or emitter) that receives triplet-triplet energy transfer from the triplet excited state of sensitizer and emits high-energy light through its singlet excited state populated by TTA between two triplet states. Heavy metal-ligand complexes are one of the reliable classes for sensitizer. Due to triplet states involved in the energy relay, protection of constituent molecules from O2 is of a great importance in the design of nanoprobes for bioimaging. It was reported that soybean oil droplets stabilized by bovine serum albumin-dextran enabled efficient TTA upconversion of the loaded sensitizer/annihilator pair (Pt(II)-tetraphenyl-tetrabenzoporphyrin/BODIPY) by providing an stable inner dispersion matrix in addition to O2 protection, allowing for lymph node mapping [27].
2.2. Chemiluminescence imaging
Limited tissue penetration of light is a major obstacle limiting in vivo applications of FL imaging. As mentioned in section 2.1, fluorophores with absorption and emission in the NIR region have been widely employed for in vivo FL imaging to minimize light attenuation during tissue penetration. Nonetheless, there is still a need to improve contrast and spatial resolution for deep tissue imaging. Chemiluminescence (CL) imaging suggests one possible solution for the light attenuation issues. While FL imaging requires an external photoexcitation that causes autofluorescence background noises, CL signals that are generated by a chemical reaction of biological metabolites such as reactive oxygen or nitrogen species, do not need photoexcitation to allow for background-free high imaging sensitivity [28]. According to the working principle, CL imaging can be utilized to monitor the biological molecules related to various diseases at molecular level. Moreover, CL imaging that displays improved contrast and spatial resolution with minimized noises, is relevant to in vivo applications.
Since the early report of oxygen-triggered CL by 2,4,5-triphenylimidazole (lophine) in 1877 [29], various types of chromophores and their chemical triggers have been studied for CL. Among the chemical triggers realized, reactive oxygen species (ROS) is a representative example. ROS includes superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (∙OH), which are related to the progress of inflammatory diseases and aging, etc [30]. Thereby, ROS-mediated CL imaging can serve as an efficient diagnostic tool for monitoring change or progress of inflammatory diseases. For example, Lee et al. devised a nanoformulation integrated with a peroxalate polymer and pentacene as a fluorophore [31]. As shown in Fig. 4, peroxalate groups in the polymer backbone are converted to a dioxetane intermediate through chemical reaction with the external hydrogen peroxide (H2O2), followed by dissociation of the intermediate to generate chemical energy. The energy is transferred to emissive pentacene, resulting in CL emission without external photoexcitation. It should be noted that the close proximity between peroxalate group and dye molecule is a critical factor determining the efficiency of chemical energy transfer. The H2O2-responsive CL nanoprobe successfully enabled in vivo CL imaging in a peritoneal inflammatory model. Further, oxalate-backboned polymeric nanoformulations incorporated with small molecular fluorophores were devised and successfully applied to in vivo H2O2-mediated CL imaging of ischemia-reperfusion injury [32,33].
Fig. 4 a) Schematic diagram of the peroxalate-backboned polymer nanoparticle as a ROS-activated CL nanoprobe. b) In vivo CL imaging of peritoneal inflammatory model after administration of CL nanoprobe. (Reprinted by permission from Macmillan Publishers Ltd, Nature Materials [31], ©2007)
Recently, an aqueous dispersion of Pluronic nanoformulation integrated with bis[3,4,6-trichloro-2-(pentyloxycarbonyl) oxalate (CPPO) and 3,3′-diethylthiadicanocyanine iodide (Cy5, NIR fluorophore) was devised as a CL nanoprobe with ultrahigh in vivo sensitivity to H2O2, detectable at around ~10−8 M [34]. In this approach, hydrophobic oxalate species was physically integrated with dye molecules into the biocompatible nanoformulation, in which chemical energy transfer to fluorophore is feasible, allowing in vivo CL imaging in an inflammatory mouse model. However, typical small molecular fluorophores may undergo ‘concentration quenching’ phenomenon when they are loaded in a high local concentration [8], limiting the brightness of CL nanoprobes. In this regard, CL nanoprobes with high brightness were developed by dense integration of CPPO and antracence-derived fluorophores with the aggregation-enhanced fluorescence (AEF) property into a nanoscopic space. Indeed, highly sensitive in vivo CL imaging was achieved with such dye-concentrated CL nanoprobes by local and systemic administration in murine disease models of acute inflammation, rheumatic arthritis, and cancer [35,36] (Fig. 5).
Fig. 5 a) Schematic diagram of Pluronic nanoparticle with dense co-integration of CPPO and antracence dye (BDSA) as a ROS-activated CL nanoprobe. b-d) In vivo CL images of murine disease models of acute inflammation (a), rheumatic arthritis (b), and cancer (c) after systemic administration of CL nanoprobe. (Reprinted with permission from [36], ©2015, American Chemical Society)
2.3. Photoacoustic imaging
Photoacoustic (PA) imaging is a hybrid signaling modality of optical and ultrasound (US) imaging. In the typical PA system, a contrast agent absorbs electromagnetic energy in the form of light from a non-ionizing short-pulsed laser beam and generates US waves through thermal expansion [37,38]. The photo-induced US waves are received by a wide-band ultrasonic transducer and generate PA images. Due to low attenuation of US waves during tissue penetration, PA imaging can provide high spatial resolution with deep penetration in the order of centimeters [37,38]. Since some biological molecules such as hemoglobin, myoglobin, and melanin are capable of generating PA signals upon light energy absorption, biological information of tissue or organ can be obtained through PA imaging without use of exogenous contrast agent. For example, hemoglobin (Hb) changes photo-absorption behavior and generates PA signals after binding to oxygen (HbO2), by which in vivo imaging of oxygen saturation-related physiological processes or disease is possible [39]. There have been impressive advances in the development of exogenous PA contrast agents, by which contrast enhancement significantly exceeds inherent background generated by endogenous PA agents in blood and muscle. A variety of light-absorbing materials including small molecular dyes, gold nanoparticles (AuNPs), and carbon nanotubes (CNTs) have been explored as PA contrast agents, where nanostructured contrast agents are favored over small molecules for in vivo PA applications due to their high light absorptivity, in vivo stability, and targetability [2].
Among various types of PA nanoprobes, AuNPs are a representative example of exogenous PA agent [40–44]. Typically, AuNPs exhibit light absorption in the region of visible to NIR due to localized surface plasmon resonance (LSPR), where the absorption band can be tuned by modifying morphology, size, surface nature, etc. In addition to optical absorption, AuNPs have several attractive features as an in vivo PA nanoprobe such as biocompatibility, resistance to photobleaching or fading, and in vivo stability [40–44]. Srivalleesha et al. reported spherical AuNP (Dia. 50 nm) whose surface is decorated with either anti-EGFR, a targeting ligand, or poly(ethylene glycol) (PEG), an antifouling polymeric segment, and compared their PA activities in a tumor-mimicking ex vivo mouse tissue [40]. While the PEG-decorated AuNPs exhibited strong absorption at around 520 nm, the anti-EGFR-decorated AuNPs exhibited a widely extended absorption spectrum (in the range of visible to NIR) resulting in amplification of PA signals under irradiation of NIR laser (Fig. 6(a)). It should be noted that amplification of PA signal with NIR light dose is more favorable for in vivo PA applications in terms of penetration depth. Morphology of AuNPs also affects optical properties and the related efficiency of PA imaging. Cage-shaped AuNPs reported by Jingyi et al. exhibited a significantly wider and more red-shifted absorption band, in the range of 600-1200 nm (λmax ≈800 nm), than spherical AuNP with a similar size [41]. Rod-shaped AuNPs also exhibited favorable absorption spectrum in the region of 700-840 nm, allowing NIR light dose-mediated in vivo PA imaging in mouse models [42]. (Fig. 6(b)) Further, hollow-shaped Au nanoshells (Outer Dia. 43-46 nm) displayed absorption spectrum at around of 800 nm [43]. Besides morphological modulation, the absorption spectrum of AuNPs can be tuned to NIR region by heteroatom doping. For example, the AuNP doped with semiconductor domain (p-type copper selenide, Cu2-xSe) showed broader and flatter absorption spectrum in the NIR region (750-1150 nm) due to plasmonic interaction between semiconductor and metallic nanostructures, allowing non-invasive deep tissue visualization in PA imaging of a rat [44].
Fig. 6 a) Normalized absorption spectra of A431 cells labeled with the spherical AuNPs with or without the targeting ligand (left) and US and PA images of tumor-mimicking ex vivo mouse tissue (right) under laser illumination (right). (Reprinted with permission from [40], ©2009 American Chemical Society) b-c) TEM image (b, left) and optical absorption efficiency (b, right) of the rod-shaped AuNPs and US and PA image (c) of the AuNP-implanted mouse hind limb. Arrows show the locations of implanted gelled nanorod solution. (Reprinted with permission from [42], with permission from AIP Publishing)
CNTs have also been explored as a promising exogenous PA agent [45,46]. Many researches have devoted to exploring the potential of CNTs, and it was well demonstrated that they possess strong optical activity over a wide range of spectral window and the capability of chemical modification on the surface for in vivo applications. For example, Adam et al. employed single-wall carbon nanotubes (SWCNTs) with or without conjugation with RGD, a tumor targeting moiety, on the surface as a PA nanoprobe [45]. The RGD-conjugated SWCNTs exhibited more enhanced PA response than unmodified SWCNTs, elucidating chemical modification on the surface is another key governing factor for in vivo applications in addition to optical activity (Fig. 7). Recently, hybrid nanoprobes such as Au-coated CNT and mesoporus silica-coated AuNPs have been devised to enhance inherent optical properties of typical PA nanoprobes or to impart additional functionalities for using combined diagnostic modalities of PA and other imaging techniques [47,48].
Fig. 7 US and PA images (a) and time dependent-PA signals (b) of SWCNT-injected mice. The images show the plane of one vertical slice (white dotted line) through the tumor. The US show the skin and tumor boundaries. (Reprinted by permission from Macmillan Publishers Ltd, Nature Nanotechnology [45], ©2008)
2.4. Other optical imaging
2.4.1. Cerenkov luminescence imaging
Cerenkov luminescence imaging is one of the emerging imaging tools in medicine, which utilizes the optical signal generated from radioisotopes. Cerenkov luminescence results from charged particles, such as positrons and electrons, traveling faster than light through a dielectric medium with a refractive index over 1 [49]. It generally produces continuous wave light in the range of 250–600 nm without external photoexcitation. Since Cerenkov luminescence was recently visualized using 18F-fluoro-2-deoxyglucose (18F-FDG) for in vivo tumor imaging [50], the combination of radioisotopes and nanomaterials has drawn great interest as multifunctional nanoprobes for cancer imaging.
Conventionally, radioisotopes are labelled on the surface of nanomaterials via ligands or chelating agents. However, this approach has potential dissociation problems of radioisotopes, leading to providing false pharmacokinetics of imaging agents and misdiagnostic information. In this regard, Wang et al. reported a facile synthetic route for 198Au-doped Au nanocages (AuNCs) as a Cerenkov luminescence imaging agent without additional radiolabeling or dye conjugation [51]. They developed 198Au-doped AuNCs via direct incorporation of 198Au into the walls of nanocages (Fig. 8(a)). The AuNCs are stably integrated with 198Au atoms, ensuring highly accurate in vivo information and imaging. Similar to other radioisotopes for Cerenkov luminescence, the 198Au-doped AuNCs displayed a continuous luminescent spectrum in the maximum range of 500–550 nm. After surface modification with poly(ethylene glycol) (PEG, MW ≈5000) to enhance the half-life in the bloodstream, 198Au-doped AuNCs exhibited excellent tumor accumulation, presenting potential for cancer imaging as well as photothermal treatment (Fig. 8(a)). Furthermore, they synthesized several 198Au nanostructures with a similar size but different morphologies, such as nanospheres, nanodisks, nanorods, and nanocages, to compare their biodistribution, tumor uptake, and intratumoral distribution using Cerenkov luminescence [52]. Among four Au nanostructures, the 198Au-doped nanospheres and nanodisks showed higher Cerenkov luminescence signal in tumor region than nanorods and nanocages (Fig. 8(b)). Interestingly, while nanorods and nanocages are localized in the core of the tumor, nanospheres and nanodisks are mostly distributed in the periphery region of the tumor, contributing high Cerenkov luminescence signal in tumor region.
Fig. 8 a) Schematic diagram of radioluminescent 198Au-incorporated NCs and Cerenkov luminescence images of EMT-6 tumor-bearing mice at 2 and 24 h post-injection after tail vain injection of the PEGylated 198Au-doped AuNCs. (Reprinted with permission from [51], ©2013, American Chemical Scoiety) b) Co-registered in vivo luminescence and X-ray images of the tumor-bearing mice at 24 h post-injection of the different types of 198Au-doped nanostructures. (Reprinted with permission from [52], ©2014, American Chemical Society)
Due to the relatively short-wavelength emission range, however, Cerenkov luminescence imaging has inherent limitations for in vivo imaging. To overcome this, long-wavelength spectral tuning by Cerenkov resonance energy transfer (CRET) has been tried by combining radioisotopes and emissive energy acceptors that can absorb the short-wavelength Cerenkov emission and downconvert it to the NIR. In particular, Q-dots are one of the most promising candidates as an acceptor for Cerenkov luminescence due to the distinctive optical properties, such as broad absorption, large stokes shift, high quantum yield, and high photostability. Recently, Sun et al. developed self-illuminating Q-dot by direct doping of 64Cu into CdSe/ZnS Q-dots via cation-exchange reaction [53]. 64Cu-doped Q-dots showed more efficient Cerenkov luminescence than free 64Cu or the mixture (64Cu/Q-dots) with no dissociation of 64Cu during in vivo circulation. By the enhanced permeability and retention (EPR) effect, 64Cu-doped Q-dots were highly accumulated in tumor region up to 12.7% ID/g at 17 h post-injection as revealed by positron emission tomography (PET), which is well correlated with Cerenkov luminescence imaging (Fig. 9(a)). They reported another self-illuminating Q-dots, [64Cu]CIS/ZnS Q-dots, prepared by direct incorporation method using 64CuCl2 as a precursor [54]. The 64Cu was stably incorporated into CIS/ZnS Q-dots and less than 3% of 64Cu was dissociated from the Q-dots even in serum and EDTA condition for 24 h. Consistent with the PET imaging, PEGylated GSH-[64Cu]CIS/ZnS Q-dots (MW of PEG ≈5000) exhibited the highest tumor accumulation compared with bare GSH-[64Cu]CIS/ZnS Q-dots or free 64Cu (Fig. 9(b)). Taken together, dual-imaging performance (PET and Cerenkov luminescence) of 64Cu-containing Q-dots holds great promise for multimodal cancer imaging.
Fig. 9 a) Representative whole-body coronal PET (upper) and luminescence (bottom) images of U87MG tumor-bearing mice at the predetermined time points after intravenous injection of 64Cu-doped Q-dot 580. White and black arrows indicate the locations of tumor and liver, respectively. (Reprinted with permission from [53], ©2014, American Chemical Society) b) CRET images of U87MG tumor-bearing mice at 6 h post-injection of 64CuCl2, GSH-[64Cu]CIS/ZnS, and PEGylated GSH-[64Cu]CIS/ZnS Q-dots, respectively. Circles indicate the location of tumor area. (Reprinted with permission from [54], ©2015, American Chemical Society)
2.4.2. Multimodal imaging
In the past few decades, various imaging modalities have been developed for improved visualization and localization of disease sites to result in better prognosis. However, in spite of remarkable advances in probe design and instrumentations of individual imaging technology, each imaging modality suffers from its own intrinsic limitations. For instance, while positron emission tomography (PET) and single-photon emission computed tomography (SPECT) exhibit high target sensitivity, they have low spatial resolution. Magnetic resonance imaging (MRI) and computed tomography (CT) provide three-dimensional anatomical information, but they have poor target sensitivity. To compensate drawbacks of individual imaging modality, there have been attempts to combine two or more modalities, and it has shown significant progresses [5,55]. Indeed, dual imaging systems of PET-CT and SPECT-CT are already widely used in the clinic.
Complementary combination of optical signals, such as fluorescence (FL) and photoacoustic (PA), with other imaging modalities including MRI, PET, and other radionuclide imaging has been explored and offered simultaneous structural and functional information of disease sites with high resolution and sensitivity. For example, superparamagnetic iron oxide nanoparticles (IONPs) incorporated with NIR fluorescence dyes, such as DiI [56] and Cy5.5 [57,58], or quantum dots [59,60] have been prepared and shown potentials as a dual-imaging nanoprobe. Further, there have been studies using silica nanoparticles labeled with 124I and NIR fluorophores including Cy5 [61] or DY776 [62] to prepare PET/FL nanoprobes for complementary dual-imaging with high resolution and sensitivity in animal models.
Besides development of multimodal imaging nanomaterials using different imaging techniques, synergistic combination of different types of optical imaging modalities has also been attempted to overcome the weakness of individual modalities. A representative example would be combination of FL and PA imaging since FL can provide images with high target sensitivity while PA can offer images with high spatial resolution and tissue penetration. Although fluorescent emission and photoacoustic characteristics are contradictory because the former requires high fluorescent quantum yield while the latter needs efficient conversion of the absorbed photon energy into heat, proper tuning of the concentration and aggregate state of FL dye in a nanocomposite can confer an optimal balance between the dual characteristics (Fig. 10). For instance, indocyanine green (ICG)-incorporated nanoparticles constructed by PEGylated hyaluronic acid [63] and human serum albumin [64] showed feasible FL and PA signals in tumor-bearing mouse models. Liposomes composed of L-α-phosphatidylcholine/cholesterol and lysophosphatidylcholine, encapsulating squaraine [65] and pyropheophorbide [66], demonstrated great potential in tumor and lymph node visualization by FL/PA combination imaging with high sensitivity and resolution. In a recent study by Liu et al [67], it was shown that Cy754-conjugated silica nanoparticles can be utilized as a FL/PA dual imaging probe with appropriate adjustment of dye concentration in the particle. Above-mentioned examples used a single dye to generate both FL and PA signals, whereas there have been reports that exploit separate signal sources in the same nanoformulation. For instance, polyacrylamine nanoparticles containing silver nanoplate and fluorescent dye Rhodamine 6G [68] and HER2-targeting affibody functionalized IONP encapsulated with NIR-830 dye [69], where noble metal clusters and IONPs are PA sources, have been prepared for FL/PA dual-modality in vivo tumor imaging.
Fig. 10 Utilization of NIR dye as a dual-modality (FL/PA) probe. (Reprinted with permission from [65], ©2014, American Chemical Society)
3. Recent progress on clinical applications
Diagnostic imaging has helped improve prognostics in various diseases. For instance, MRI and radionuclide-based imaging technologies have grown significantly in the past several decades, being an essential part of medical imaging-based diagnosis. Recently, by virtue of advances in probes and instrumentation, optical imaging has emerged as an alternative and/or complementary modality in the clinic. The instrumentation of optical imaging is relatively simple, inexpensive and portable, which makes it more efficient, convenient and economic than traditional medical imaging that requires large and expensive settings [70]. Indeed, several small-molecule dyes for FL and PA have been approved by FDA for intraoperative surgery-guiding agent. For example, indocyanine green (ICG) is being utilized as an intraoperative surgical guide for gastric cancer [71,72], liver cancer [73], liver metastases from pancreatic cancer [74], and also exploited as a lymph node mapping agent for breast cancer [75–77], vulvar cancer [78], lung cancer [79] and cervical cancer [80]. As a NIR dye precursor for photodynamic diagnosis (PDD), 5-aminolevulinic acid (5-ALA) has been successfully applied to fluorescence-guided surgery in glioma [81] and bladder cancer [82,83]. Recently, methylene blue (MB) has shown potential as a PA contrast agent for sentinel lymph node detection in breast cancer patients [84].
Despite the clinical success of small dye molecules in image-guided surgery or lymph node detection as mentioned above, they are target-nonspecific and have issues of rapid body clearance, bleaching, quenching and autofluorescence in soft tissues [85]. To overcome such intrinsic drawbacks of small-molecule probes, a wide range of nanoformulations with improved specificity, circulation time and photostability as well as ease of multiple functionalization have been devised, e.g. dye-incorporated polymeric nanoparticles/liposomes/silica nanoparticles, quantum dots, carbon dots, etc., and shown great potential in preclinical studies [86,87]. However, there are hurdles for the nanoprobes to be applied to the clinics because of their structural complexity. It is hard to manufacture nanoprobes in industrial scale with quality control. Further, toxicity of each component in nanoformulations, as well as pharmacokinetics, need to be fully examined in appropriate animal models before clinical applications [88]. Therefore, only a handful of optical imaging nanomaterials have been evaluated in clinical settings. An example is ICG-incorporated human serum albumin colloid labelled with 99mTc (ICG-99mTc-Nanocolloid), which was formulated to improve intraoperative visualization of SLN using sensitive NIR fluorescence after preoperative localization of SLN via SPECT/CT (Fig. 11). It was demonstrated that ICG-99mTc-Nanocolloid was able to precisely identify SLN intraoperatively in patients with prostate cancer [89], head and neck melanoma [90], and breast cancer [91]. Another optical imaging nanoprobe evaluated in a clinical setting is “C dots”. It is Cy5-incorporated silica nanoparticles whose surface is modified with PEG conjugated with 124I-labelled cyclic Arg-Gly-Asp-Tyr peptide (cRGDY) for targeting integrin αvβ3 overexpressed in various tumors. In a pilot clinical study to evaluate eligibility of C dots as an intraoperative optical imaging probe [92], it was shown that systemic administration of C dots is safe with no toxicity, and favorable pharmacokinetics was observed with fast renal clearance and low uptake by reticuloendothelial system (RES). Although the clinical studies mentioned above utilized optical imaging modality as an ancillary component to radionuclide-based imaging techniques for target visualization, it is anticipated that the role of optical imaging would increase with rapid development of nanomaterials and imaging instrumentations.
Fig. 11 Procedure of pre- and intra-operative identification of sentinel lymph node using ICG-99mTc-Nanocolloid. (Reprinted from [89], ©2011, with permission from Elsevier)
4. Concluding remarks
A myriad of nanoprobes have been developed to improve the performances of optical bioimaging. As such, the nanoprobe-mediated optical imaging provides specific information of biological events or progresses at the cellular and molecular levels by employing FL, CL, PA, or combination of multiple modalities. The engineered structural parameteres of nanoprobes, i.e. morphology, size, surface nature, and composition, offer suitable optical properties with target specificity and favorable pharmacokinetic profiles. Moreover, recently devised multimodal nanoprobes composed of optical and other conventional imaging techniques have offered improved diagnostic accuracy. In spite of their potentials as a diagnostic agent, there have been only a few clinical researches on the nanoprobe-mediated optical imaging due to the unclear biological effects of nanoprobes and a limited application range of current optical imaging techniques. However, rapid developments in nanoprobes and instrumentation are expected to extend the applicability of optical imaging for early and accurate diagnosis as well as real-time image-guided treatment in the foreseeable future, followd by well-designed researches for clinical translation.
Acknowledgments
This work was supported by the grants from the National Research Foundation of Korea (No. 2014M3A9E5073316, 2014M3C1A3054141), the Korea Health Industry Development Institute (No. HI15C1540), and the Intramural Research Program of KIST.
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