Nanoprobes for biomedical imaging in living systems

Nano Today (2011) 6, 204—220

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nanotoday

REVIEW

Nanoprobes for biomedical imaging in living systems Heebeom Koo 1, Myung Sook Huh 1, Ju Hee Ryu, Dong-Eun Lee, In-Cheol Sun, Kuiwon Choi, Kwangmeyung Kim, Ick Chan Kwon ∗ Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, South Korea Received 15 December 2010; received in revised form 31 January 2011; accepted 17 February 2011 Available online 21 March 2011

KEYWORDS Nanoprobe; Nanoparticle; Imaging; Diagnosis; Sensor; Nanotechnology

Summary Recent progress in bio and nanotechnology enables the development of various nanoprobes for biomedical imaging. The detection and imaging of specific changes in biological microenvironments play key roles in many fields such as biological mechanism study, drug screening, diagnosis of many diseases, and monitoring of therapeutic responses. Especially to obtain fine images in living systems, there are many requirements such as stability in complex condition, efficient accumulation in target site, and high sensitivity to the target molecule. For this purpose, many researchers have developed a number of nano-size probes sensitive to the external changes which are valuable from biomedical point of view. Herein, we will review the on-going challenges of developing novel nanoprobes to detect chemical or biological changes such as enzyme, oxygen, and pH, and applying them to biomedical imaging in living systems. © 2011 Elsevier Ltd. All rights reserved.

Introduction Recent several decades are explosion of nanotechnology for biomedical applications. Nanotechnology is emerged as an integrated research field aimed at investigating and controlling nano-materials by combining concepts from chemistry, biology, engineering, pharmaceutics, and so on [1]. Importantly, it provides the driving force of many small and great changes in the biomedical research and clinical fields [2]. The rapid growing applications of nanotechnology in the biomedical field is evidenced by the fact that this market is increasing by more than about 17% annually and could grow to about $53 billion by 2011 [3].

∗ 1

Corresponding author. Tel.: +82 2 958 5912; fax: +82 2 958 5909. E-mail address: [email protected] (I.C. Kwon). Both the authors contributed equally.

In biomedical fields, there are various different parts including drug delivery, tissue engineering, imaging, and medical device, etc., and nanotechnology currently exerts great influence in overall parts [4,5]. Especially the application of nanotechnology in biomedical imaging is highly active and exciting for there are wide range of targets for imaging and many hurdles to overcome [6]. The impact of nanotechnology on this field does not only assist to improve the efficacy of conventional imaging moieties and but also make entirely new imaging methods [7]. A lot of challenging developments and clinical applications of novel nano materials and techniques have been developed with the help of nanotechnology [8,9]. Among various applications of nanotechnology for biomedical imaging such as cell tracking, diagnostic kit, harvested tissue analysis, and live imaging, the present article aims to describe about the real-time live imaging with nanoprobes and detecting particular changes of microenvironments in living systems.

1748-0132/$ — see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.nantod.2011.02.007

Nanoprobes for biomedical imaging in living systems

Why ‘nano’ probe? At present, a lot of nanoprobe systems are being developed to explore their potential in biomedical fields, with many applications to the diagnosis or monitoring of various diseases [10]. At this time, we first address a question in a flood of so many nanoprobes for biomedical imaging and therapy: Why ‘nano’ probe? To answer this question, we must check the advantages of nano-size probes for the application in biomedical field. First, the scale of cell and tissue structures is meaningful. Since most nanoprobes are about 10—500 nm which are generally over 100 fold smaller than cells, their sizes are of the same or lower order of magnitude compared with that of the pores and openings in vasculature and tissues of human body [4]. Their uptake by kidney (renal clearance) was lower than single molecules and the uptake by reticularendothelial system (RES) of liver was lower than micro-size materials [11]. Therefore, nano materials show increased circulation time and have more chances to reach the target site than single molecules. In addition, they efficiently accumulate at the vascular angiogenic sites like tumor and arthritis because of the fenestrated vasculature structures resulting in the enhanced permeability and retention (EPR) effect [12]. These features allow nanoprobe systems to be more preferable for targeted delivery of imaging agent or drug. Second, the building blocks of nanoprobes like proteins, carbohydrates, nucleic acids, synthetic polymers or small inorganic particles are generally smaller than about several scores of nanometers [13]. Researchers fabricate their nanoprobes by the assembly of these blocks on rational design for particular purpose. Therefore the scale of most nanoprobes is inevitably determined to several scores or hundreds of nanometers. Third, nanoprobes have potential for wide application because they can easily contain useful molecules such as imaging agents, active targeting moieties, or drugs by simple loading or conjugation. Once well-designed nanoprobe was developed and its feasibility was confirmed, it can be used to other purpose by small changes of containing functional molecules [2]. Thus innovate design of nanoprobes is valuable in multiple targets and they can be simply modified or improved for various purpose on both imaging and therapy. Moreover target cell specificity can be equipped by simple conjugation of active targeting moieties like antibody or peptide [14]. And at last, switching ability or multi-functions can be adapted

Table 1

205 to nanoprobes by smart design and development. Several nanoprobes can be degraded or dissembled to release their containing molecules at specific conditions, and some are based on the fluorescence quenching systems which can be activated at specific condition [10,15]. Moreover, when both imaging molecules and drugs are adapted to same nanoprobe, simultaneous imaging and therapy are enabled [16]. These properties can be hardly achieved with single molecule.

Various materials used for nanoprobes Scientists have found various useful materials from different origins, and developed many nanoprobes with them for their purpose. Representative materials generally used in development of nanoprobes and their advantages are summarized in Table 1. Polymers are most general building blocks of nanoprobes with easily tunable characters [17]. Their physicochemical properties such as size, charge, hydrophobicity, branching and degradation can be controlled by changing their chemical structure or physical fabrication method. A lot of natural or synthetic polymers have been used for nanoprobe fabrication. Carbohydrates like hyaluronic acid, dextran, chitosan, pulluran and their derivatives can be adjusted to proper sizes and widely applied to the clinical fields [18]. Proteins such as albumin, transferrin, and antibodies are used as both building blocks and targeting moieties for nanoprobe development [19]. Recently, nucleic acids are also paid many attentions and used for its target specific binding ability [20]. In addition, biocompatible synthetic polymers such as polyetheyleneglycol (PEG), polyvinylpyrrolidone (PVP) and poly(lactic-co-glycolic acid) (PLGA) have approved by FDA for their biomedical usage to human body and usefully applied for many probes [21,22]. Quantum dots (QDs) are nano-size fluorescent semiconductor crystals with unique optical and electrical properties [7]. Compared with other organic dyes and fluorescent proteins, QDs show much greater brightness, broad absorption/narrow emission bands, and negligible photobleaching [23]. These properties make QDs advantageous especially for real-time imaging in living systems when properly delivered to target site [24]. Along with the studies about their biodistribution and clearance and the development of less toxic indium—gallium QDs, applications of QDs are predicted to keep growing [25,26].

Various materials used in nanoprobe fabrication.

Materials

Types

Advantages

References

Polymer

Carbohydrate, protein, nucleic acid, synthetic polymer

[13,17]

QD

Cd/Pd QD, Id/Ga QD

Iron oxide Gold

SPION, CLIO Nanoparticle, nanoshell, nanorod

Most general building blocks. Various kinds of polymers are applicable. Easy synthesis and modification Strong fluorescence than single molecule dye. Negligible photo-bleaching Biocompatible. MR imaging Biocompatible. Raman, SERS, and CT imaging. Photothermic effect. Quenching effect

[23,25] [27,28] [33,35]

206 Iron oxide nanoparticles have received considerable attention for their potential in optical, magnetic and electronic fields [27]. They are expected to show biocompatibility at low concentration and high magnetic property [28,29]. Importantly, superparamagnetic iron oxide nanoparticles (SPION) have large magnetic moments and can be applied as T2 contrast agents in MRI [30]. Highly superparamagnetic iron oxide can be coated by biocompatible polymers such as dextran to develop the crosslinked iron oxide nanoparticles (CLIO) with increased stability [31]. This type of iron oxide nanoparticles like Feridex, Combidex, Resovist, and AMI-288/gerumoxytrol are currently used in clinical fields for MRI diagnosis [32]. Gold nanoparticles were discovered more than 100 years ago, and have been extensively studied for both biomedical imaging and therapy [1]. They have several attractive characteristics for diagnostic applications, such as biocompatibility, optical properties, strong binding affinity toward thiol groups, high atomic number and high X-ray absorption coefficient [33]. Especially, its optical property is tunable by its size and shape, and its surface plasmon enables quenching effect to near fluorophores [34]. Moreover, facile synthetic methods were developed for producing them with precise control over the particle size and shape [35]. Therefore, gold nanoparticles have been applied as an imaging agent with various techniques such as dark-field microscopy, two-photon luminescence, optical coherence tomography, Raman scattering and computed tomography (CT) [36,37].

Representative imaging targets in body During the disease progress, inflammation, or therapeutic treatment cause some physiological and biochemical changes within human body microenvironment such as temperature or pH. Thus to know the exact site and the degree of these changes are significantly valuable in the biomedical perspective. To detect and image these changes inside live systems, various smart nanoprobes were developed by many researchers [38]. Diagnosis, imaging and monitoring of therapeutic response with these nanoprobes are very challengeable and exciting project in both academic and clinical fields. Representative features of human body in abnormal conditions are highly related with temperature, enzyme, oxygen, pH and so on. High fever is the most general symptom of illness, and specific enzyme secretion is the core metabolism of diseases and therapeutic responses in body [39]. Reactive oxygen and hypoxia is highly attractive themes for biomedical researchers, and acidic or basic conditions also show the significant imbalance of homeostasis [40]. Among them, temperature is most easily accessible subject and studied for long time, and nowadays, this can be simply detected by thermometers and infrared cameras [41]. However, the other three factors are not fully understood and there are still many obstacles to detect and image in living systems. Therefore in following contents, we will focus on these three factors (enzyme, oxygen, and pH) and examine recent developments and applications of novel nanoprobes for detecting and imaging them in living systems.

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Nanoprobes for enzyme imaging Design concepts of nanoprobes for enzyme imaging Enzymes play key roles in many pathophysiological processes, such as degradation of extracellular matrix, invasion, metastasis, and angiogenesis in cancer and several inflammatory diseases like arthritis [42]. Some kind of enzymes are relatively abundant in diseased tissues (even at early stages), which are attractive targets for both imaging and therapy [43]. Among various enzymes, large amount of them are proteolytic enzymes that degrade specific peptide sequence within proteins that participate in many biological mechanisms. Therefore this sequence specific recognition and cleavage has been usefully applied to design contrast agents or molecular probes [44—46]. Additionally, well-known solid phase peptide synthesis (SPPS) allows reproducible construct synthesis with accurate chemical structures, and increases scale-up feasibility for the mass production of peptide-based construct [10]. Two different designs of peptide-based probes are available depending on the type of quencher used. The first design relies upon self-quenching mechanism, where the dye and quencher/acceptor are the same or similar fluorophores [43]. This probe offers advantages such as relatively simple synthesis process, while they can generally represent less efficient fluorescent quenching. Bremer et al. reported NIR fluorescent probe consisting of multiple Cy5.5 bound to a long circulating graft copolymer containing poly-lysine backbone and methoxy polyethylene glycol graft [47]. Multiple Cy5.5 on the graft copolymer results in self-quenching because of the close proximity between the fluorophores. Unmodified lysines on the poly-lysine backbone were served as a substrate peptide (Lys-Lys) for proteases including cathepsin B. The cathepsin B-responsive probe can determine cathepsin B enzyme activity in optical imaging of mouse breast cancer. The second design is based upon fluorescence resonance energy transfer (FRET) as well as self-quenching mechanism, where another kind of quencher is used to suppress fluorescence signal of fluorophore [48]. This design is advantageous with respect to high quenching efficiency, which can improve both the sensitivity and specificity of optical imaging. The basic structure of peptide-based probe for imaging of targeted enzymes is very simple. Peptide-based probe is comprised of fluorophore and quencher covalently linked to opposite terminus of a short peptide linker that is the substrate of certain specific enzymes [10]. In the native condition, the emitting light from the fluorophore is absorbed into the quencher by FRET. Once target enzymes degrade the peptide linker, the extent of fluorescence remarkably enhances by increasing the physical distance between the fluorophore and the quencher. These peptide linkers may be selected by modification of known native peptides within proteins, peptide libraries such as phage-displayed peptide libraries, or molecular modeling of receptor-peptide in the binding pocket of enzymes [49,50]. Especially for in vivo imaging, fluorophore generally use the near-infrared (NIR) fluorescence light (650—900 nm). Advantages of NIR fluorescence light include high tissue penetration (up to several centimeters deep) and low autofluorescence providing suffi-

Nanoprobes for biomedical imaging in living systems cient signal-to-background ratio [51]. Quencher, an acceptor fluorophore in FRET mechanism, can be selected after considering emission spectrum of used fluorophores. However, several drawbacks including inherent fluorescence of the quencher may reduce high signal-to-background ratio. Dark quenchers have no native fluorescence and act only as a strong absorber. Introducing dark quencher into the peptidebased probes would further reduce the background signal of fluorescence [52]. Peptide-based probes can need targeting property and cell permeability, and have short circulation time in itself [10]. Modifying the peptide-based probes with cellpenetrating peptides, targeting moiety and biocompatible nanoparticles (e.g., polymeric nanoparticles, gold nanoparticles) can be accompanied to overcome these drawbacks. Use of polymeric nanoparticles (NP) as imaging probes offers several advantages over small molecules: (i) NPs allow easy surface modification and large surface area-to-volume, which is beneficial for the attachment of fluorophores or targeting moiety [1]. (ii) NPs can increase circulation time and preferentially accumulate at tumor sites because of the EPR effect [12]. These features of NPs as imaging probes contribute extended localization of larger amount of imaging probes, which is critical to high signal-to-background ratio [43]. Gold nanoparticles (AuNPs) are also attractive candidates for attachment of peptide-based probe due to their excellent biocompatibility, controllable size, high photostability, strong absorbing and scattering properties, sensitive fluorescence quenching efficacy, and easy bioconjugation and preparation [53].

Various enzyme imaging with nanoprobes Apoptosis is the process of programmed cell death that occurs in multicellular organisms to maintain homeostasis. Apoptosis is highly related with human diseases including cancer and neurodegenerative or autoimmune disorders, and the therapeutic methods inducing or inhibiting apoptosis has been widely studied. Therefore, imaging tools capable of detecting apoptosis progression would become increasingly important. One strategy for imaging apoptosis is to monitor specific apoptosis signaling molecules like the caspases [54]. Jun et al. reported AuNP assemblies that detected earlystage of caspase-3 activation at the single-molecule level using plasmon coupling property (Fig. 1) [55]. The distinctive plasmon coupling property in AuNPs provides excellent potential for the optical characterization of many cellular and biomolecular processes. In this construct, the AuNPs are assembled to nanostructure where one AuNP is surrounded by several others and attached together by caspase-3 substrate peptide linker (DEVD). Initial intense red-colored spots gradually progressed yellow and then dim green spots as time elapsed by addition of caspase-3 enzyme. Caspase-3 substrate peptide linker-attached AuNP assemblies allowed continuous monitoring of caspase-3 activity in live cells for over 2 h and provided clearly detectable signal of early-stage caspase-3 activation in apoptotic cells. These nanoprobes allow excellent potential to provide increased signal intensity required for direct visualization of apoptosis in the single cell level, and prolong the monitoring time necessary for imaging cellular and biomolecular processes in live cells

207 due to strong scattering property in visible light and high photostability without blinking or photobleaching. Urokinase plasminogen activator (uPA) has been implicated in several cancers including prostate and breast, by involving tumor formation, local invasion, and metastasis [56]. Mu et al. presented the utility of uPA-responsive gold nanoprobe as an imaging agent in a mouse tumor model and reported on optimal formulation of parameters important to probe performance for making protease-responsive fluorogenic nanoprobes based on AuNPs (Fig. 2) [48]. In this construct, AuNPs (20 nm diameter) were assembled with fluorophore (Quasar 670) and dark-quencher (BHQ-2)-labeled peptide substrates and poly(ethylene glycol) using a AuNPthiol modification. The results demonstrated that the AuNPs using dark quencher further reduce the background signal of fluorescence, beyond the signal suppression offered by the AuNPs, resulting in optimal fluorescence signal and image contrast. These gold nanoprobes with the optimal formulation exhibited extended circulation time and high image contrast in a mouse tumor model. Matrix metalloproteinases (MMPs) are a class of zincdependent endopeptidases that are involved in certain inflammatory diseases and cancer progression [57,58]. Because of their significant role in various diseases, MMPs have long been interesting as a crucial target for imaging and therapy. A variety of imaging modalities are utilized for the detection and imaging of MMPs in vivo. Recently, our group developed NIR fluorescent nanoprobes consisting of polymeric nanoparticles and dark-quenched peptide-based probes for detection of MMPs (Fig. 3) [59]. The dark-quenched MMP-specific peptide-based probes (Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-GlyLys(BHQ-3)-Gly-Gly) were conjugated onto the surface of glycol chitosan-based polymeric nanoparticles (HGC) as a tumor targeting carrier. Among the peptide sequence, the specific substrate, Pro-Leu-Gly-Val-Arg-Gly could be degraded by various MMP enzymes. This nanoprobe formed spherical structures with diameters ranging from 200 to 400 nm in aqueous conditions and demonstrated the linear proportional recovery of fluorescence signals according to MMP concentrations in vitro. It was investigated the utility of in vivo imaging by treating them to MMP-2/9-positive SCC7 tumor and caused the high recovery of fluorescence signals in SCC7 tumor. In contrast, the recovery of fluorescence signal was suppressed when the nanoprobe was treated with MMP-2/9 inhibitor. When the peptide-based probe itself without nanoparticle was administered, reduced fluorescence signals was shown in SCC7 tumor compared to that of the nanoparticle conjugated probe, which means nanoprobes could be preferred for delivery of the imaging agents to the target region. This NIR fluorescent nanoprobe was also administered to colon cancer, and the ex vivo fluorescence image demonstrated that all tumors gained high signals of fluorescence, comparing to adjacent mucosa. Moreover, these MMP-responsive nanoprobes were applied for the imaging of atherosclerotic plaque by Kim et al. [60]. Fluorescence signals reflected MMP activities from macrophages in complex human atheromata. In addition, fluorescence signals in carotid atheromata or in emboli show the pathophysiologic changes of plaque inflammation. These results suggest that use of NIR fluorescent nanoprobe in molecular imaging provides molecular information on patho-

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Figure 1 Gold nanoprobe with responsive assembly and caspase-3 imaging in live cells. (a) AuNP assemblies were immobilized on a glass flow chamber via avidin—biotin chemistry. AuNPs are assembled to nanostructure where one AuNP is surrounded by several others and attached together by caspase-3 peptide linker (DEVD). (b) Scattering color changes (intense red colored spots progress to yellow spots and then dim green spots) by addition of caspase-3 enzyme (c) Schematic diagrams of AuNP assemblies. (d) Cellular delivery of AuNP assemblies in live cells. Bright red-colored spots correspond to individual AuNP assemblies. Source: Figure adapted, with permission, from [55].

logical processes such as inflammatory protease activities, and complements anatomic imaging like angiography or ultrasound.

Nanoprobes for oxygen imaging Reactive oxygen species (ROS) imaging The environmental stress from the exposure of toxin and drugs as well as endogenous factor like inflammations may induce the production of reactive oxygen species (ROS), including hydrogen peroxide (H2 O2 ), superoxide anion (• O2 − ), and hydroxyl radical (• OH− ), and so on [40]. Over production of ROS can make oxidative stress and damage to

the intracellular DNA, protein and lipid layers. This result cause cellular damage and increase the sensitivity to the further treat of chemotherapeutic agents, thus it can be an important indicator for the therapeutic procedure. Furthermore, change of ROS level provides physiological and biological information about the state of the living system, because it is believed that ROS generation during the oxygen metabolism is a signal that it is associated with aging and most diseases such as cancer, heart disease and atherosclerosis [61—64]. Previous studies revealed that cancer cells generally produce more ROS than normal cells [65,66]. On the other hands, generation of ROS leads not only the oxidative stress, but it can be utilized for a tool of cancer therapy. ROS could be actively generated from the anti-cancer drug treatment, such as photosensitizer. When the photosensi-

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Figure 2 PEGylated gold nanoprobe for in vivo urokinase imaging. (a) Schematic diagrams of gold nanoprobe synthesis and activation. (b) In vivo NIR fluorescence imaging of subcutaneous tumor mouse with molar equivalents of uncomplexed Q670-labeled and BHQ2-labeled peptide substrate with and without unlabeled AuNPs (upper) and gold nanoprobe with and without trypsin as compared to unlabeled AuNPs (lower). Source: Figure adapted, with permission, from [48].

Figure 3 Fluorescence activatable nanoprobe and in vivo MMP imaging. (a) Schematic diagrams of MMP-sensitive nanoprobe. (b) Chemical structures of polymeric nanoparticle and MMP-specific peptide-based probe. (c) Fluorescence image of a 96-well microplate containing the nanoprobes in the presence of various concentrations of activated MMP-2. (d) TEM image of the nanoprobes. (e) In vivo NIR fluorescence imaging of subcutaneous SCC7 tumor-bearing mice after intravenous injection of the nanoprobes with or without the inhibitor and unbounded peptide-based probes without inhibitor and histology analysis of SCC7 tumors (right). (f) Photo image of colon tumors from an A/J mouse treated with azoxymethane (upper), NIR fluorescence image of colon tumors after intravenous injection of the nanoprobes (lower), and histology analysis of colon tumors (right). Source: Figure adapted, with permission, from [59].

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Figure 4 Functionalized hyaluronic acid immobilized gold nanoprobes (HHAuNPs) for ROS detection. (a) Schematic illustration of NSET-based on/off mechanism in NIRF dye (HilyteFluorTM 647) labeled oligo-HA immobilized AuNP. (b) NIRF dye labeled oligo-HA fragments for surface immobilization to the AuNPs. (c) Monitoring the over production of ROS from the LPS (1 mg/ml) or PMA (100 ␮M) stimulated THP-1 cells. (d) Evaluation of cellular HAdase secretion from three different cell lines. (e) In vivo fluorescence images of tail vein injection of HHAuNPs (10 pmol) in normal and tumor-bearing mice. Source: Figure reproduced, with permission, from [72].

tizer is irradiated with a light of specific wavelength, drug is excited to singlet states and returned to the ground singlet states resulting in release of singlet oxygen molecules during the time [67,68]. As the monitoring of the ROS concentration by imaging probes, many cellular events can be understood, and that’s what researcher has been interested in development of probes for monitoring the production and effect of ROS. Many probes are developed to amplify the signal upon specific biochemical stimuli or environmental change for real-time imaging. As for the ROS probes, it should be designed to reveal specific ROS generation and change with a high selectivity, and can elucidate the spatial and temporal reactive oxygen behavior. A number of ROS detection probes and methods are developing and using, but conventional ROS probes have some limitation such as auto-oxidation, poor specificity and less sensitivity for in vivo analysis of ROS generation [69,70]. Recently several attractive nanoprobes that exhibit a high selectivity toward the ROS have been reported, and these nanoprobes are expected to have great potential to unveil the unknown physiological mechanisms related with ROS generation.

Fluorescent probes are versatile tools for imaging the biological event within in vitro as well as in vivo system. Several strategies are tried to monitor the production of ROS and evaluate the fate of ROS. Fluorescent proteins are commonly used for genetically modified as an indicator for specific cellular events and circumstance. Belousov et al. constructed fluorescence probe HyPer, for detecting H2 O2 within living cells [71]. This probe composed with cpYFP (circularly permuted yellow fluorescent protein) inserted into H2 O2 specific sensing OxyR protein and in vitro assay represented a specific and sensitive for detecting H2 O2 . Recently, Lee et al. developed novel nanoprobes with gold nanoparticle and NIRF dye labeled hyaluronic acid for ultra-sensitive detection of ROS (Fig. 4) [72]. Hyaluronic acids are easily degraded from the gold nanoparticles by intracellular superoxide anions and hydroxyl radicals, and simultaneously NIRF fluorescent dye recovered from the quenched states for a real-time imaging. The advantage of fluorescence conjugated with AuNPs is that they can exhibit the highly sensitive nanoparticle surface energy transfer (NSET) for ROS detection than commercial fluorescent probes, like DCFH and APF. Using this nanoprobe, they

Nanoprobes for biomedical imaging in living systems evaluated enhanced in vivo NIRF fluorescence signal with increasing ROS concentration from the induced rheumatoid arthritis or tumor-bearing mice model through local or systemic injection. Also they increased the intracellular stability of the nanoprobe by introducing the DOPA (L-3, 4-dihydroxy-L-phenylalanine) which is responsible for the much strong adhesive properties to the inorganic/organic surfaces via non-reversible binding [73]. DOPA functionalized HA was tightly linked on the surface of gold nanoparticle via Au-catechol bond and it serves for more stability of nanoprobe for intracellular ROS detection. One of the needs for ideal nanoprobe for in vivo imaging is minimal autofluorescene interference with a high specificity and sensitivity. Chemiluminescence (CL) can be introduced for the detection and imaging modality for ROS concentration as emission intensities of luminescence by the result of chemical reaction. The advantage of CL is a reduced interference, because there are no external excitation light sources, which cause scattering back-ground such as autofluorescene [74]. One of the most interested ROS, hydrogen peroxide (H2 O2 ) is revealed as a crucial signaling molecule to control cell death and growth by mitochondria. It is considered that each kind of ROS has different properties. Among the ROS, H2 O2 is generally known for the highest stability and the least reactivity with the highest intracellular concentration [75]. Lee et al. introduced peroxyoxalate chemiluminescence (POCL) nanoparticles for in vivo imaging of H2 O2 with high specificity [76]. After that, Lim et al. demonstrated that advanced water-dispersed multifunctional nanoprobes can achieve the in vivo visualization of disease related hydrogen peroxide and glucose level using optimized POCL (peroxyoxalate chemiluminescence) reaction (Fig. 5) [77]. They produced FPOC nanoparticles (FPOC NPs), which are composed of biocompatible Pluronic (F-127), poly(lactic-co-glycolic acid) (PLGA) for a polymeric stability, bis[3,4,6-trichloro-2(pentyloxycarbonyl)phenyl] oxalate (CPPO) as for a optimum POCL reagent with enhanced reactivity, and Cy5 as a NIR dye. These nanoprobes generate dramatic luminescence signal from the chemically triggered by H2 O2 with high sensitivity as low as 10−8 M which is useful for detecting disease caused hydrogen peroxide level, which is also below the normal physiological fluids around 10−7 M. FPOC NPs demonstrated the great capability to visualization H2 O2 level from the inflammatory disease model at early stage and blood glucose level at tumor bearing mice model.

Tumor hypoxia imaging In solid tumors, hypoxia represents a tumor-specific microenvironment condition in which tissues possess lower oxygen levels than physiological levels and show difference in oxygen concentration between intra- and inter-tumoral regions (Fig. 6) [78]. It is well known that the vasculature in a solid tumor is often inadequate to satisfy the demand of oxygen and nutrition from blood vessels for growing beyond a size of 1—2 mm, mainly due to rapid proliferation and abnormality of blood vessels, which result in making some areas of tumors more hypoxic compared to other normal tissues [79,80].

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Figure 5 Optimized ROS detection nanoprobes based on peroxyoxalate fuel and a cyanine dye. (a) Schematic illustration of nanoprobes formulation (FPOC NPs) and CL generation by hydrogen peroxide (CPPO as a POCL reagent) input. (b) Mechanisms of POCL reaction in FPOC NPs. (c) In vivo CL imaging of LPS induced arthritis model. FPOC NPs were injected into bilateral ankles by intra-articularly 48 h after with or without LPS treatment. (d) Time-dependant in vivo image of CL intensity (total flux of imaging signal) from FPOC NPs-injected ankle joints taken from (c). Source: Figure adapted, with permission, from [77].

Tumor hypoxia has shown to have a poor prognosis on response to various tumor therapies, and also been known as important in an aggressive tumor phenotype, such as invasion, growth, and metastasis [81]. Under these hypoxic conditions, tumor cells with low oxygen partial pressure (pO2 ) <5 mmHg are generally resistant to radiation therapy since oxygen is a potent radiosensitizer which enhance the radiation therapeutic effect [82]. Furthermore, hypoxia refer to decreased susceptibility for chemotherapy because tumor regions located at some distance from blood vessels is known to impair therapeutic agents delivery to tumor cells at an effective concentration [83]. For these reasons, many techniques for measuring hypoxia in the tumor region are emerging to provide invaluable information in tumortreatment planning. Since Gray et al. first proposed the presence of hypoxia in lung cancer model, there has been increasing interest and

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Figure 6 The representative features of hypoxia in solid tumors. Tumor tissues possess impaired oxygen and drug delivery from the capillary and show difference in oxygen concentration between normoxia (black) and hypoxia tumoral regions (red). Hypoxia regions with pimonidazole (red) are located far from the blood vessels (green). The figure also shows a resistance to therapy in solid tumors against increasing distance from the blood vessels. Source: Figure adapted, with permission, from [78].

development efforts to detect the hypoxic region in tumors for predicting drug resistance and radioresistance [82]. For the detection and quantification of tumoral hypoxia, polarographic needle electrodes have been advanced, but this method have limitation to their practical application due to their invasive nature, especially referring to disrupt tissues by insertion of an electrode, partial information in the whole tumor region, and the inability to monitor oxygenation during treatment [84,85]. Therefore, variety of noninvasive imaging methods including photoacoustic tomography (PAT), near-infrared spectroscopy (NIRS) and blood oxygen level-dependent (BOLD) MRI have been interested to detect hypoxia (in tumor region) based on the distinct oxygen-dependent contrast mechanisms between oxy- and deoxy-hemoglobin [86—88]. However, although these methods have considerable potential to measure tumor hypoxia, they only provide partial pressure of oxygen in vasculature but not in the tumor tissues. Nitroimidazole derivatives such as pimonidazole and EF5 are used for both invasive immunohistochemical (IHC) detection and non-invasive PET/SPECT imaging of hypoxia [89,90]. These exogenous markers usually form stable adducts for in vitro quantitative measurement or in vivo non-invasive hypoxia imaging. Radiolabeled PET and SPECT tracers with nitroimidazole derivatives have been intensively investigated and this type of radiotracers for imaging tumor hypoxia are summarized in several review articles [91—93]. Imaging techniques based on the phosphorescence or near-infrared fluorescence quenching of an oxygen sensitive probe have been used for oxygen sensing in biological systems. The use of albumin bound metal—porphyrin complex (Oxyphor) has shown to provide real-time measurement of oxygenation in the vasculature [94]. Recently, Lebedev et al. reported the preparation of non-invasive dendritic phosphorescent probes for oxygen imaging [95]. They demonstrated the probes can be used for measurement of oxygen level in the vasculature of the rate brain with a high selectivity. The oxygen-dependent quenching of chromophores is reversible so that the probes are useful for monitoring oxygenation status. Luminescent optical probes using oxygen sensitive near-infrared (NIR) dye have also been used for measuring oxygen. Lee et al. prepared NIR luminescent oxy-

gen nanosensors with nanoparticle matrix to be applied for in vivo oxygen imaging [96]. The ratiometric probe-loaded oxygen nanosensors have been used for measuring of oxygen levels under normal and hypoxic conditions. Recently, Zhang et al. described a simple strategy to construct dualemissive boron biomaterial with tuning relative fluorescence and room-temperature phosphorescence (RTP) intensities [97,98]. In this study, they demonstrated that nanoparticles with iodide-substituted difluoroboron dibenzoylmethanepoly(lactic acid) (BF2dbm(I)PLA) serve as optical tumor hypoxia imaging agents (Fig. 7) [97,98]. Biological activity of hypoxia-inducible factor 1 (HIF-1) consisting of HIF-1a and HIF-1b subunit is specifically regulated by hypoxic condition in the cell [99,100]. While the normoxia leads to the proteasomal degradation of HIF-1a, the hypoxia condition stabilizes HIF-1a to form heterodimer HIF-1 with HIF-1b. HIF-1 binds to hypoxia responsive element (HRE) in the promoter of target genes that are involved survival and aggressiveness of hypoxia tumor cells. Optical imaging strategies by using hypoxia-response promoter coupled with reporter genes such as green fluorescent protein (GFP) or luciferase have been studied by many researchers [101,102]. Kizaka-Kondoh et al. prepared protein transduction domain (PTD)- oxygen-dependent degradation domain (ODD)-enhanced GFP labeled with near-infrared fluorescent dye Cy5.5 as an imaging probe for HIF-1 active cells [102,103]. This fusion protein containing the oxygendependent degradation domain (ODD) of HIF-1a showed that it permeated tumor cells with high efficiency and accumulated in the hypoxia condition after intravenous injection of the labeled probe, resulting in imaging of HIF-1 active cells in vivo. For the effectiveness of gene therapy, it is needed to develop non-invasive visualizing method that could monitor gene delivery. Recently, Han et al. have constructed PEG-b-P[Asp(DET)] polyplex micelles containing HRE-driven fluorescent reporter genes (Fig. 8) [104]. These polyplex micelles with reporter genes showed hypoxia-selective gene expression in the multicellular tumor spheroids (MCTSs) models and intratumoral injection into subcutaneous tumor models. These results suggested that they can be used to treat the hypoxic region of the tumor tissue by gene therapy and to provide non-invasive monitoring for gene delivery. Furthermore, these nanoprobes show improved efficiency

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Figure 7 Stereocomplexed poly(lactic acid)—poly(ethylene glycol) nanoparticles with dual-emissive boron dyes (BF2dbm(I)PLLA) for tumor hypoxia imaging. (a and b) Schematically chemical structures BF2dbm(I)PLLA and mPEG-b-PDLA for boron nanoparticles (BNPs). (c) The emission spectra provide green room temperature phosphorescence (RTP) decrease as a function of increasing O2 concentration. (d) Dual-emissive BNPs show blue fluorescence (left) and green—yellow phosphorescence (right) measured under air and nitrogen atmosphere, respectively (ex = 365 nm). (e) In vivo tumor hypoxia imaging with BNPs under the bright-field (i) and while breathing carbogen (95% O2 ) (ii), air (21% O2 ) (iii) and nitrogen (0% O2 ) (iv). The images indicate oxygenated regions in the fluorescence/phosphorescence intensity (yellow—red) with less-oxygenated tumor region (blue). Source: Figure reproduced, with permission, from [97,98].

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Figure 8 PEGylated polyplex micelles containing hypoxia responsive element (HRE)-driven fluorescent reporter genes for enhanced permeation and gene expression in tumor hypoxia. (a) Chemical structures of P[Asp(DET)] and PEG-b-P[Asp(DET)] polymers for PEGylated polyplex micelles. (b) Gene expression of pCAcc + Venus at the inner region of MCTS transfected with PEGylated polyplex micelles. Source: Figure adapted, with permission, from [104].

of delivery and transfection, thereby achieving enhanced fluorescence signal in hypoxia.

Nanoprobes for pH imaging pH imaging inside cells Intracellular pH change is highly important factor in the regulation of different cellular processes and provides much valuable information in biomedical imaging. For instance, the imaging of whole endocytosis procedure helps for the development of efficient drug carriers. Especially, in case of biological drugs like proteins or nucleic acids, their structural change or degradation crucially affect on their therapeutic ability and their large size prohibits fast movements across cellular organelles. Thus the spatial or temporal information about endocytosis is highly considered for the use of biological drugs [105]. Many papers also showed that intracellular acidification is highly related multi-drug resistance or apoptosis [106]. Moreover, the change of cytosolic pH is sometimes one of the responses to drug administration [107]. Therefore, imaging of the change in pH in live cells is crucial to our understanding and quantification of cellular metabolism, disease, and response to therapy. This means that there is a significant need for noninvasive, sensitive, quantitative, stable, and real-time pH imaging and nanotechnology showed very interesting improvements in this field. Peng et al. used ratiometric fluorescence for sensing of intracellular pH changes from six to eight [108]. Their nanoprobe is made of biocompatible polyurethane polymer nanogel that was made pH sensitive by loading the pH indicator bromothymol blue (BTB). To this nanogel, two standard fluorophores, coumarin 6 (C6) and Nile Red (NR) were added and they show efficient fluorescence resonance energy transfer (FRET) inside the nanogel. They were selected to give two different fluorescent signals (green and red) and their ratio in pH-responsive nanogel indicates pH. Their feasibility of intracellular pH sensing was

demonstrated in epithelial normal rat kidney (NRK) cells and showed pH imaging at moderate levels. More precise intracellular pH imaging was accomplished by Modi et al. with ‘DNA nanomachine’ (Fig. 9) [109]. DNA nanomachines are nucleotide assemblies that switch between rational molecular conformations in response to external stimuli. The combination of pH-responsive nucleotide assemblies and multiple color fluorophores enables pH-triggered fluorescence resonance energy transfer (FRET) inside live cells. With this DNA nanomachine, spatial and temporal pH changes associated with endosome maturation was successfully detected and imaged. Moreover, when this nanoprobe was linked to particular protein, they can track and analyze the pH change of given protein. They have demonstrated proof of concept by labeling the receptor-mediated endocytic (RME) pathway of transferrin inside cell with this nanoprobe. The fine performance of DNA nanomachines inside live cells showed the great potential of them in diagnostics and targeted therapies in living systems.

In vivo pH imaging Intracellular lysosomal pH sensitivity can be also applied to improve the specificity of in vivo imaging. Urano et al. developed novel pH-activatable nanoprobes for viable cancer cell imaging [110]. They conjugated multiple pH-sensitive fluorescence dyes with trastuzumab, monoclonal antibody. Trastuzumab binds to target the human epidermal growth factor receptor type 2 (HER2) in tumor cells and this nanoprobe internalized by receptor-mediated endocytosis. During this pathway, conjugated dyes are activated by lysosomal acidic pH and produce fluorescence signal. Therefore this probe can yield a highly specific signal inside tumor cells with greatly reduced background signal. The most important advantage of this probe is its specificity to work only live tumor cells, not dead cells due to its active cellular uptake and lysosomal activation. In HER2+ lung metastatic tumor-bearing mice model, in vivo detection and monitor-

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Figure 9 Intracellular pH imaging with DNA nanomachine. (a) Schematic diagram of the DNA nanomachine (I-switch) in the ‘open’ state at high pH and in the ‘closed’ state at low pH. FRET occurs in the ‘closed’ state. (b) Fluorescence profile of the Alexa-488/647 labeled I-switch at pH 4—7.3 in vitro. Inset graph is the time-resolved fluorescence spectra of the I-switch at pH 7.3 and 5. (c) Spatial and temporal imaging of pH changes during endocytosis with the I-switch in living cells. Scale bar is 5 mm. Source: Figure adapted, with permission, from [109].

ing of viable cancer cells was enabled with this nanoprobe of high sensitivity and tumor specificity. The in vivo pH change of large scale in tumor tissue or ischemic site as well as cellular change is very attractive for both biomedical imaging and therapy. In rapidly growing tumor tissue, large amount of oxygen and nutrients are needed but they are insufficient for relatively slower vascular generation as mentioned [79]. In this condition, the rate of glycolysis is up to about 200 times higher than that of normal tissues and it is called the ‘Warburg effect’ [111]. For this increased glycolysis, the concentration of lactic acid is highly increased causing microenvironmental acidosis. Importantly, it has been reported that this acidic pH about 6.5—7.0 in tumor tissues are closely related to the malignancy of cancer, the response to drugs or radiation, and finally the death rate of patients [112]. Furthermore, acidosis is a fundamental factor of ischemic stroke in brain tissue and is known to cause severe injury of neuronal cells [113]. The low extracellular pH in ischemic tissue increases inward currents of acid-sensing ion channels (ASICs) and intracellular calcium concentration in human neurons. Activation of these ion channels plays a crucial role in acidosis mediated injury of neurons in human brain [114]. Therefore in vivo extracellular pH imaging in target tissue provides highly valuable information to biomedical researchers. For the acidic pH imaging in tumor tissues, some imaging probes have been developed. However, most of them use relatively low pH responsive range below pH 6.5 targeting endosomal pH after cellular uptake or did not accomplish the fine visualization of the tumoral low pH under in vivo condition. Recently, our group rationally designed pH-responsive

nanoprobe suitable for tumoral lower pH imaging in live body (Fig. 10) [115]. We used pH-responsive block copolymer micelles with controllable pH range according to the change of alkyl chain length. By containing fluorescence dye and quenchers together in the core of micelle, very narrow and specific pH-responsive fluorescence generation was enabled in both in vitro and in vivo condition. It has nano-size structure that enables high accumulation in tumor tissues through fenestrated vascular, and it can be disrupted and produce strong fluorescence signal at the target acidic tumor tissue with high specificity and sensitivity. This pH-sensitive ‘nanoflash’ showed great potential for non-invasive in vivo tumor imaging of low pH environment for research and diagnosis. Novel nanoprobe for MR imaging of ischemic region of brain tissue was also developed with same pH-responsive block copolymer micelle [116]. To produce T2 MR signals, Fe3 O4 nanoparticles were encapsulated into this micelle without any targeting moiety. Its stable micelle structure at the physiological condition of pH 7.4 was disassembled in an acidic environment (pH < 6.8) of the ischemic site causing the release and accumulation of Fe3 O4 nanoparticles. Efficient accumulation of Fe3 O4 nanoparticles produces significant T2 MR contrast within ischemic tissue. Successful in vivo MR imaging with this nanoprobe was accomplished in middle cerebral artery occlusion (MCAO) treated mouse model. With this result, the MR imaging ability of this nanoprobe was confirmed and its application to other ischemic or pathologic acidic areas as well as the ischemic brain tissue is expected. Moreover, this type of nanoprobes could enable both imaging and drug delivery simultaneously, by changing the containing molecules in micelle structure [15].

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Figure 10 In vivo pH imaging in tumor and brain ischemia with pH-responsive micelle. (a) Chemical structure of pH-responsive MPEG-PAE block copolymer. (b) Illustration of pH-dependent fluorescence recovery system in acidic condition prepared from ‘nanoflash’. Upon pH lowering, the strong fluorescence intensity is recovered by disassembly of micelle. (c) Non-invasive real-time fluorescent imaging of tumoral pH in MDA-MB-231 tumor-bearing mice. (d) Schematic mechanism of pH-sensitive Fe3 O4 -encapsulated PEG-PAE micelles. (e) pH-dependent precipitation of Fe3 O4 -encapsulated PEG-PAE micelles in aqueous media at 37 ◦ C. (f) In vivo T2-weighted MRI scan of the rat brain with Fe3 O4 -PEG-PAE micelles. Middle cerebral artery occlusion (MCAO) was done in the right hemisphere of the rat brain. Source: Figure reproduced, with permission, from [115,116].

Figure 11 Tumoral pH activated charge-conversional nanogel. (a) Hydrolysis of PAMA—DMMA at pH 6.8 and illustration of increased tumor cell uptake. (b) Fluorescence images in tumor tissue after intratumoral injection of FITC-labeled PAMA—DMMA nanogels. DAPI-stained nuclei (blue), FITC-labeled nanogel (green), rhodamine phalloidin labeled F-actin (red). The white arrows indicate the location of the nanogels. Source: Figure adapted, with permission, from [117].

Nanoprobes for biomedical imaging in living systems Another pH-responsive nano material which can be used for both imaging and drug delivery was developed by Du et al. (Fig. 11) [117]. They developed pH-responsive polymeric nanogel and controlled its surface charge by acidic pH induced bond cleavage. The surface of this nanogel has negative charges at physiological pH and its cellular uptake is inhibited by charge repulsion with the anionic cell surface. However, at tumoral acidic pH, the amide bond between 2,3-dimethylmaleic anhydride (DMMA) and amine groups is degraded and its surface charge turn to positive causing fast internalization into tumor cells [118]. With fluorescein isothiocyanate (FITC), the efficient uptake and of this nanogels and their accumulation inside tumor cells were determined by green fluorescence signals in MDA-MB-435 tumor bearing mouse model. This type of nanogels can be used as nanoprobes for imaging and diagnosis and as nanocarriers for drug delivery and therapy.

Conclusion In the past decades, remarkable progress was accomplished in nanotechnology and their biomedical application for drug delivery, in vitro diagnosis, in vivo imaging, therapy techniques, and tissue engineering [5]. In this review, we focused on the challenges in detecting and biomedical changes of microenvironments in body and introduced recent developments of various novel nanoprobes for enzyme, oxygen, and pH imaging in living systems. During designing and developing nanoprobes for biomedical imaging, there are several demands for successful imaging in living systems [119]. First, their building blocks and final product should be biocompatible. The usage of body materials, biodegradable materials or FDA approved materials will be advantageous for fast application to clinical fields. Second, sufficient stability of nanoprobes are essentially required. Physical obstacles like blood flow and chemical obstacles like serum proteins or enzymes can be a cause for the unintended disruption, degradation, or aggregation of nanoprobes in living systems [120]. Third, the distribution of nanoprobes is also important for strong signaling. High amount of nanoprobes at the target site is needed than the required concentration for imaging, and size, surface property, and delivery method should be well determined [121]. Finally, for strong intensity and low background signals, target specificity is highly required. Its designated task should be performed with the same efficacy as it does in vitro within the molecularly crowded and dynamically changing living systems. Smart quenching system or stimuli-responsive activation with nanotechnology is very helpful in this point of view [10,115]. To meet these demands of biomedical imaging in live systems, the wide application of nanotechnology is essential and continuous efforts are necessary in developing novel nanoprobes with high efficiency and specificity. The integration of nanotechnology and biomedical imaging introduced new direction to the field of diagnosis of disease and monitoring of therapy [1,2]. We believe and expect that new intelligent nanoprobes will be developed by many researchers and more precise and efficient biomedical imaging in living systems will be realized in near future.

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Acknowledgements This research was supported by Real-Time Molecular Imaging Project, M.D.-Ph.D. Program (2010-0019863, 2010-0019864) of MEST, Fusion Technology Project (2009-0081876) of MEST, and National R&D Program for Cancer Control of Ministry for Health and Welfare from Republic of Korea (1020260).

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219 Chan Kwon. His research interests include molecular imaging and drug/gene delivery for diagnosis and therapy. Myung Sook Huh received her Ph.D. in department of microbiology and immunology, Seoul National University, College of Medicine, Korea under the supervision of Dr. Ik-Sang Kim. Her previous work included the development of novel anti-bacterial vaccine development and drug delivery systems. She joined Dr. Kwon’s group at Biomedical Research Center at Korea Institute of Science and Technology, KIST in 2008 as a postdoctoral researcher. Currently her research interests are development of molecular imaging nanoprobes and novel gene therapeutics for cancer therapy. Ju Hee Ryu is a Ph.D. candidate in School of Chemical and Biological Engineering from Seoul National University under the guidance of Dr. Byung-Soo Kim. Her previous work includes the development and characterization of smart optical sensor at the Biomedical Research Center of Korea Institute of Science and Technology (KIST) under the mentorship of Dr. Ick Chan Kwon. With a background in molecular imaging and nanotechnology, she is focused on development of novel nanoplatforms for diagnosis and therapy. Dong-Eun Lee received his Ph.D. from the department of biological sciences at Korea Advanced Institute of Science and Technology (KAIST) in Korea, under the supervision of professor Hak-sung Kim. He joined the Radioisotope Research Division, Basic Science and Technology Department, Korea Atomic Energy Research Institute (KAERI) as a postdoctoral researcher. He then moved to Biomedical Research Center, Korea Institute of Science and Technology (KIST). Based on the background in radiodiagnostic imaging techniques for nuclear medicine, he is focused on the development of radiolabeled nanoparticles for molecular imaging and therapy of cancer. In-Cheol Sun is currently a research scientist in Biomedical Research Center at Korea Institute of Science and Technology (KIST). He received his B.S. and M.S. degrees in the department of materials science and engineering of Seoul National University in 2008 under the guidance of Dr. Cheol-Hee Ahn. His works include designing and developing of molecular imaging probes and therapeutic agents using nanoparticles. Kuiwon Choi is currently a principal research scientist in Biomedical Research Center at Korea Institute of Science and Technology (KIST). He received his B.S. and M.S. degrees from the college of engineering at Seoul National University and his Ph.D. in Bioengineering from University of Michigan in 1991. After a post-doctoral training in Bone and Joint Center at Henry Ford Hospital in Detroit, he joined KIST in 1993. He served as a Head of Biomedical Research Center for 4 years and as an Editor-in-chief of the Journal of Biomedical Engineering Research for 6 years (1998—2003). He served as a president of the Korean Society of Biomechanics (2006—2007), and also as an International

220 Advisory Committee Member of Asian Pacific Association of Biomechanics. His main research interest is medical device systems and is now expanding to the development of new diagnostic and therapeutic systems utilizing molecular imaging techniques. Kwangmeyung Kim is a senior research scientist at Biomedical Research Center at Korea Institute of Science and Technology, KIST. He received his Ph.D. from the department of materials science and engineering at Gwangju Institute of Science and Technology (GIST) under the supervision of Dr. Youngro Byun in 2003 in Korea. Then he joined Dr. Kwon’s group at KIST and developed cancer specific optical imaging systems. His research are focused on non-invasive cancer specific molecular imaging and therapeutic/diagnostic nanoprobe by development of smart nano-platform technology for future diagnosis and

H. Koo et al. therapy of various diseases. He has published over 80 peer-reviewed papers and 20 patents. Ick Chan Kwon is currently Head of the Biomedical Research Center at Korea Institute of Science and Technology (KIST). He received his Ph.D. in pharmaceutics and pharmaceutical chemistry at the University of Utah in 1993. He serves as a president of the Korean Society of Molecular Imaging, an Associate Editor of the Journal of Controlled Release, Asian Editor of the Journal of Biomedical Nanotechnology and a member of several editorial boards. His current research interests are targeted drug delivery with polymeric nanoparticles and development of smart nanoplatforms for theragnosis. He has published over 180 peer-reviewed papers and has given over 60 national and international invited lectures.